The Gut Microbiota-Associated Metabolite Network in Ischemic Stroke: From Pathophysiological Imbalance to Potential Precision Intervention Strategies.

Introduction

Stroke is one of the leading causes of death and long-term disability worldwide, with an estimated 13 million new cases occurring annually. Among all stroke types, Ischemic Stroke (IS) accounts for approximately 80–85% of cases.1 According to the 2021 Global Burden of Disease Study, there were approximately 7.8 million new cases of IS worldwide in 2021, with an age-standardised incidence rate of 92.4 per 100,000, representing a decrease from 109.8 per 100,000 in 1990.2 Despite this decline in incidence, population ageing and the growing prevalence of risk factors such as hypertension, diabetes, and obesity have led to a continued increase in the absolute number of IS cases. China, in particular, faces a substantial and expanding burden of IS, characterized by rising incidence rates and a trend toward a younger age at onset, making stroke prevention and management a major public health priority.3 Although acute reperfusion therapies, including intravenous thrombolysis and endovascular thrombectomy, have significantly improved stroke treatment, their overall effectiveness remains constrained by narrow therapeutic windows, strict eligibility criteria, incomplete reperfusion, and the risk of haemorrhagic transformation.2 Consequently, many patients continue to experience severe neurological deficits, long-term disability, recurrent stroke, or death.

A deeper understanding of the mechanisms underlying IS and the identification of novel therapeutic targets therefore remain essential.

The pathological mechanisms of IS are currently recognized as involving multiple interconnected processes, including excitotoxicity, calcium overload, oxidative stress, mitochondrial dysfunction, apoptosis, neuroinflammation, and immune dysregulation. However, the complex underlying pathophysiological network of IS has not yet been fully clarified.4 In recent years, mounting evidence has highlighted the important role of the microbiota-gut-brain axis (MGBA) in the onset, progression, and prognosis of IS. The MGBA is a bidirectional communication network that links the gut microbiota with the central nervous system through neural, immune, endocrine, and metabolic pathways.

Through these interconnected mechanisms, the axis helps maintain physiological homeostasis. Growing evidence indicates that gut microorganisms and their metabolites play significant roles in post-stroke pathological processes and may offer promising targets for future therapeutic intervention.5–8

The relationship between IS and the gut microbiota is reciprocal. On the one hand, IS can profoundly disrupt intestinal homeostasis. Following the onset of stroke, autonomic dysfunction, neuroendocrine alterations, systemic inflammation, and immune imbalance may impair gastrointestinal motility, increase intestinal permeability, and compromise epithelial barrier integrity.

These changes can lead to gut microbial dysbiosis.9 Clinically, patients with IS frequently exhibit reduced microbial diversity, depletion of beneficial bacterial populations, enrichment of opportunistic pathogens, altered dietary intake, and increased susceptibility to infection. These changes may further promote bacterial translocation, peripheral inflammatory activation, and systemic metabolic disturbances, ultimately aggravate neurological injury and impair neural repair. On the other hand, alterations in gut microbiota composition and function may in turn influence the severity and prognosis of IS.10 These observations suggest that the interaction between stroke and the gut microbiome is not a simple one-way process but rather a dynamic cycle of mutual regulation.

It is currently widely accepted that, following an IS, brain injury may initially induce gut dysbiosis and intestinal inflammation via autonomic, neuroendocrine, and immune pathways. The resulting microbial imbalance may subsequently exacerbate neuroinflammation, impair barrier function, and amplify peripheral immune activation, thereby aggravating brain injury. This creates a self-reinforcing cycle of “brain injury-gut dysbiosis-metabolic disruption-inflammation amplification-further neurological damage”, which may hinder post-stroke recovery.

Consequently, maintaining gut microbial homeostasis and targeting the MGBA have emerged as promising approaches for stroke prevention, treatment, and rehabilitation.

Gut microbiota-associated metabolites, including short-chain fatty acids (SCFAs), trimethylamine N-oxide (TMAO), bile acids (BAs), tryptophan (Trp) metabolites, and lipopolysaccharide (LPS), may regulate neuroinflammation, immune responses, blood-brain barrier (BBB) integrity, vascular endothelial function, neurotransmitter synthesis, and host metabolism. SCFAs generally exert anti-inflammatory and neuroprotective effects, maintain intestinal barrier integrity, and promote immune homeostasis.4,11 Conversely, TMAO and LPS are often associated with endothelial dysfunction, thrombosis, vascular inflammation, and the amplification of neuroinflammatory responses. Additionally, BAs and Trp metabolites may participate in immune modulation, neuronal signaling, and metabolic regulation through multiple receptor-mediated pathways.4,12,13

Before discussing individual pathways, the terminology employed in this review is clarified. It is important to note that all components of the proposed network are direct microbial metabolites: SCFAs and several tryptophan-derived indoles are produced directly by gut microorganisms; TMAO is a host-microbial co-metabolite generated when microbial trimethylamine (TMA) is absorbed and oxidized in the liver by flavin-containing monooxygenase 3 (FMO3); primary BAs are host-derived and subsequently modified by gut bacteria into secondary BAs; and LPS is a structural component of Gram-negative bacterial cell walls rather than a metabolite per se. In this review, we therefore use the broader term “gut-associated metabolites” to describe this heterogeneous group.

In addition, the biological effects of these molecules are highly context-dependent and vary with disease stage, biological compartment, concentration, diet, renal function, comorbidities, and treatment exposure. Finally, we explicitly distinguish interactions that have been directly demonstrated in ischemic stroke—in patients or in experimental stroke models—from interactions inferred from shared downstream pathways or from evidence obtained in other cardiovascular, metabolic, or inflammatory diseases.

Notwithstanding the rapid expansion of research on gut microbiota-associated metabolites in IS, several limitations remain. The majority of extant studies have focused on individual metabolites and have classified them within relatively simplistic frameworks. For example, SCFAs are commonly regarded as beneficial metabolites, whereas TMAO is often considered harmful.

Despite the fact that this reductionist approach has enhanced comprehension of particular metabolic functions, it fails to capture the complexity of microbial metabolic interactions. In reality, microbial metabolites operate within an integrated and highly interconnected network characterized by synergistic, antagonistic, compensatory, and feedback mechanisms. Their biological effects are influenced by multiple factors, including disease stage, host metabolic status, microbial composition, treatment exposure, and pathological context.

Consequently, the impact of a given metabolite may vary substantially under different conditions.

A number of recent reviews have furnished exhaustive discourse on the roles of gut microbial metabolites, immune signaling pathways, barrier dysfunction, and translational approaches in IS. The present review makes a distinct contribution to the existing literature by focusing on metabolite-metabolite crosstalk as the organising principle. Rather than merely enumerating individual metabolite pathways, the review explores the synergistic, antagonistic, compensatory, and feedback interactions among SCFAs, TMAO, BAs, Trp metabolites, and LPS.

The proposed concept of “gut microbiota-associated metabolite network imbalance” should thus be regarded as an integrative, hypothesis-generating framework rather than an established mechanistic model-one that may help explain discrepancies among previous studies, facilitate the discovery of composite biomarkers and improved risk-stratification tools, and inform the design of multi-target therapeutic strategies.

Within this framework, the present review systematically summarizes current evidence on gut-associated metabolites and their involvement in IS in the context of the MGBA, emphasizing their roles in inflammation, immune homeostasis, vascular function, intestinal and BBB integrity, neuronal injury, and neurological recovery. We further propose a theoretical model of metabolite network imbalance in IS and explore potential interactions among microbial metabolites. The central objective is not to categorize individual metabolites as beneficial or harmful but to elucidate how disturbances of the gut microbiota metabolic network collectively contribute to the occurrence, progression, and prognosis of IS, thereby providing a systematic framework for understanding its complex pathophysiology and a theoretical foundation for future precision diagnosis, prevention, and therapy targeting the gut microbiota metabolic network.

Search Strategy

This is a narrative review based on a structured search of the English-language literature. We searched PubMed, Web of Science for articles published up to 30 June 2026, using combinations of terms related to ischemic stroke, the microbiota-gut-brain axis, gut-associated metabolites, short-chain fatty acids, trimethylamine N-oxide, bile acids, tryptophan metabolism, and lipopolysaccharide. Original research articles, systematic reviews, and meta-analyses addressing the gut microbiota or gut-associated metabolites in ischemic stroke or closely related cerebrovascular conditions were considered for inclusion; conference abstracts, case reports, and non-English-language publications were excluded.

Throughout the review, clinical investigations, experimental animal models, and in vitro mechanistic studies are distinguished by evidence type, and the strength of supporting evidence-whether a finding has been directly demonstrated in ischemic stroke or is inferred from other disease settings-is explicitly indicated.

Gut Microbiota Dysbiosis and Risk Factors in IS

Gut Microbiota Dysbiosis in IS

Gut microbiota dysbiosis following IS is characterized by alterations in microbial composition, diversity, and functionally relevant bacterial taxa. Evidence from both clinical studies and experimental models generally suggests that IS is associated with disturbances in intestinal microecological homeostasis. These alterations typically include an increased abundance of opportunistic pathogens and pro-inflammatory bacterial taxa, reductions in beneficial commensal bacteria and SCFA-producing microorganisms, impairment of intestinal barrier integrity, enhanced systemic and neuroinflammatory responses, and dysregulation of host metabolism.

Although variations exist across studies, the overall findings support the presence of gut microbiota dysbiosis following IS (Table 1 and Table 2). Table 1Summary of Clinical and Cerebrovascular-Risk Studies Examining Gut Microbiota Alterations Relevant to Ischemic StrokeStudyStroke TypeCountrySampling Time After Stroke onsetSequencing MethodAlpha DiversityIncreased TaxaDecreased TaxaKarlsson et al,14Atherosclerotic cardiovascular diseaseSwedenNot reported16S rRNA sequencingNot reportedCollinsellaEubacterium, Roseburia,BacteroidesYin et al,15LAA/TIAChina48 h16S rRNA sequencingIncreasedMegasphaera, Enterobacter,Desulfovibrio, OscillibacterPrevotella, Faecalibacterium,BacteroidesYamashiro et al,16AISJapan24 h16S rRNA sequencingNot reportedLactobacillus ruminis,Atopobium clusterLactobacillus sakei subgroup,lactic acid–producing bacteriaXu et al,17AIS/TIAChina24 h16S rRNA sequencingNot reportedBifidobacterium, Megamonas,Holdemanella, ClostridiumEscherichia–Shigella,BlautiaHaak et al,18AISNetherlands24 h16S rRNA sequencingIncreasedTMAO–producing bacteriaButyrate–producing bacteriaLi et al,19CIChinaNot reported16S rRNA sequencingNo significant changeLactic acid–producing bacteria, Proteobacteria, Actinobacteria,Lactobacillus, LactococcusButyrate–producing bacteria,Firmicutes,Bacteroidetes,Faecalibacterium,Subdoligranulum,Eubacterium rectale group,Roseburia, Lachnoclostridium,ButyricicoccusTan et al,20AISChina48 h16S rRNA sequencingNot reportedLactobacillaceae,AkkermansiaSCFA–producing bacteriaXia et al,21AISChina7 d16S rRNA sequencingNot reportedEnterobacteriaceae, Oscillospira, Parabacteroides, BacteroidaceaeFaecalibacterium,Clostridiaceae, Lachnospira,butyrate-producing bacteriaWang et al,22AISChina7 d16S rRNA sequencingNo significant changeBacteroides,Cronobacter,MegasphaeraBifidobacteriumWang et al,23CIChinaNot reported16S rRNA sequencingDecreasedGammaproteobacteriaBacteroidiaLledós et al,24IS outcomeSpain1–4 d16S rRNA sequencingNo significant changePathogensCommensalsMarsiglia et al,25IS recoveryItalyNot reported16S rRNA sequencingDecreasedSCFA–producing bacteriaRuminococcaceae,ClostridialesLuo et al,26AISChina24 h16S rRNA sequencingNo significant changeEnterococcusFaecalibacterium,Lachnospiraceae,RuminococcaceaeBao et al,27CISChinaNot reported16S rRNA sequencingNo significant changeStreptococcusEscherichia–Shigella, Bacteroides, AgathobacterNotes: (1) Increased and decreased entries include bacterial taxa, bacterial groups, or microbiota-derived indices reported in the original studies. The entry of Karlsson et al (ref.

25) investigated patients with symptomatic atherosclerotic cardiovascular disease rather than overt ischemic stroke. (2) It is retained in this table because symptomatic atherosclerosis constitutes the principal pathological substrate of large-artery atherosclerotic stroke and provides directly relevant human gut-microbiota evidence along the cerebrovascular-risk continuum; it should not be interpreted as an ischemic stroke cohort.Abbreviations: IS, ischemic stroke; AIS, acute ischemic stroke; CI, cerebral infarction; CIS, cerebral ischemic stroke; TIA, transient ischemic attack; LAA, large-artery atherosclerotic ischemic stroke.Table 2Gut Microbiota Changes in Experimental Ischemic Stroke ModelsStudyAnimal ModelSampling TimeSequencing MethodAlpha DiversityIncreased TaxaDecreased TaxaWu et al,28MCAO/R ratsDay 316S rRNA sequencingAlteredProteobacteria,DeferribacteresFirmicutesKurita et al,29MCAO in db/db miceDay 716S rRNA sequencing; qRT-PCRNot reportedNot reportedBifidobacterium,Enterococcus,Atopobium cluster,Clostridium perfringens,EnterobacteriaceaeStanley et al,30MCAO mice24 h16S rRNA sequencingNot reportedAkkermansia muciniphila, Clostridia spp.Not reportedSpychala et al,31MCAO miceDay 716S rRNA sequencingNot reportedIncreased Firmicutes/Bacteroidetes ratioNot reportedJeon et al,32MCAO pigsPre-stroke, days 1–516S rRNA sequencingDecreased (Day 1)ProteobacteriaLactobacillus,Decreased Firmicutes/Bacteroidetes ratioYang et al,33MCAO miceDays 1–916S rRNA sequencingDecreasedLactobacillus, LactococcusDecreased Firmicutes/Bacteroidetes ratioDeng et al,34ALHI pig modelNot reported16S rRNA sequencingNo significant changeBacteroides, Acetitomaculum, Colidextribacter, Corynebacterium, Muribaculaceae, DesulfovibrioButyricicoccus, Parabacteroides, Lactobacillus, BifidobacteriumNotes: Increased and decreased entries include bacterial taxa, bacterial groups, or microbiota-derived indices reported in the original studies. “Not reported” indicates that the data were not provided in the original study.Abbreviations: MCAO, middle cerebral artery occlusion; ALHI, acute large hemispheric infarction; qRT-PCR, quantitative real-time polymerase chain reaction.

Main Characteristics in Clinical Studies

Most clinical investigations have focused on patients with acute IS, cerebral infarction, large-artery atherosclerotic stroke, or transient ischemic attack (TIA), and fecal microbiota profiles have mainly been analyzed using 16S rRNA amplicon sequencing. Overall, these studies generally indicate that gut microbial composition in patients with IS differs significantly from that in healthy controls.

With respect to microbial diversity, findings remain inconsistent. Some studies report reduced α-diversity,22,25 whereas others observe no significant differences19,21,24,26,27 or even increased diversity.15,18 These discrepancies suggest that post-stroke microbial diversity is influenced by multiple factors, including stroke subtype, disease severity, sampling time, comorbidities, diet, and medication use.15,18,20,25,26 In terms of microbial composition, clinical studies have consistently reported an increased abundance of opportunistic pathogens and pro-inflammatory bacterial taxa, including Megasphaera, Enterobacter, Desulfovibrio, Oscillibacter, Enterobacteriaceae, Enterococcus, Streptococcus, Gammaproteobacteria, and Proteobacteria.15,19–22,26,27 In addition, increases in Lactobacillus and Lactococcus have been observed in some cohorts, although these findings are not consistent across all studies.16,19,20 These alterations suggest a shift toward a pro-inflammatory intestinal environment characterized by impaired epithelial barrier function and enhanced systemic inflammation.16,18,20,26

Conversely, reductions in beneficial commensal bacteria and SCFA-producing taxa represent another hallmark of post-stroke dysbiosis. Decreased abundances of Faecalibacterium, Roseburia, Blautia, Bacteroides, Prevotella, Lachnospiraceae, Ruminococcaceae, Subdoligranulum, and Eubacterium rectale group are frequently reported.14,15,17,19,21,23,26,27 Many of these microorganisms are associated with the production of short-chain fatty acids (SCFAs), especially butyrate, as well as the maintenance of intestinal epithelial barrier integrity and the regulation of anti-inflammatory responses. A reduction in their abundance may weaken intestinal barrier protection and exacerbate inflammatory processes following stroke.18–21,25,26

Emerging evidence further suggests that gut microbiota dysbiosis is associated with stroke severity and prognosis.20,25–27 A higher Stroke Dysbiosis Index has been associated with increased Enterobacteriaceae, Parabacteroides, and Bacteroidaceae, along with reduced butyrate-producing bacteria such as Faecalibacterium, Clostridiaceae, and Lachnospira.21 Moreover, patients with poor outcomes tend to exhibit increased abundances of pathogenic taxa and decreased abundances of beneficial commensals.20,25–27

Main Characteristics in Animal Models

Experimental studies demonstrate that cerebral ischemia can induce rapid and dynamic alterations in gut microbiota. Common models include MCAO mice, MCAO/R rats, diabetic stroke models, and porcine stroke models. Microbiota changes can occur within 24 hours after stroke onset and persist for several days.30,32,33

Similar to clinical findings, microbial diversity changes are inconsistent. Some studies report reduced α-diversity,32,33 whereas others observe no significant changes.34 These discrepancies likely reflect differences in model type, age, stroke severity, and sampling time.31–34

In terms of composition, animal studies consistently show increases in potentially pathogenic taxa following stroke. For example, IS is associated with increased Proteobacteria and Deferribacteres and decreased Firmicutes in rodent models.28 Porcine models also exhibit increased Proteobacteria and reduced Lactobacillus.32 In addition, changes in mucosa-associated microbiota, including increased Akkermansia muciniphila, have been reported.30

Reductions in beneficial bacteria are also observed. Decreased abundances of Butyricicoccus, Parabacteroides, Lactobacillus, and Bifidobacterium are reported in several models.29,32,34 However, some taxa, such as Enterococcus, show inconsistent trends across studies, indicating model-dependent variability.29,32–34

Furthermore, aging and stroke severity significantly influence gut microbiota dysbiosis. Aged animals exhibit more pronounced microbial alterations and worse outcomes.31 Recurrent stroke models show greater reductions in diversity and altered Firmicutes-to-Bacteroidetes ratios compared with single-stroke models.33

Overall Summary

Overall, gut microbiota dysbiosis is a common feature following IS. Key characteristics include rapid disturbance of microbial homeostasis, increased pro-inflammatory taxa, reduced SCFA-producing bacteria, and impaired intestinal barrier function.18–21,26,28,30,32,34 Importantly, the severity of dysbiosis is closely associated with stroke severity, aging, and prognosis, suggesting that gut microbiota alterations may actively contribute to disease progression rather than merely representing a secondary consequence of stroke.20,21,25–27,31,33

Risk Factors in IS

IS is a multifactorial disease arising from complex interactions among genetic susceptibility, environmental exposures, metabolic abnormalities, and lifestyle-related factors.35,36 Epidemiological studies conducted worldwide demonstrate that risk factors for IS can generally be categorized into non-modifiable and modifiable factors.37 Non-modifiable risk factors include age, sex, ethnicity, genetic background, and family history, whereas modifiable risk factors include hypertension, diabetes mellitus, dyslipidemia, atrial fibrillation (AF), obesity, hyperhomocysteinemia, smoking, alcohol consumption, and dietary habits.36,38,39 In addition, environmental factors such as air pollution are increasingly recognized as important contributors to stroke risk.40

In recent years, growing evidence suggests that the gut microbiota and its metabolites may contribute to the development and progression of stroke-related risk factors.41–43 Through modulation of inflammatory responses, immune homeostasis, vascular endothelial function, and host metabolic pathways, the gut microbiota may influence susceptibility to IS and modulate stroke outcomes.

Aging

Aging is one of the most important non-modifiable risk factors for IS. Aging profoundly alters the composition, diversity, and metabolic function of the gut microbiota. Compared with young mice, aged mice generally exhibit poorer neurological outcomes after stroke.44 Experimental studies have shown that aged animals exhibit more severe intestinal injury and gut microbiota dysbiosis after stroke, characterized by exacerbated disruption of the intestinal epithelial barrier, extensive translocation of intestinal bacteria to the liver and lungs, and a consequent increase in the risk of sepsis-related mortality.44 In addition, aged stroke mice display a more pronounced intestinal inflammatory response, accompanied by reduced expression of mucins and tight junction proteins, both of which play critical roles in maintaining intestinal barrier integrity.45

Aging is further associated with dysregulated microbial metabolites. Previous studies have shown that SCFA levels decline progressively with age. Spychala et al31 reported that aged mice exhibited an approximately nine-fold increase in the Firmicutes/Bacteroidetes ratio compared with young mice, accompanied by marked reductions in fecal acetate, propionate, and butyrate levels.

Importantly, aging-associated dysbiosis appears to be partially reversible. FMT studies show that transplantation of microbiota from aged donors aggravates stroke severity and increases mortality, whereas transplantation of young microbiota can significantly improve neurological recovery after middle cerebral artery occlusion (MCAO) in aged mice.46 Lee et al47 further demonstrated that administration of SCFA-producing bacterial strains, including Bifidobacterium longum, Clostridium symbiosum, Faecalibacterium prausnitzii, and Lactobacillus fermentum, combined with inulin supplementation, could restore enteric nervous system function, boost mucin production, maintain intestinal barrier integrity, alleviate stroke-induced intestinal injury, and improve stroke outcomes in aged mice.

From an immunological perspective, γδ T cells can exacerbate neuronal injury after cerebral ischemia through the secretion of IL-17. Aged stroke mice receiving FMT from young donors exhibited lower cerebral IL-17 protein expression than those receiving microbiota from aged donors. Moreover, transplantation of a youthful microbiota significantly reduced the number of IL-17-positive γδ T cells in the brain, indicating an attenuation of neuroinflammatory responses.

Although these findings derive exclusively from animal models, they provide proof-of-concept evidence that transplantation of a young gut microbiota can ameliorate aging-related dysfunction of the gut-brain axis and further suggest that targeting age-associated gut dysbiosis may represent a promising therapeutic strategy for improving stroke outcomes in elderly individuals **that warrants evaluation in prospective clinical studies.

Hypertension

Hypertension is one of the most important modifiable risk factors for IS and contributes to stroke pathogenesis through multiple mechanisms, including atherosclerosis, thrombosis, endothelial dysfunction, oxidative stress, microvascular injury, and chronic inflammation. Increasing evidence suggests that gut microbiota dysbiosis and microbially derived metabolites play important roles in blood pressure regulation and the development of hypertension.

Both animal and clinical studies consistently demonstrate a depletion of SCFA-producing bacteria in hypertension.48 Li et al49 compared the gut microbiota composition of individuals with prehypertension, untreated hypertension, and healthy controls, and found significantly lower microbial gene richness and α-diversity in hypertensive subjects. In particular, beneficial bacterial taxa such as Faecalibacterium, Roseburia, and Bifidobacterium were markedly reduced. Because these microorganisms are major SCFA producers, their depletion may contribute to impaired vascular and immune homeostasis.

SCFAs regulate blood pressure through multiple complementary mechanisms. They induce vasodilation via G protein-coupled receptors, modulate renin secretion, and suppress systemic inflammation by maintaining intestinal barrier integrity.50

TMAO has also been implicated in hypertension-related pathophysiology. A meta-analysis involving 6,176 participants reported that circulating TMAO levels were positively associated with hypertension risk in a dose-dependent manner, with each 5 μmol/L increase in circulating TMAO levels associated with a 9% higher risk of hypertension.51 In experimental rodent models, excessive salt intake increased circulating and central nervous system TMAO levels, thereby enhancing oxidative stress and inflammatory signaling in the hypothalamic paraventricular nucleus and promoting sympathetic activation and the development of hypertension.52

In addition to SCFAs and TMAO, BAs may also participate in blood pressure regulation. Animal studies have demonstrated that BAs can attenuate vascular reactivity and lower blood pressure in spontaneously hypertensive rats.53 Furthermore, clinical studies have shown that ursodeoxycholic acid supplementation significantly reduced diastolic blood pressure in healthy individuals.54 Collectively, these findings suggest that bile acid metabolism represents an additional mechanistic link between the gut microbiota and blood pressure homeostasis.

Diabetes

Diabetes mellitus represents a major independent risk factor for IS and contributes to stroke development through multiple mechanisms, including endothelial dysfunction, accelerated atherosclerosis, insulin resistance, oxidative stress, and coagulation abnormalities. Patients with type 2 diabetes mellitus (T2D) exhibit approximately 1.5-to 3-fold higher stroke risk than non-diabetic individuals and generally experience poorer outcomes after stroke.55,56

SCFAs exert multiple protective effects on glucose metabolism. Experimental studies show that butyrate supplementation can improve insulin sensitivity, reduce inflammatory responses, alleviate hepatic steatosis, enhance glucose tolerance, and promote β-cell proliferation and insulin secretion in diabetic models.57,58 In high-fat diet-fed mice, butyrate administration can prevent obesity-associated insulin resistance and improve metabolic homeostasis.59

TMAO is increasingly implicated in diabetes-related metabolic abnormalities. A large Chinese cohort study reported that elevated serum TMAO levels were associated with higher fasting blood glucose levels and an increased risk of T2D, even after adjustment for dietary and lifestyle factors.60 Chen et al61 reported that TMAO activates the endoplasmic reticulum stress kinase PERK, leading to upregulation of FOXO1 expression and thereby promoting diabetes progression. In addition, TMAO impairs glucose tolerance by inducing β-cell apoptosis, inhibiting calcium signaling, and reducing SERCA2 expression, ultimately contributing to pancreatic β-cell dysfunction and metabolic deterioration.62

Dyslipidemia

Dyslipidemia contributes to IS primarily by promoting atherosclerosis and vascular occlusion. Increasing evidence indicates that gut microbiota dysbiosis is closely associated with disturbances in lipid metabolism.

A depletion of beneficial commensals, such as Bifidobacterium and Blautia, is frequently observed in individuals with hypertriglyceridemia, which may promote the development of a pro-inflammatory microenvironment in both the gut and the brain.63 Under hyperlipidemic conditions, increased levels of certain metabolites may reflect a compensatory response to inflammation. However, when TG levels remain persistently elevated, pro-inflammatory processes may become predominant, thereby exacerbating neurological injury and leading to higher NIHSS scores.27,64

Multiple studies in mouse models and cellular experiments have demonstrated that supplementation with SCFAs can reduce total cholesterol and TG levels.65–67 SCFAs exert hypolipidemic effects by regulating adipocyte metabolism, enhancing cholesterol clearance, and improving energy homeostasis.65–67 In parallel, BAs act as key regulators of lipid metabolism through nuclear receptor signaling pathways, including the farnesoid X receptor (FXR) pathway.68 Upon activation, hepatic FXR can downregulate the transcription factor SREBP-1c, thereby inhibiting de novo lipogenesis and reducing TG levels.68 It can also promote free fatty acid oxidation via the PPARα pathway, induce HDL synthesis, increase ApoC-II activity, and decrease very-low-density lipoprotein (VLDL) secretion.69,70 While activation of FXR may improve certain metabolic parameters, it can also produce adverse lipid changes.71 For example, obeticholic acid may improve hepatic fibrosis, but it can simultaneously increase LDL-C and total cholesterol levels while reducing HDL-C levels. These findings indicate that therapeutic targeting of bile acid signaling pathways will require precise and individualized approaches.

AF

AF is one of the most prevalent cardiac arrhythmias and markedly increases the risk of cardioembolic stroke through promotion of intra-atrial thrombus formation and embolic events. AF accounts for approximately 15% of all strokes and increases stroke risk by nearly five-fold.72

Accumulating evidence suggests that gut microbiota-associated metabolites contribute to the initiation and maintenance of AF through modulation of atrial electrical remodeling, autonomic remodeling, structural remodeling, and calcium homeostasis, thereby increasing the likelihood of ectopic firing and facilitating the formation and maintenance of reentrant circuits.73 TMAO has emerged as a key mediator in this process. Experimental studies have demonstrated that TMAO can aggravate atrial electrical remodeling by activating the p65/NF-κB signaling pathway and increasing inflammatory cytokine production within atrial ganglionated plexi, thereby promoting AF maintenance.27 Furthermore, TMAO may enhance atrial fibrosis through activation of the Wnt3a/β-catenin signaling pathway and exacerbate diabetes-associated AF by promoting atrial inflammation and connexin remodeling.74,75

Environmental factors further modulate these pathogenic effects. For example, experimental studies in rodents have reported that stressors such as cold exposure alter gut microbiota composition, increase TMAO levels, and exacerbate inflammatory and structural remodeling processes, thereby increasing AF susceptibility.

Although several studies have reported associations between elevated TMAO levels and thromboembolic risk in patients with AF, the current evidence remains limited. Therefore large-scale prospective clinical studies are warranted to further elucidate the prognostic value of TMAO in AF-related stroke risk stratification.

Obesity

Obesity constitutes a well-established independent risk factor for atherosclerotic cardiovascular disease. Obese individuals frequently exhibit gut microbiota dysbiosis, characterized by an increased Firmicutes-to-Bacteroidetes ratio and reduced abundances of SCFA-producing bacteria and Bifidobacterium.76

Gut microbiota-associated metabolites contribute to obesity through multiple mechanisms. SCFAs regulate adipogenesis and energy homeostasis through activation of carbohydrate response element-binding protein (ChREBP) and sterol regulatory element-binding protein 1 (SREBP1), while inhibition of fasting-induced adipose factor (FIAF) may enhance triglyceride storage.77 In addition, excessive acetate production may promote hepatic lipogenesis and metabolic dysfunction.77

Plasma TMAO levels positively correlate with body mass index, visceral adiposity index, and fatty liver index.78 Mechanistically, TMAO promotes adipocyte differentiation, increases pro-inflammatory cytokine production in adipose tissue macrophages, and impairs reverse cholesterol transport.79,80 Inhibition of TMAO-generating pathways, including targeting microbial cutC enzymes or FMO3 expression, may alleviate obesity-related metabolic abnormalities and improve energy expenditure.15

Experimental studies have demonstrated that supplementation with ursodeoxycholic acid alleviates diet-induced obesity in mice subjected to maternal undernutrition during gestation,81 whereas dietary acetate inhibits high-fat diet-induced obesity in mice through regulation of taurine-conjugated bile acid metabolism.82 Furthermore, Watanabe et al83 demonstrated that bile acid supplementation can increase brown adipose tissue energy expenditure, prevent obesity, and improve insulin resistance. These findings suggest that bile acid metabolism represents an important mechanism through which the gut microbiota modulates obesity-associated stroke risk.

Diet

Diet is a pivotal environmental factor that shapes gut microbiota composition and metabolic activity. Different dietary patterns may influence stroke through their effects on gut microbiota structure, microbial metabolite production, inflammation, and metabolic homeostasis.

High-fat and pro-inflammatory diets can reduce the abundance of SCFA-producing bacteria, promote inflammatory responses,84 and increase circulating TMAO levels through alterations in gut microbiota composition and FMO3 expression.85 In contrast, the Mediterranean diet has been consistently associated with a reduced risk of stroke. Characterized by high intake of fruits, vegetables, legumes, whole grains, nuts, and olive oil, this dietary pattern may reduce inflammation, improve endothelial function, regulate lipid and glucose metabolism, and promote a beneficial gut microbial profile.86,87

Dietary fiber plays a particularly important role because it serves as a primary substrate for microbial SCFA production.88 Increased fiber intake has been shown to improve blood pressure regulation, lipid metabolism, and systemic inflammation while promoting intestinal barrier integrity and gut microbial homeostasis.89 Dietary fiber may reduce TMA and TMAO production by limiting the availability of choline-related precursors within the intestinal lumen.90 Collectively, these findings highlight the critical role of diet-gut microbiota interactions in stroke pathogenesis and suggest that dietary modulation of the gut microbiota may represent a promising strategy for stroke prevention and post-stroke rehabilitation.

Pharmacological Interventions

Pharmacological modulation of the gut microbiota has emerged as a pivotal area of research in studies of the MGBA. Under specific clinical conditions, antibiotics may help control severe infections and indirectly influence post-stroke inflammatory responses.91 However, indiscriminate antibiotic use may disrupt microbial homeostasis and precipitate secondary dysbiosis.92

Several gut-targeted therapeutic agents have shown potential neuroprotective effects. Rifaximin has been reported to increase beneficial bacterial taxa, including Ruminococcaceae and Lachnospiraceae, elevate cerebral butyrate levels, and promote anti-inflammatory microglial responses.93 In addition, novel microbiota-targeted nanomedicine approaches have demonstrated promising therapeutic potential. Turmeric-derived nanovesicles have been shown to alleviate inflammation by reshaping gut microbiota composition, restoring intestinal barrier integrity, and regulating macrophage polarization.94

Traditional Chinese medicine (TCM) has also attracted increasing attention because of its capacity to modulate gut microbiota composition and intestinal immune homeostasis. Several TCM formulations have been reported to promote gut microbial homeostasis by enriching beneficial bacterial taxa and reducing pathogenic colonization, while simultaneously exerting antioxidant, anti-inflammatory, and neuroprotective effects. For instance, Ganlu Yin (GLY) ameliorates gut dysbiosis through enrichment of Firmicutes and reduction of Proteobacteria- and Bacteroidetes-associated taxa, enhances intestinal barrier integrity, and suppresses gut-derived LPS/TLR4/NF-κB signaling.95 Similarly, Coptidis Rhizoma and Magnoliae Officinalis Cortex have been shown to increase the abundance of beneficial genera, including Akkermansia, Allobaculum, Alloprevotella, and Blautia, while reducing potentially pathogenic bacteria such as Shigella and Clostridium sporogenes.96 Collectively, these findings suggest that microbiota-targeted pharmacological interventions may represent promising strategies for stroke prevention, treatment, and rehabilitation, although none of these interventions has yet been validated for improving stroke outcomes in prospective randomized controlled trials.

Protective Metabolite Network

SCFAs

Sources and Receptors

SCFAs are major microbial metabolites produced through the fermentation of indigestible carbohydrates, particularly dietary fiber, by the gut microbiota. Acetate, propionate, and butyrate are the predominant SCFAs in the human intestine, with an approximate molar ratio of 60:20:20 in the colon.97 Acetate is primarily generated by anaerobic bacteria via glycolysis-related pathways and acetyl-CoA metabolism.76,88 Beyond its role in intestinal carbohydrate metabolism, acetate enters systemic circulation and contributes to lipid synthesis, glucose metabolism, and energy homeostasis in peripheral tissues and the central nervous system.76,88 Propionate is mainly produced through the succinate pathway during carbohydrate fermentation and is largely taken up by the liver after absorption, where it participates in gluconeogenesis and lipid metabolic regulation.76,88 Butyrate is synthesized from acetate, lactate, amino acids, and various carbohydrates via pathways such as the butyryl-CoA:acetate CoA-transferase pathway and the phosphotransbutyrylase/butyrate kinase pathway. Notably, butyrate serves as the primary energy source for colonocytes and plays essential roles in maintaining intestinal epithelial homeostasis, promoting mucosal repair, and preserving barrier integrity.76,88

SCFAs have received increasing attention as key mediators linking gut microbiota dysbiosis to the pathogenesis of IS. Human studies consistently report that circulating and fecal SCFA levels are altered in patients with IS, although the direction and magnitude of change are not uniform across cohorts and depend on sampling time, stroke subtype, and host inflammatory status. Yamashiro et al16 reported significant changes in acetate and valerate concentrations in stroke patients, with acetate levels negatively correlated with glycated hemoglobin and low-density lipoprotein cholesterol, whereas valerate levels were positively associated with C-reactive protein and leukocyte counts.

Henry et al98 found that elevated circulating SCFA levels during the acute phase of stroke were linked to increased inflammatory markers, delayed functional recovery during hospitalization, and poorer functional outcomes at discharge. Similarly, Chou et al99 observed that abnormal increases in circulating SCFAs were associated with severe post-stroke complications. Complementary human evidence is now emerging from acute cerebrovascular injury more broadly.

A recent two-timepoint LC-MS study of patients with aneurysmal subarachnoid hemorrhage (aSAH) reported that circulating SCFAs and tryptophan-derived metabolites measured serially were independently associated with functional outcome and delayed cerebral ischemia, and that serial measurements added prognostic value beyond established clinical predictors.100 Another clinical study found that butyrate-producing bacterial taxa differed between ruptured and unruptured intracranial aneurysms.101 Although these data derive from hemorrhagic rather than ischemic stroke and therefore cannot establish causality in IS, they provide direct human support for the prognostic relevance and temporal dynamics of SCFA-related metabolites in acute cerebrovascular disease, and motivate analogous prospective studies in IS.

The biological activities of SCFAs are primarily mediated through G protein-coupled receptors (GPCRs), particularly FFAR2 (GPR43) and FFAR3 (GPR41).57 All three SCFAs activate these receptors, although their tissue distribution and downstream signaling differ.76 GPR43 is highly expressed in intestinal epithelial cells, adipose tissue, and immune cells, functioning as a key metabolic sensor. Experimental studies show that GPR43-deficient mice develop obesity even under normal dietary conditions, whereas adipose tissue-specific overexpression of GPR43 confers resistance to diet-induced obesity.102 Activation of GPR43 by SCFAs inhibits insulin signaling in adipocytes, reduces lipid accumulation, and enhances systemic glucose and lipid metabolism. Furthermore, GPR43 activation suppresses oxidative stress and NF-κB signaling, partly by enhancing β-arrestin-2 and IκBα interactions, thereby alleviating inflammatory injury.102,103

In contrast, GPR41 is more closely associated with neural and immune regulation. SCFA-mediated activation of GPR41 regulates lipid metabolism and promotes IL-22 production by CD4⁺ T cells and innate lymphoid cells, thereby preserving intestinal epithelial integrity and maintaining mucosal immune homeostasis.104 In addition to GPR41 and GPR43, HCAR2 (GPR109A) is another important SCFA receptor, predominantly activated by butyrate and β-hydroxybutyrate.76 HCAR2 is expressed on colonic epithelial cells, macrophages, and dendritic cells and mediates multiple anti-inflammatory and immunomodulatory effects, including induction of regulatory Tregs and promotion of IL-10 production.105,106 These pathways are essential for maintaining intestinal immune tolerance and preventing excessive inflammatory activation.

Pleiotropic Protective Mechanisms

Anti-Inflammatory and Immunomodulatory Effects

Neuroinflammation is a hallmark of IS pathology. Cerebral ischemia rapidly triggers activation of microglia and astrocytes, accompanied by infiltration of peripheral immune cells, including neutrophils, monocytes/macrophages, and T lymphocytes, into the ischemic brain.107 Although a controlled inflammatory response facilitates debris clearance and tissue repair, excessive or prolonged activation contributes to neuronal damage and worsens neurological outcomes.107

The anti-inflammatory properties of SCFAs are mainly mediated through activation of GPCRs and inhibition of histone deacetylases (HDACs). Engagement of GPR41 and GPR43 suppresses pro-inflammatory signaling pathways, particularly NF-κB, thereby reducing the production of cytokines such as TNF-α, IL-1β, and IL-6. In middle cerebral artery occlusion (MCAO) models, sodium butyrate exerts neuroprotective effects through activation of the GPR41/Gβγ/PI3K/Akt pathway.108 Acetate and butyrate have also been shown to improve endothelial function by limiting oxidative stress and enhancing nitric oxide bioavailability.109 Importantly, most of these mechanistic insights derive from experimental models and in vitro studies, and direct confirmation of GPCR- and HDAC-dependent SCFA effects in patients with IS remains limited.

Among SCFAs, butyrate is recognized as a potent HDAC inhibitor. HDAC suppression promotes histone hyperacetylation and alters the transcription of genes involved in inflammatory responses.110 Experimental evidence indicates that sodium butyrate attenuates microglial activation through epigenetic mechanisms, enhances IL-10/STAT3 signaling, and reduces LPS-induced expression of TNF-α, IL-6, Nos2, and Stat1.111

Microglia are particularly responsive to metabolites derived from the gut microbiota. Distinct phenotypic and functional characteristics have been reported between microglia isolated from germ-free mice and those from specific pathogen-free animals. SCFAs, predominantly acetate, restore the maturation-related phenotypes of microglia by remodeling their morphology, transcriptome profiles, and mitochondrial metabolism to correct innate immature states during development.112,113 In inflammatory microenvironments, acetate-derived acetyl-CoA elevates histone acetylation, suppresses the pro-inflammatory MAPK and NF-κB signaling cascades, reduces the expression of pro-inflammatory mediators (IL-1β and TNF-α), and increases the levels of anti-inflammatory and homeostatic factors (TGF-β1 and IL-4).114 Such modifications reverse LPS-triggered excessive M1 polarization and normalize microglial immune surveillance functions.

Notably, although acetate supplementation effectively rescues microglial immaturity in germ-free mice, it fails to fully recapitulate the complete microglial characteristics of specific-pathogen-free mice. Furthermore, current evidence is mainly obtained from animal and in vitro experiments, with insufficient clinical data to validate these findings in humans. Comparable neuroprotective effects have been observed with butyrate, largely through HDAC inhibition and enhancement of anti-inflammatory signaling cascades.111

Beyond innate immunity, SCFAs also influence adaptive immune responses after stroke. Following cerebral ischemia, T lymphocytes infiltrate the central nervous system and participate in secondary brain injury.115 γδ T cells are particularly detrimental during the acute phase because of their production of IL-17.116 Administration of SCFA-producing bacteria or prebiotics such as inulin strengthens intestinal barrier function, expands Treg populations in both the gut and brain, and limits the accumulation of IL-17-producing γδ T cells within ischemic lesions in aged stroke mice.47 These immunoregulatory effects are thought to involve modulation of the mTOR-S6K signaling axis together with HDAC-dependent epigenetic mechanisms.116

Notably, however, clinical evidence on the relationship between circulating SCFA levels and IS outcomes remains conflicting, and some findings appear to contradict the predominantly protective role described above. In a study using the Blood and Clot Thrombectomy Registry and Collaboration, Henry et al,98 measured plasma concentrations of acetate, propionate, isobutyrate, butyrate, and 2-methylbutyrate in arterial blood collected at mechanical thrombectomy from 53 patients with acute ischemic stroke. Circulating SCFA levels at thrombectomy were not associated with stroke severity at presentation, as no significant correlations were found with admission NIHSS scores, infarct volume, or edema volume.

Acetate was the only SCFA that differed significantly (n = 12, P = 0.02) and the only one positively correlated with time from stroke onset. Regarding subacute outcomes, isobutyrate was positively correlated with discharge NIHSS scores (P = 0.018), whereas propionate was negatively correlated with the percentage change in NIHSS from admission to discharge (P = 0.036), indicating worse symptom burden at discharge and less functional recovery during hospitalization. Mechanistically, multiple SCFAs were positively correlated with systemic and intracranial IL-6 levels-which themselves correlated with admission NIHSS scores-representing the most consistent associations.

These findings suggest that elevated circulating SCFAs in the acute phase of stroke may not be uniformly protective, and that their biological effects are highly context-dependent, varying with SCFA subtype, concentration, disease stage, and host inflammatory status. The authors proposed that the early rise in plasma SCFAs may reflect displacement of these metabolites from the gut into the circulation following acute gut dysbiosis, or a compensatory anti-inflammatory host response, although sampling at thrombectomy may be too early to fully capture these dynamics. Collectively, this study highlights the discrepancy between experimental and clinical findings and cautions against directly extrapolating the neuroprotective effects of SCFAs from animal models to human IS patients.

Preservation of BBB Integrity

The BBB is a critical component of central nervous system homeostasis. Disruption of gut microbial composition and intestinal inflammation can increase circulating levels of pro-inflammatory cytokines, including TNF-α, IL-1β, and IL-6, which impair endothelial tight junctions and promote BBB leakage. As a consequence, inflammatory mediators and peripheral immune cells gain greater access to ischemic brain tissue, amplifying neuronal injury.

SCFAs can penetrate the BBB through monocarboxylate transporters, particularly MCT-1, and have been detected in both cerebrospinal fluid and brain tissue.117 Growing evidence suggests that these metabolites contribute to BBB stabilization following cerebral ischemia. In experimental models, butyrate supplementation promotes angiogenesis and strengthens barrier integrity, at least in part through enhanced IL-22 production.118 Activation of IL-22 signaling increases the expression of tight-junction proteins, including occludin, claudin-5, and ZO-1, facilitating restoration of BBB function.118

Studies in aged female rats have shown that delayed administration of sodium butyrate attenuates blood-brain barrier permeability after ischemic stroke, suppresses inflammatory responses, reduces infarct volume, and ameliorates sensorimotor deficits.119 Matrix metalloproteinases (MMPs), especially MMP-9, are major contributors to BBB breakdown because they degrade tight-junction proteins. By suppressing NF-κB signaling and stimulating Nrf2-dependent antioxidant responses, SCFAs limit MMP expression and help preserve barrier structure.120

Beyond their effects on the BBB, SCFAs also protect neuronal viability. Activation of the GPR41/PI3K/Akt signaling cascade has been linked to reduced neuronal apoptosis following SCFA treatment (especially sodium butyrate).108 Furthermore, supplementation with short-chain fatty acids upregulates the expression of tight junction proteins (occludin and ZO‑1), preserves the integrity of intestinal and blood‑brain barriers, mitigates neuroinflammation, and ameliorates behavioral dysfunction in mice with sepsis-associated encephalopathy.121 Studies also reported that propionate can act on human brain microvascular endothelial cells, remodel the transcriptome and regulate BBB-related genes, thereby strengthening the capacity of the blood-brain barrier against inflammatory injury.122

Notably, the above conclusions are predominantly derived from animal models and in vitro studies, and sufficient clinical evidence is still lacking to validate the essential role of SCFAs in maintaining intestinal barrier and blood-brain barrier integrity after ischemic stroke.

Enhancement of Neural Plasticity

Cerebral ischemia disrupts energy metabolism and causes neuronal injury, resulting in functional impairment. Brain-derived neurotrophic factor (BDNF) is a key regulator of neurogenesis, synaptic remodeling, and neuronal survival, and decreased BDNF levels are associated with adverse functional outcomes.

Butyrate serves as a crucial mediator linking gut permissive metabolism to BDNF expression.64,123 By inhibiting HDAC activity, butyrate maintains an open chromatin configuration and enhances BDNF transcription.63 Animal studies show that butyrate alleviates depression-like behaviors and promotes neurogenesis.124 In addition, microbiota-derived neurotransmitters, including serotonin, γ-aminobutyric acid, and dopamine, can indirectly influence neuroplasticity through neuroimmune and neuroendocrine pathways.125

Synaptic remodeling is another critical component of recovery. Cerebral ischemia reduces the expression of synaptic proteins such as PSD-95 and synapsin-I.126 Animal experiments show that FMT and SCFAs supplementation can reverse the downregulation of the aforementioned synapse-related proteins, alleviate microglia-mediated synaptic loss, preserve synaptic plasticity, and thereby promote synaptic repair and the recovery of cognitive function.127

Notably, these findings are derived exclusively from animal studies, and no human clinical trials have confirmed that SCFAs improve post-stroke neurological deficits by upregulating BDNF and modulating synaptic plasticity. Furthermore, the synaptic-related evidence originates from the chronic cerebral hypoperfusion model, whose pathological process differs from that of acute ischemic stroke.

BAs

Sources and Receptors

BAs are cholesterol-derived metabolites involved in a broad spectrum of physiological processes, including lipid absorption, glucose homeostasis, energy metabolism, and immune regulation.128 Primary BAs, namely cholic acid (CA) and chenodeoxycholic acid (CDCA), are synthesized from cholesterol in the liver and then conjugated with glycine or taurine prior to secretion into the intestinal lumen. Within the gut lumen, microbial bile salt hydrolases (BSHs) hydrolyze conjugated BAs to liberate free BAs, which may undergo further transformation via 7α/β-dehydroxylation to form secondary BAs such as deoxycholic acid (DCA) and lithocholic acid (LCA).129

Accumulating evidence suggests that perturbations in BA metabolism are closely associated with the onset and progression of cerebrovascular diseases. Elevated BA excretion may correlate with slower atherosclerotic progression, while prospective studies have reported that low BA excretion could serve as an independent predictor for stroke incidence and stroke-related mortality.130

The biological activities of BAs are largely mediated via FXR and G protein-coupled bile acid receptor 1 (GPBAR1/TGR5).131 While these receptors exhibit high expression levels in the liver and gastrointestinal tract, they can also be detected in the central nervous system, where they contribute to the modulation of neuroinflammation, neuronal survival, neurogenesis, and synaptic plasticity. In this context, the BAs-FXR/TGR5 signaling axis may represent one important mechanism linking gut microbiota-associated metabolites to stroke pathogenesis and post-stroke neurological recovery.

Pleiotropic Protective Mechanisms

Anti-Inflammatory and Immunomodulatory Effects

BAs exert broad anti-inflammatory and immunomodulatory effects through signaling pathways mediated by the FXR and the GPBAR1/TGR5. FXR activation may inhibit the NF-κB signaling pathway, thereby reducing the production of pro-inflammatory mediators such as interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and interleukin-8 (IL-8), as demonstrated in HepG2 cells, primary hepatocytes, and LPS-treated Fxr-null mice.132 In addition, FXR signaling also limits NLRP3 inflammasome activation and decreases the release of IL-1β and IL-18, as shown in in vitro and rodent studies, contributing to the attenuation of inflammatory responses after ischemic injury.133 Of note, direct evidence regarding the anti-inflammatory effects of FXR activation in ischemic stroke remains limited.

TGR5 signaling provides an additional mechanism through which BAs regulate inflammation. Upon activation, TGR5 elevates intracellular cyclic adenosine monophosphate (cAMP) levels and stimulates protein kinase A (PKA), ultimately suppressing NF-κB-dependent inflammatory signaling.134 Through this pathway, BAs restrain NLRP3 inflammasome activity and mitigate downstream inflammatory damage. In non-stroke cerebrovascular models (SAH), TGR5 activation has been shown to alleviate oxidative stress and neuronal apoptosis via the cAMP/PKCε/ALDH2 signaling cascade, and the synthetic TGR5 agonist INT-777 reduces neuronal oxidative injury and apoptosis following SAH135 However, these protective effects have been observed mainly in SAH models; whether TGR5 activation confers comparable benefits in ischemic stroke cannot be directly verified and warrants dedicated investigation.

Beyond their direct effects on inflammatory signaling, BAs influence innate immune responses by regulating macrophage and microglial polarization. Tauroursodeoxycholic acid (TUDCA), a bile acid derivative, activates TGR5 in microglia, leading to increased intracellular cAMP levels, enhanced expression of anti-inflammatory markers, and suppression of pro-inflammatory pathways.136 Activation of TGR5 also favors macrophage polarization toward the M2 phenotype while limiting the pro-inflammatory M1 state, thereby reducing cytokine production and tissue injury.137 In addition, secondary BAs, including DCA and LCA, have been shown to suppress NLRP3-dependent inflammatory responses in several pathological conditions, including systemic inflammation and metabolic disorders.135,138,139 Nevertheless, most of the above evidence originates from in vitro studies and animal models of systemic inflammation or metabolic disease; its direct translatability to ischemic stroke remains to be established.

Bile acid metabolites also participate in the regulation of adaptive immunity. Emerging evidence indicates that secondary bile acid derivatives, such as isolithocholic acid and isodeoxycholic acid, promote the differentiation and expansion of regulatory T cells (Tregs), either directly or indirectly through modulation of dendritic cell function and immunogenicity.139 Given the critical role of Tregs in restraining excessive post-stroke inflammation and facilitating tissue repair, bile acid-mediated immune regulation is likely to contribute substantially to the remodeling of the post-stroke immune microenvironment.140 It should be emphasized, however, that these conclusions are largely inferred from animal experiments, and whether bile acid-mediated immune modulation exerts the same effects in ischemic stroke patients requires further clinical validation.

Dual Regulation of BBB Integrity

The effects of BAs on BBB integrity are complex and exhibit bidirectional regulation that depends on the bile acid subtype, concentration, and pathological state. Hydrophilic BAs, including TUDCA and ursodeoxycholic acid (UDCA), confer pronounced neuroprotective effects and help maintain BBB integrity. Activation of TGR5 enhances BBB function in ischemic brain injury models via BRCA1/Sirt1 signaling and stabilization of tight junction proteins.141

TUDCA has been shown to exert potent neuroprotection in experimental cerebral ischemia. Rodrigues et al142 reported that intravenous administration of TUDCA significantly reduced infarct volume and ameliorated neurological deficits following reperfusion injury. Subsequent studies demonstrate that UDCA suppresses NLRP3 inflammasome-mediated inflammatory signaling through TGR5/PKA pathways, thereby improving both neurological and cognitive outcomes.143 By protecting endothelial cells and attenuating inflammatory activation, TUDCA and UDCA indirectly preserve the structural integrity of the BBB.144

In contrast, highly hydrophobic BAs, such as DCA, may exert cytotoxic effects at elevated concentrations due to their detergent-like properties. Certain BAs can disrupt endothelial cytoskeletal architecture through Rac1-dependent signaling, resulting in increasing BBB permeability.145 For instance, studies have confirmed that taurocholic acid (TCA) can increase blood-brain barrier permeability after ischemic stroke by activating sphingosine-1-phosphate receptor 2 (S1PR2), thereby mediating cerebrovascular endothelial barrier injury.146,147

Nevertheless, most relevant findings are derived from preclinical animal or in vitro endothelial cell experiments. The concentration gradient and dynamic changes of endogenous BAs in the brain parenchyma after ischemic stroke remain unclear, and the dual regulatory characteristic of BAs also brings challenges for clinical transformation, as it is difficult to precisely control the type and local concentration of BAs to selectively activate protective pathways while avoiding barrier-damaging effects.

Synergistic Network Effects of SCFAs and BAs

SCFAs and BAs do not act independently; rather, they appear to form an interconnected metabolic network that is proposed to coordinately regulate intestinal homeostasis, immune responses, barrier integrity, and systemic metabolism. Through multilayered crosstalk within the “gut microbiota-metabolite-host immunity-cerebrovascular injury” axis, SCFAs and BAs are suggested to synergistically influence stroke progression and neurological recovery (Figure 1). It should be noted, however, that this integrated framework is largely assembled from independent lines of evidence obtained in different experimental systems, rather than demonstrated within a single integrated model.

Figure 1Synergistic neuroprotective mechanisms of short-chain fatty acids (SCFAs) and bile acids (BAs) through the gut-brain axis after ischemic stroke. Dietary fiber is fermented by gut microbiota to generate SCFAs (acetate, propionate, butyrate), whereas cholesterol-derived primary BAs (cholic acid, chenodeoxycholic acid) are converted by gut microbiota into secondary BAs. Both metabolite classes cooperatively maintain intestinal barrier homeostasis.

Circulating SCFAs cross the BBB via MCT-1 and engage GPRs while inhibiting HDACs, thereby suppressing NF-κB-driven transcription of NLRP3 inflammasome components and pro-inflammatory cytokines (TNF-α, IL-1β, IL-6). Concurrently, UDCA and TUDCA activate TGR5/cAMP/protein kinase A signaling to promote microglial polarization toward the anti-inflammatory M2 phenotype; FXR signaling modulates the pro-inflammatory M1 phenotype. SCFAs also stimulate CD4⁺ T-cell-derived IL-22, which acts on IL-22 receptors to upregulate tight junction proteins and attenuate ischemic BBB disruption.

At the immune and synaptic levels, SCFAs expand regulatory Tregs, suppress γδ T-cell-driven Th17 differentiation, and promote BDNF expression and synaptic protein (Synapsin-1, PSD-95) synthesis, supporting neuronal survival and synaptic reconstruction. Line-style conventions: Solid green arrows indicate pathways directly demonstrated in experimental ischemic stroke models (eg, transient/ permanent middle cerebral artery occlusion). Blue dashed arrows indicate mechanisms supported by in vitro studies or non-stroke disease models (eg, colitis, cholestatic liver disease, metabolic syndrome) but not yet directly confirmed in stroke.

Red T-shaped (⊥) lines indicate inhibitory relationships.Schematic pathway linking gut microbiota metabolism to blood brain barrier and brain immune signaling. Left section labeled Gut microbiota metabolism contains SCFA Pathway and BAs Pathway. In SCFA Pathway, Dietary Fiber points to Gut microbiota, which points to SCFAs shown as three chemical structures; SCFAs connect by an arrow toward the intestinal epithelium barrier at the center divider.

In BAs Pathway, a liver shape labeled Primary BAs contains Cholesterol and CA, CDCA; arrows lead through Gut microbiota to Secondary BAs. Right section labeled Brain shows BBB at the boundary. SCFAs are shown crossing BBB through a transporter labeled MCT-1, with SCFAs labeled on both sides of the BBB.

SCFAs connect to GPRs and to HDAC, with an inhibitory T shaped line from SCFAs to HDAC. GPRs and HDAC connect by inhibitory lines into NF-kappaB. Below NF-kappaB, upward arrows precede NLRP3, TNF-alpha, IL-1beta and IL-6.

At the top of the BBB region, SCFAs connect to CD4 plus T cell, which connects to IL-22, then to IL-22R, then to Tight junctions proteins with an upward arrow. A labeled arrow, Ischemic BBB disruption, points toward the BBB. BAs connect to FXR and to TGR5.

FXR connects by an inhibitory line toward NF-kappaB. TGR5 connects to cAMP and then to Protein kinase A. Dashed connections from this receptor signaling area point toward two labeled microglia states, M1 pro-inflammatory and M2 anti-inflammatory.

Lower right outputs show SCFAs pointing to Tregs. A gammadeltaT cell is shown next to Th17 with a downward arrow. Dashed arrows lead to BDNF and to Synaptic Proteins, Synapsin-1, PSD-95.

These connect to neuronal survival with an upward arrow and to Synaptic reconstruction with an upward arrow.Pathway diagram of gut microbiota SCFAs and BAs acting on BBB, immune cells and brain after stroke.Abbreviations: SCFAs, short-chain fatty acids; BAs, bile acids; BBB, blood-brain barrier; MCT-1, monocarboxylate transporter 1; CA, cholic acid; CDCA, chenodeoxycholic acid; GPRs, G protein-coupled receptors; HDAC, histone deacetylase; NF-κB, nuclear factor-κB; NLRP3, NOD-like receptor family pyrin domain-containing 3; TNF-α, tumor necrosis factor-α; IL-1β, interleukin-1β; IL-6, interleukin-6; UDCA, ursodeoxycholic acid; TUDCA, tauroursodeoxycholic acid; TGR5, G protein-coupled bile acid receptor; FXR, farnesoid X receptor; cAMP, cyclic adenosine monophosphate; PKA, protein kinase A; M1/M2, classically/alternatively activated microglial phenotypes; IL-22, interleukin-22; IL-22R, interleukin-22 receptor; Tregs, regulatory T cells; BDNF, brain-derived neurotrophic factor; PSD-95, postsynaptic density protein 95.

First, SCFAs and BAs are thought to collaboratively maintain intestinal barrier integrity. Butyrate has been reported to directly fuel epithelial cell metabolism and promote the expression of tight junction proteins via activation of GPCRs and modulation of HDAC activity.118,148 In parallel, BAs have been shown to regulate the expression of barrier-related genes through FXR signaling, decrease epithelial permeability, and suppress local inflammatory activation. These complementary mechanisms are believed to collectively prevent intestinal barrier disruption (“leaky gut”) and reduce systemic inflammatory burden following stroke, although direct evidence causally linking intestinal barrier protection to improved stroke outcomes remains limited.

Second, SCFAs and BAs are proposed to synergistically modulate immune and inflammatory responses. SCFAs have been shown to enhance the differentiation of regulatory Tregs, suppress Th17 responses, and inhibit the activation of macrophages and neutrophils. In contrast, BAs have been reported to suppress NF-κB signaling and NLRP3 inflammasome activation in macrophages, microglia, and dendritic cells via FXR- and TGR5-dependent pathways.

Together, these metabolites may attenuate both peripheral and central inflammatory amplification after stroke. Of note, most of these immunomodulatory effects have been documented primarily in in vitro or non-stroke experimental settings, and their in vivo relevance to ischemic stroke warrants further investigation.

Third, SCFAs and BAs may exert cooperative effects on intestinal endocrine signaling and systemic energy metabolism. Both classes of metabolites have been reported to regulate glucagon-like peptide-1 (GLP-1) secretion through GPCR- and TGR5-mediated mechanisms, thereby potentially influencing insulin secretion, glucose homeostasis, and energy balance.149,150 Additionally, SCFAs are suggested to modulate bile acid synthesis via the FXR-FGF15-CYP7A1 axis, suggesting bidirectional interactions between SCFA and bile acid metabolic pathways-an inference largely derived from downstream pathway analyses that awaits direct validation.151

Finally, SCFAs and BAs are believed to indirectly reinforce each other through modulation of the gut microbial ecosystem. SCFAs lower colonic pH and selectively enrich acid-tolerant, SCFA-producing bacteria, including members of the Lachnospiraceae and Ruminococcaceae families.152–155 Many of these bacteria possess bile acid-transforming capabilities, such as 7α-dehydroxylation.129,156,157 Conversely, secondary BAs exert antimicrobial effects against bile acid-sensitive pathogens while promoting the expansion of metabolically beneficial microbial communities.158 Through these reciprocal ecological interactions, SCFAs and BAs are proposed to collectively shape a gut microbial environment that supports metabolic and immune homeostasis, although the causal direction of these ecological relationships has not been fully resolved.

Despite these advances, direct mechanistic evidence for a clearly defined positive feedback network between SCFAs and BAs remains limited. Most current studies rely on correlation analyses and metabolic network inference, and several of the mechanistic links described above are extrapolated from other tissues or disease models, or inferred from downstream pathway activation rather than demonstrated causality. Future investigations integrating germ-free animal models, defined microbial colonization, isotope tracing, multi-omics approaches, and receptor-specific interventions are required to elucidate the causal mechanisms and key molecular nodes underlying SCFA-BAs interactions in IS.

Injury-Associated Metabolite Network

TMAO

Production and Metabolism

TMAO is a host-microbial co-metabolite generated through a two-step metabolic process. In the first step, intestinal microorganisms metabolize dietary nutrients containing TMA moieties, such as choline, phosphatidylcholine, betaine, and L-carnitine, to produce TMA.159 TMA is subsequently absorbed into the portal circulation and oxidized in the liver by flavin-containing monooxygenases, particularly flavin-containing monooxygenase 3 (FMO3), to form TMAO.159 More than 95% of circulating TMAO is reported to be ultimately excreted largely unchanged through the kidneys.160

Accumulating evidence suggests that TMAO contributes to an injury-associated metabolic network along the “peripheral vasculature-blood-brain barrier-central nervous system” axis. By promoting atherosclerosis, enhancing thrombotic risk, inducing endothelial dysfunction, and amplifying neuroinflammatory responses, TMAO has been implicated in cerebrovascular injury and may accelerate stroke progression. It should be noted, however, that much of the mechanistic evidence supporting these effects derives from experimental models, whereas direct evidence in patients with IS remains limited and requires prospective validation.

Pleiotropic Mechanisms of Injury

Promotion of Atherosclerotic Plaque Formation

Atherosclerosis represents the principal pathological substrate of large-artery atherosclerotic IS. Progressive vascular stenosis impairs cerebral perfusion, whereas plaque rupture accompanied by thrombus formation can directly lead to arterial occlusion and cerebral infarction. Clinical studies have demonstrated that, in patients experiencing very late stent thrombosis, plasma TMAO levels are significantly associated with plaque instability, neoatherosclerosis, and plaque rupture in patients experiencing very late stent thrombosis.161

Endoplasmic reticulum stress (ERS) is increasingly recognized as a critical mechanism driving atherosclerotic progression. In experimental models, TMAO selectively binds to protein kinase R-like endoplasmic reticulum kinase (PERK) and activates the PERK signaling pathway, thereby enhancing ERS responses and accelerating plaque development.162

According to the lipid infiltration hypothesis, the accumulation of circulating cholesterol within the arterial wall constitutes a critical initiating event in atherosclerosis. TMAO impairs reverse cholesterol transport (RCT), the process by which excess cholesterol is transported from peripheral tissues back to the liver for excretion, thereby promoting cholesterol deposition within vascular lesions.163 Although the precise mechanisms remain to be fully elucidated, current evidence suggests that TMAO may disrupt cholesterol homeostasis, at least in part, by interfering with BA metabolism. Experimental studies indicate that TMAO downregulates enzymes involved in BAs synthesis, including Cyp7a1 and Cyp27a1, as well as transporters such as Oatp1, Oatp4, Mrp2, and Ntcp.163 In addition, TMAO suppresses BAs synthesis through activation of the small heterodimer partner (SHP) and FXR signaling pathways, further impeding cholesterol elimination.164

Foam cell formation is another hallmark of atherosclerosis. Following the uptake of oxidized low-density lipoprotein (oxLDL), macrophages undergo lipid overload and transform into foam cells. In vitro studies have shown that TMAO upregulates the scavenger receptor SR-A1, elevates the levels of scavenger receptors including LOX-1 and CD36, and enhances the uptake of oxLDL by macrophages.

It can also downregulate ATP-binding cassette transporter A1 (ABCA1), and this dual effect promotes foam cell formation.165 Animal experiments further confirm that TMAO significantly promotes plaque progression within 8 weeks in high-fat-fed Apoe−/− mice, increases macrophage recruitment, and upregulates CD36 and pro-inflammatory cytokine expression within plaques. In these models, siRNA-mediated CD36 knockdown, as well as MAPK (SB230580) and JNK (SP600125) inhibitors, suppresses CD36 expression and reduces foam cell formation, indicating that the CD36/MAPK/JNK signaling pathway is a key pathway for TMAO-induced foam cell formation.166 Furthermore, TMAO regulates inflammatory molecules such as α2-macroglobulin, apolipoprotein C1, and transmembrane protein 106a, and promotes macrophage polarization toward a pro-inflammatory M1 phenotype through activation of the NLRP3 inflammasome.167 Collectively, these processes exacerbate vascular inflammation and accelerate plaque progression; however, the extent to which these experimentally derived mechanisms translate to human atherosclerotic IS warrants further investigation.

Promotion of Thrombosis

Platelet activation and aggregation are central events in arterial thrombosis.168 Emerging evidence indicates that TMAO enhances platelet reactivity,168–170 and elevated circulating TMAO levels are independently associated with a higher risk of cardiovascular events and all-cause mortality.168 Mechanistic studies, predominantly performed in vitro and in animal models, have clarified that TMAO can mediate abnormal platelet activation and promote thrombosis by activating intracellular Ca2⁺ mobilization in platelets.168

TMAO upregulates the expression of tissue factor (TF) and vascular cell adhesion molecule-1 (VCAM-1) in vascular endothelium in a dose-dependent manner, mediating prothrombotic effects via endothelium-derived TF and exacerbating vascular inflammatory responses.171 In addition, TMAO can activate the NF-κB signaling pathway in human primary coronary endothelial cells, promote NF-κB nuclear translocation, upregulate TF expression and increase TF activity, thereby facilitating thrombin generation; TMAO can also synergistically amplify this effect together with low-dose pro-atherogenic factors such as TNF-α and HMGB1.172 Animal studies further support the role of gut microbiota-dependent TMAO production in driving platelet hyperreactivity and enhancing thrombogenic potential.173 Notably, low-dose aspirin not only suppresses platelet activation but may also reduce circulating TMAO levels, thereby attenuating its prothrombotic effects.174

Induction of Systemic Inflammation and Oxidative Stress

TMAO functions as a pivotal pro-inflammatory and pro-oxidative stress metabolite. A central mechanism involves activation of the NLRP3 inflammasome, a cytoplasmic multiprotein complex expressed in endothelial cells, fibroblasts, cardiomyocytes, and immune cells. Activation of NLRP3 promotes maturation of IL-1β and IL-18 and induces pyroptosis, apoptosis, and necroptosis, collectively contributing to vascular and inflammatory injury.175

TMAO facilitates NLRP3 inflammasome assembly and activation in endothelial cells by promoting mitochondrial reactive oxygen species (ROS) generation and enhancing thioredoxin-interacting protein (TXNIP) binding to NLRP3.176–178 This process facilitates caspase-1 activation and subsequent release of IL-1β and IL-18, thereby amplifying vascular inflammation and endothelial dysfunction; In vitro studies further confirm that TMAO can induce increased inflammation and oxidative stress in endothelial progenitor cells (EPCs), inhibit EPC function, and aggravate vascular endothelial injury.178

In addition, TMAO activates NF-κB signaling through Toll-like receptor (TLR)- and MAPK-dependent pathways, promoting nuclear translocation of NF-κB and upregulating inflammatory cytokines such as TNF-α and IL-6, as well as adhesion molecules including VCAM-1 and ICAM-1.179–182 These alterations enhance leukocyte adhesion and exacerbate vascular inflammatory responses.

Oxidative stress represents another major mechanism underlying TMAO-mediated vascular injury. TMAO escalates intracellular and mitochondrial ROS generation, activates the NLRP3-TXNIP signaling axis, suppresses nitric oxide (NO) production, and impairs endothelial progenitor cell (EPC) function.176,177 TMAO inhibits AMPK activity, thereby downregulating the expression and function of its downstream silent information regulator 1 (SIRT1), resulting in the accumulation of intracellular ROS and amplification of inflammatory responses; either SIRT1 overexpression or the AMPK activator AICAR can reverse TMAO-induced oxidative stress and inflammatory injury.183

Dual Regulation of BBB Integrity

As a small-molecule metabolite, TMAO can cross the BBB, and its effects on BBB integrity appear to be bidirectional and highly context-dependent. Under pathological conditions, elevated TMAO levels downregulate tight junction proteins, such as occludin and ZO-1, increase BBB permeability, and facilitate infiltration of peripheral inflammatory cytokines and immune cells into the brain parenchyma, thereby aggravating neuroinflammation and neuronal injury.79,184

In contrast, TMAO at physiological concentrations stabilizes BBB integrity and protects against inflammatory injury via the tight-junction regulator annexin A1, as demonstrated in integrated in vitro and in vivo models. Long-term exposure to physiological TMAO can preserve cognitive function against inflammatory challenges and restrict astrocyte and microglial reactivity in a brain-region-specific manner, whereas its precursor TMA impairs BBB function and disrupts tight junction integrity.185 These discrepant observations likely stem from differences in TMAO concentration, exposure duration, disease stage, and experimental conditions, indicating that TMAO exerts distinct regulatory effects on the BBB under physiological versus pathological states.

Clinical Relevance

Clinical evidence strongly indicates that elevated plasma TMAO levels are associated with the occurrence, severity, and prognosis of IS, although the reported strength of these associations varies across studies. A meta-analysis of 15 studies comprising 40,061 patients demonstrated that elevated TMAO levels independently predicted poor 3-month functional outcomes after cerebral infarction (OR 1.581, 95% CI 1.259–1.987), and a dose-response meta-analysis further suggested that each 1 μmol/L increment in circulating TMAO was associated with a 12.1% increase in the risk of acute ischemic stroke.186,187 In patients with acute IS, TMAO concentrations have been reported to positively correlate with National Institutes of Health Stroke Scale (NIHSS) scores and serve as an independent predictor of early neurological deterioration, long-term disability, and mortality.188–190

Elevated TMAO levels are also associated with an increased risk of stroke recurrence. A prospective multicenter study in China confirmed that circulating TMAO and choline levels are independent predictors of recurrent stroke, and this association is not significantly modified by antiplatelet or lipid-lowering therapy.191 Notably, the predictive value of TMAO varies across stroke subtypes, with stronger associations observed in patients with small-artery occlusion.

Nevertheless, several caveats warrant consideration. First, contradictory and null findings have been reported. A case-control study found lower circulating TMAO levels in patients with large-artery atherosclerotic stroke or transient ischemic attack than in asymptomatic controls, challenging the unidirectional interpretation that TMAO elevation is uniformly harmful.15 In addition, in the multicenter BIOSIGNAL cohort (n = 1,726), TMAO was not independently associated with recurrent stroke (HR 1.07, 95% CI 0.78–1.47) or major adverse cardiovascular events (HR 0.90, 95% CI 0.74–1.09) after multivariable adjustment, suggesting that the prognostic value of TMAO may be population-dependent.

192 Second, most of the mechanistic evidence linking TMAO to vascular and cerebrovascular injury derives from animal models and cellular experiments, and causal demonstration in patients with IS remains limited; accordingly, proposals that patients with elevated TMAO could be selected for specific dietary or microbiota-targeted interventions should be regarded as hypotheses requiring prospective validation rather than clinically actionable strategies. Third, whether circulating metabolite panels improve risk prediction beyond established clinical models-including age, NIHSS score, vascular risk factors, stroke subtype, and reperfusion status-remains to be determined in prospective studies.

Trp Metabolism

Trp is an essential amino acid obtained primarily from the diet. In addition to its role in protein synthesis, Trp is metabolized through several pathways, generating a wide range of bioactive metabolites. Increasing evidence indicates that Trp metabolism is closely involved in the pathogenesis and progression of IS.

It should be noted that these three pathways differ in their metabolic origin: the KYN and 5-HT pathways are predominantly host enzymatic pathways, whereas the indole pathway generates direct microbial metabolites of host Trp; the host pathways are nevertheless modulated by and interact with the gut microbiota. Three major canonical pathways are implicated in Trp metabolism: the kynurenine (KYN) pathway, the 5-hydroxytryptamine (5-HT) pathway, and the gut microbiota-mediated indole pathway.192 These pathways exert distinct, and in some cases opposing, effects on neurotoxicity, immune regulation, thrombosis, oxidative stress, and BBB stability.

KYN Pathway

The KYN pathway represents the principal route of Trp catabolism, accounting for more than 95% of Trp metabolism. The initial and rate-limiting conversion of Trp is catalyzed by tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO). TDO is predominantly expressed in the liver, whereas IDO is widely distributed in immune cells and peripheral tissues and can be strongly induced by pro-inflammatory cytokines, including IFN-γ, TNF-α, and IL-6.193,194

Following IS, inflammatory activation enhances the activity of IDO and kynurenine 3-monooxygenase (KMO), redirecting Trp metabolism toward the KYN pathway and promoting the generation of metabolites with diverse neurobiological functions. A systematic review of 45 studies (39 preclinical and 6 clinical) concluded that the KYN pathway is consistently activated after stroke and that its metabolites are associated with stroke severity and poor outcome.192 These metabolites can be broadly classified as neurotoxic or neuroprotective, and the balance between these two groups plays a critical role in determining stroke outcomes. In clinical cohorts, increased KYN/Trp ratios-a surrogate marker of IDO activity-have been associated with stroke severity, infarct volume, and long-term functional outcome in a multi-timepoint study, and high K/T ratios have been reported to predict post-stroke cognitive impairment (sensitivity 67%, specificity 86%, AUC 0.730).195,196

Among the neurotoxic metabolites, L-kynurenine (L-Kyn), 3-hydroxykynurenine (3-HK), and quinolinic acid (QA) are major injury-promoting molecules. Elevated L-Kyn concentrations and increased KYN/Trp ratios have been consistently observed following stroke and are positively associated with disease severity.195,197 In experimental MCAO models, exogenous administration of L-Kyn exacerbates histopathological damage and accelerates neurodegeneration.198 Furthermore, 3-HK promotes oxidative stress and apoptosis through excessive free radical production, whereas QA acts as an N-methyl-D-aspartate (NMDA) receptor agonist, triggering excitotoxicity, mitochondrial dysfunction, disruption of glutamate homeostasis, and neuronal death.193,199,200 In addition, QA enhances ROS generation, leading to DNA and protein damage, while simultaneously compromising BBB integrity.201 These events aggravate cerebral edema and establish a self-perpetuating cycle of inflammation, oxidative stress, and neuronal injury in experimental models; the extent to which this cascade operates in human stroke warrants further investigation. Furthermore, a clinical study demonstrated that elevated QA levels were significantly associated with 24-h neurological deterioration (β = 0.49, P = 0.0002).

The study also identified sex-related differences in Trp metabolism, including reduced Trp levels in female patients, suggesting that sex should be considered as an important stratification factor when interpreting KYN pathway-related biomarkers.202

In contrast, kynurenic acid (KYNA) and 3-hydroxyanthranilic acid (3-HAA) exhibit neuroprotective properties. KYNA functions as an NMDA receptor antagonist and modulator of α7 nicotinic acetylcholine receptors, thereby attenuating excitotoxicity, suppressing inflammatory responses, and preserving BBB integrity.203–205 Meanwhile, 3-HAA possesses antioxidant and immunomodulatory activities that may contribute to the alleviation of neuroinflammation.206,207 However, under inflammatory conditions, Trp metabolism is preferentially shifted toward the production of neurotoxic metabolites, resulting in a reduced KYNA/QA ratio and exacerbation of neuronal damage.208 Whether this neurotoxic shift reliably occurs in human stroke, and its net contribution to outcome, require prospective confirmation given the predominantly observational nature of the available clinical evidence.

Beyond its role in metabolic regulation, the KYN pathway is closely involved in immune and inflammatory responses. Following stroke, activated microglia and infiltrating immune cells release elevated levels of IFN-γ, which further stimulates IDO and KMO activity and promotes KYN pathway reprogramming.209 Although certain KYN metabolites exert anti-inflammatory effects,210 other metabolites, especially quinolinic acid (QUIN), can amplify inflammatory signals and oxidative stress and even induce apoptosis of neurons and astrocytes by overactivating NMDA-like receptors, upregulating the expression of inducible and neuronal nitric oxide synthases (iNOS, nNOS), and promoting free radical damage mediated by excess nitric oxide (NO).201 The above bidirectional effects suggest that the regulatory role of the kynurenine pathway in the pathophysiology of stroke is highly complex and markedly microenvironment-dependent, and its ultimate effect relies on the dynamic balance between neurotoxic and neuroprotective metabolites. Nevertheless, most of these mechanistic links have been established in preclinical or in vitro systems, and their direct demonstration in human ischemic stroke remains limited; the clinical relevance of KYN pathway modulation should therefore be regarded as a promising but incompletely validated hypothesis, ideally addressed through prospective, metabolomics-guided human studies.

5-HT Pathway

Trp can be metabolized into 5-hydroxytryptamine (5-HT, serotonin) under the catalysis of tryptophan hydroxylase (TPH). Among TPH isoforms, tryptophan hydroxylase 1 (TPH1) serves as the key rate-limiting enzyme for peripheral 5-HT synthesis. Preclinical animal experiments have confirmed that the mRNA and protein expression levels of TPH1 in brain tissue are markedly upregulated after cerebral ischemic injury, indicating that cerebral ischemia potently activates the peripheral 5-HT synthetic pathway and promotes robust production and release of 5-HT.211 However, since TPH1 is mainly expressed in peripheral tissues, particularly gut enterochromaffin cells, the significance of its upregulation in brain tissue remains to be determined, and the underlying source—whether peripheral or central-needs further clarification.

Functionally, 5-HT elevates thrombogenic potential by mediating abnormal platelet aggregation and inducing vascular smooth muscle contraction, thereby exacerbating cerebral vascular thrombosis and brain ischemic injury; it is therefore considered a candidate regulator contributing to ischemic stroke damage, although direct evidence in human stroke is lacking. Studies by Duan et al211 using the MCAO rat model further verified the upregulation of TPH1 expression following cerebral ischemia, supporting the activation of peripheral 5-HT synthesis under ischemic stress. Nevertheless, this study has certain limitations: it only detected alterations in TPH1 expression without quantitative measurement of the core effector molecule 5-HT and its primary metabolite 5-hydroxyindoleacetic acid (5-HIAA).

Therefore, the hypothesis that TPH1 upregulation promotes thrombosis and aggravates ischemic injury lacks direct experimental evidence, and the reliability of this regulatory mechanism requires further validation. Moreover, the effects of 5-HT are pleiotropic and likely dependent on the biological context, including factors such as vascular bed, disease stage, and platelet activation status. Therefore, its overall impact on ischemic injury and repair cannot be determined based solely on the available preclinical evidence.

In summary, the specific regulatory effects, temporal sequence of action, and molecular mechanisms of 5-HT and its downstream metabolite 5-HIAA throughout the initiation and progression of stroke remain incompletely elucidated, and more systematic investigations are warranted for in-depth exploration.

Indole Pathway

Gut microbiota can metabolize host Trp to produce a variety of indole derivatives with broad biological activities, mainly including indole-3-acetic acid (IAA), indole-3-propionic acid (IPA), and indoxyl sulfate. Unlike the KYN and 5-HT pathways, this pathway generates direct microbial metabolites of host Trp. These gut-derived metabolites act via the gut-brain axis to regulate multiple pathophysiological processes such as oxidative stress, inflammatory response, thrombosis, and neural injury repair, exerting bidirectional regulatory effects on the initiation and progression of IS.

However, direct clinical evidence connecting indole metabolites with human ischemic stroke remains limited. A recent two-timepoint LC-MS analysis in patients with aneurysmal subarachnoid hemorrhage provided preliminary human evidence by demonstrating that serum indole derivatives, together with SCFAs, were associated with functional outcomes and delayed cerebral ischemia. These findings support the potential clinical relevance of microbiota-derived Trp metabolites in cerebrovascular injury.100 Nevertheless, current understanding is still largely based on animal studies and investigations in other disease settings, and the precise direction and functional implications of these metabolic changes require further clinical validation.212

Among them, IPA is a potent antioxidant and neuroprotective mediator with prominent protective effects against cerebral ischemic injury.213–215 Animal studies have demonstrated that IPA levels are markedly decreased in mice subjected to middle cerebral artery occlusion.216 Regulation of gut microbiota or exogenous IPA administration via gavage improves the diversity and structural stability of gut microbiota, reduces infarct volume, and alleviates neurological deficits. Mechanistic investigations confirm that IPA ameliorates ischemic brain injury by optimizing mitochondrial function and upregulating the expression of core antioxidant proteins including peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) and uncoupling protein 2 (UCP2).213 Whether these protective effects translate to human stroke remains to be established, and no prospective human study has yet confirmed IPA as a protective biomarker in patients with IS.

In contrast, indoxyl sulfate and IAA are detrimental indole metabolites with prominent procoagulant, prothrombotic, and microcirculation-impairing effects, which aggravate the pathological progression of ischemic stroke.217,218 These conclusions rest predominantly on experimental studies and on evidence from other disease contexts-particularly chronic kidney disease, in which indoxyl sulfate is a well-characterized uremic toxin-and direct demonstration in human IS remains limited. Moreover, circulating indoxyl sulfate concentrations are strongly dependent on renal function, and its effects are likely dose- and compartment-dependent, underscoring the need for caution when extrapolating these findings to stroke populations. Indoxyl sulfate and IAA establish a prothrombotic microenvironment and increase thrombotic risk by upregulating the tissue factor/Factor VII complex, activating platelets, and damaging the vascular endothelial barrier.217,218 Indoxyl sulfate possesses more extensive pathogenic mechanisms.

On the one hand, it induces phosphatidylserine externalization on cell membranes to enhance the procoagulant activity of erythrocytes, and promotes abundant release of endothelial microparticles (EMP), further accelerating thrombus formation.219,220 On the other hand, indoxyl sulfate triggers erythrocyte shrinkage and disruption of membrane phospholipid asymmetry, mediates programmed erythrocyte death, and severely impairs cerebral microvascular perfusion.221 Moreover, indoxyl sulfate specifically stimulates robust ROS production. By activating the ROS-dependent p38 MAPK signaling pathway, it persistently activates platelets, amplifies prothrombotic effects, and exacerbates microcirculatory disturbance and brain tissue damage after cerebral ischemia.222,223 Overall, the indole pathway exemplifies the context-dependent nature of microbial Trp metabolism, with a “double-edged” profile in which the same metabolic route can produce beneficial metabolites such as IPA or harmful mediators such as indoxyl sulfate and IAA, depending on the tissue environment, metabolite levels, and host physiological state.110

Aryl Hydrocarbon Receptor (AhR) Signaling Pathway

The AhR is a highly conserved ligand-activated transcription factor and serves as the core receptor mediating the regulation of central pathological injury by Trp metabolites. It is widely expressed in vascular endothelial cells, central neurons, and various glial cells including astrocytes and microglia, exerting pivotal regulatory roles in immune differentiation and neuroinflammatory responses.224 Multiple Trp derivatives metabolized by gut microbiota, such as KYNA, IAA, and IPA, can act as endogenous ligands to specifically activate AhR signaling, thereby participating in the regulation of immune disturbance and inflammatory cascades following IS.225,226 Notably, disrupted gut-brain axis homeostasis after stroke triggers gut microbiota dysbiosis, resulting in disturbed Trp metabolism and abnormal secretion of AhR agonists. This further perturbs the activation status of central AhR signaling, forming a vicious cycle that continuously aggravates central nervous injury.

During the pathological progression of ischemic stroke, AhR activation exhibits prominent microenvironment-dependent bidirectional regulatory effects.227 Moderate AhR activation positively maintains immune homeostasis, promotes regulatory Treg differentiation, induces secretion of the anti-inflammatory cytokine IL-10, effectively suppresses excessive inflammatory responses, and confers neuroprotection.227 However, under acute cerebral ischemic injury and secondary neuroinflammatory stress, central AhR is persistently and abnormally activated, initiating a cascade of damaging events. Overactivated AhR markedly upregulates the NADPH oxidase subunit p47, continuously activates the NOX2 signaling pathway, triggers massive burst of ROS in brain tissue, and induces oxidative stress injury.228 Meanwhile, aberrantly high AhR expression in microglia is a core driver of pathological neuroinflammation; it polarizes microglia toward pro-inflammatory phenotypes, overactivates resident myeloid cells in the central nervous system, induces severe vasogenic edema and neurotoxicity, and exacerbates secondary brain damage.229,230 These opposing effects underscore that the net outcome of AhR signaling is highly dependent on ligand identity and concentration, disease stage, cell type, and the inflammatory microenvironment; extrapolation of these findings to human stroke should therefore be cautious.

AhR signaling disturbance mediated by gut microbiota dysbiosis represents an important peripheral mechanism that exacerbates post-stroke inflammatory injury. Imbalanced microbial homeostasis disrupts intestinal immune equilibrium and induces aberrant differentiation and polarization of intestinal pro-inflammatory Th17 cells, a process regulated by pro-inflammatory cytokines including TGF-β, IL-6 and IL-23. Polarized Th17 cells migrate across the BBB into ischemic brain tissue and further activate central AhR signaling, which stimulates robust secretion of IL-17 and IL-22 from Th17 cells.231 Among them, IL-17 acts as a key effector molecule mediating secondary stroke injury.

Mechanistically, IL-17 (predominantly its IL-17A and IL-17F isoforms) binds to the IL-17 receptor on target cells, activates the adaptor protein Act1, recruits TRAF6, and initiates multiple classical pro-inflammatory signaling cascades downstream including NF-κB and MAPK. This significantly promotes the release of pro-inflammatory cytokines and chemokines such as TNF-α, IL-6, CXCL1/2.231,232 These inflammatory mediators recruit abundant neutrophils to infiltrate the central nervous tissue, directly compromise BBB integrity, aggravate inflammatory injury of cerebral vessels and brain parenchyma, and drive continuous progression of IS.

Aberrant AhR activation is a critical trigger for BBB damage and microcirculatory dysfunction. AhR is highly expressed in brain microvascular endothelial cells. Under cerebral ischemia and hypoxia stress, excessive AhR activation in endothelial cells directly induces endothelial dysfunction, which not only markedly increases BBB permeability but also triggers endothelial apoptosis and shedding, destroying the structural integrity of vascular endothelium.

Numerous studies have confirmed that genetic knockout or pharmacological inhibition of AhR activity effectively preserves the expression of core tight junction proteins such as claudin-5, substantially alleviates BBB breakdown after cerebral ischemia and reduces vasogenic injury.233 Meanwhile, AhR hinders normal cerebrovascular vasodilation by inhibiting NO synthesis, exacerbating hypoperfusion in ischemic lesions, further reducing cerebral blood flow and worsening hypoxic-ischemic brain injury.234

Targeted inhibition of AhR signaling has been validated as a promising therapeutic strategy for ischemic stroke. In the MCAO animal model, the specific AhR antagonist TMF effectively upregulates CREB transcriptional activity and modulates the expression of apoptosis-related proteins. It suppresses neuronal apoptosis by reducing the levels of pro-apoptotic proteins p53 and Puma while elevating the anti-apoptotic protein Bcl-X.235 Moreover, TMF treatment significantly reduces infarct volume in mice, inhibits excessive astrogliosis and microglial overactivation, and relieves glia-mediated neuroinflammation and secondary brain injury.235 Furthermore, exogenous supplementation of AhR ligands such as IPA and IAld can effectively normalize aberrant AhR signaling after stroke, ameliorate neurological deficits in aged stroke mice and mitigate neuroinflammatory damage, further supporting the critical regulatory value of AhR signaling in stroke pathogenesis.226

LPS

LPS, also known as endotoxin, is a key structural component of the outer membrane of Gram-negative bacteria. Although LPS is technically a bacterial structural component rather than a metabolite produced by microbial metabolism, it is discussed here because its role in IS injury, mediated through the “leaky gut-systemic translocation-systemic inflammation” axis, is closely interconnected with microbiota-derived metabolic pathways. During the acute phase of IS, gut microbiota dysbiosis promotes the overgrowth of opportunistic Gram-negative pathogens such as Escherichia coli, leading to increased intestinal permeability, a phenomenon known as “leaky gut”.236 Consequently, LPS translocates into the systemic circulation, induces systemic inflammatory responses, and may aggravate cerebral ischemic injury through gut-brain axis-mediated mechanisms.

Of note, this cascade is primarily supported by experimental studies, and direct evidence in human stroke remains limited.

Innate Immune Activation

LPS is a canonical activator of innate immunity. By binding to Toll-like receptor 4 (TLR4), LPS activates the MyD88-dependent signaling pathway and subsequently triggers activation of NF-κB and interferon regulatory factors (IRFs).237,238 This signaling cascade triggers the production of pro-inflammatory cytokines, including TNF-α, IL-1β, and IL-6.239

The systemic release of these inflammatory mediators contributes to host defense. However, it also amplifies vascular inflammation, promotes atherosclerotic progression, enhances platelet activation, and increases thrombogenic risk, thereby plausibly contributing to the occurrence and progression of IS at the mechanistic level.

Disruption of BBB

Although LPS cannot readily cross an intact BBB due to its high molecular weight, systemic LPS elevation indirectly disrupts BBB structural and functional integrity through multiple mechanisms.240 LPS induces downregulation and disorganization of endothelial tight junction proteins such as occludin and claudin-5, while simultaneously upregulating adhesion molecules, including ICAM-1 and VCAM-1. These alterations facilitate leukocyte adhesion and transendothelial migration, leading to increased BBB permeability.

In addition, LPS suppresses endothelial nitric oxide synthase (eNOS) activity, reduces nitric oxide (NO) bioavailability, and impairs vascular relaxation, thereby contributing to endothelial dysfunction.107 Clinical studies show that plasma endotoxin, lipopolysaccharide-binding protein (LBP), and soluble CD14 levels remain significantly elevated during the first week after stroke and correlate with unfavorable short-term outcomes.241 These findings provide indirect clinical evidence supporting a potential role for LPS in post-stroke injury. However, the specific molecular mechanisms underlying BBB disruption described above have yet to be directly validated in patients with stroke.

Dual Effects of LPS Preconditioning

Under specific experimental conditions, low-dose LPS preconditioning elicits ischemic tolerance and exerts neuroprotective effects.242 Animal studies demonstrate that repeated low-dose LPS administration attenuates BBB disruption and inflammatory responses through inhibition of the TLR4-MMP-2/MMP-9 signaling pathway, thereby reducing ischemia-reperfusion injury.243

However, these protective effects are markedly diminished in the presence of metabolic disorders, including type 2 diabetes mellitus. Mechanistic studies suggest that diabetes impairs LPS-induced ischemic tolerance by suppressing interferon-related signaling pathways, including JAK-STAT and IRF7, and is accompanied by enhanced neutrophil infiltration.243

Proteomic analyses indicate that LPS preconditioning may confer neuroprotection via modulation of type I interferon signaling in microglia.244

Synergistic Network Effects

TMAO, KYN pathway metabolites, and LPS do not act in isolation in the pathology of IS. Rather, they appear to constitute a highly interconnected gut inflammation-metabolism axis. It is important to note that these components do not share identical origins.

TMAO is generated through host-microbial co-metabolism, KYN pathway metabolites arise from host Trp catabolism with modulation by the gut microbiota, and LPS is a bacterial structural molecule rather than a microbial metabolite. Accordingly, this axis represents a conceptual framework integrating diverse microbiota-associated molecules, rather than a single metabolic pathway with a common origin. Through multilayered signaling interactions involving oxidative stress, endothelial injury, thrombosis, neurotoxicity, and inflammatory amplification, these microbiota-associated molecules may collectively aggravate cerebral ischemic injury (Figure 2).

Nevertheless, this network-level view should be interpreted with caution, as it currently represents a conceptual framework rather than a fully validated model. Direct evidence for only a limited number of these interactions exists in human ischemic stroke, while much of the supporting data comes from experimental studies or other cardiovascular, metabolic, and inflammatory contexts. Figure 2Gut microbiota-derived trimethylamine N-oxide (TMAO), lipopolysaccharide (LPS), and tryptophan metabolites exacerbate neuronal injury after ischemic stroke.

Dietary choline and L-carnitine are metabolized by gut microbiota into TMA, which is absorbed into the portal circulation and oxidized by hepatic FMO3 to generate TMAO. In parallel, gut-derived LPS translocates across a compromised intestinal barrier (“leaky gut”) into the systemic circulation and activates the TLR4/MyD88/TLR-MAPK cascade, leading to NF-κB nuclear translocation, NLRP3 inflammasome priming, and transcription of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6). NF-κB also upregulates IDO and KMO, shifting tryptophan metabolism toward the kynurenine pathway and generating neurotoxic metabolites (QA, 3-HK, L-kynurenine).

In the circulation, TMAO synergizes with LPS to promote foam cell formation and atherosclerotic plaque progression; tryptophan-derived indole metabolites (IS, IAA) and 5-HT pathway products promote TF/VCAM-1 expression and thrombus formation. Following ischemic stroke, BBB disruption permits circulating inflammatory mediators and neurotoxic metabolites to enter the brain parenchyma and further degrade tight junction proteins. Within the central nervous system, microbial products activate microglia and astrocytes and stimulate NLRP3 inflammasome signaling.

Elevated QA drives NMDA receptor overactivation, excitotoxicity, and mitochondrial dysfunction, culminating in neuronal death and progressive neurodegeneration. Line-style conventions: Solid green arrows indicate pathways directly demonstrated in experimental ischemic stroke models (eg, transient/ permanent middle cerebral artery occlusion). Blue dashed arrows indicate mechanisms supported by in vitro studies or non-stroke disease models (eg, chronic kidney disease for IS/IAA prothrombotic effects; atherosclerotic cardiovascular disease for TMAO) but not yet directly confirmed in stroke.

Red T-shaped (⊥) lines indicate inhibitory relationships.Compartmented pathway schematic labeled Gut, Liver, Blood, BBB and Brain, showing arrows from diet and gut microbiota products to vascular and brain outcomes. Gut compartment: Text reads, Diet (Choline or L Carnitine) with a dashed arrow to Gut microbiota. From Gut microbiota, a dashed arrow points to TMA and a solid arrow points to Trp.

LPS is labeled above the intestinal lining with a vertical arrow pointing downward across the lining. Liver compartment: A liver icon contains labels FMO3 and TMAO. A dashed arrow from TMA enters the liver region and a dashed arrow points downward from the liver to a labeled TMAO structure below.

Blood compartment: TLR4 is labeled at the left with a vertical arrow down to MyD88, then a vertical arrow down to TLR or MAPK, then a dashed arrow right to NF kappa B. From NF kappa B, a solid arrow points right to a cytokine label cluster with upward arrows and the text TNF alpha, IL 1 beta and IL 6. A dashed arrow from the cytokine cluster points down toward the KYN Pathway area.

At the right, IDO and KMO are labeled above a vertical arrow down into KYN Pathway, next to a chemical structure and red text with upward arrow, QA, 3 HK and L Kyn. Additional labeled items in Blood include a starburst labeled ROS connected by dashed arrows to NLRP3 inflammasome and dashed arrows to foam cell and to Atherosclerosis. A red inhibitory T bar line runs from the left side of the Blood region toward the lower left, ending near the label RCT.

Bottom of Blood: Labels read 5 HT Pathway and Indole Pathway, with dashed arrows leading toward labels 5 HT, IS and IAA. A solid arrow points right to TF and VCAM 1, then a solid arrow continues right to Thrombi. BBB region: A vertical barrier is labeled BBB.

To its right, Tight Junctions proteins is labeled next to a green junction icon. Text reads ischemic stroke near a brain icon, with a solid arrow pointing toward Tight Junctions proteins. A vertical arrow points downward along the BBB boundary.

Brain compartment: At the top are labels Microglial activation, Astrogliosis and NLRP3 activation. A vertical arrow points down to a cytokine label cluster with upward arrows and the text TNF alpha, IL 1 beta and IL 6. A vertical arrow continues down to an upward arrow with QA and the word Neurotoxicity.

A vertical arrow continues down through NMDA overactivation, then to Excitotoxicity and Mitochondrial dysfunction, then to Neurodegeneration. Neuronal death is labeled near a neuron drawing on the right.Compartment pathway diagram of gut to liver to blood to BBB to brain via TMAO, LPS and Trp routes.Abbreviations: TMAO, trimethylamine N-oxide; LPS, lipopolysaccharide; TMA, trimethylamine; FMO3, flavin-containing monooxygenase 3; TLR4, Toll-like receptor 4; MyD88, myeloid differentiation primary response 88; NF-κB, nuclear factor-κB; NLRP3, NOD-like receptor family pyrin domain-containing 3; TNF-α, tumor necrosis factor-α; IL-1β, interleukin-1β; IL-6, interleukin-6; IDO, indoleamine 2,3-dioxygenase; KMO, kynurenine monooxygenase; QA, quinolinic acid; 3-HK, 3-hydroxykynurenine; IS, indoxyl sulfate; IAA, indole-3-acetic acid; 5-HT, 5-hydroxytryptamine; TF, tissue factor; VCAM-1, vascular cell adhesion molecule-1; BBB, blood-brain barrier; NMDA, N-methyl-D-aspartate.

At the initial stage of this axis, gut microbiota dysbiosis promotes the expansion of Gram-negative bacteria and compromises intestinal barrier integrity, thereby facilitating systemic translocation of LPS. The activation of the TLR4/MyD88 signaling pathway by circulating LPS has been demonstrated to induce the substantial release of pro-inflammatory cytokines, including TNF-α, IL-1β, and IL-6. Although these innate immune mechanisms are well characterized in experimental models, their direct demonstration in human stroke remains incomplete.

It has been hypothesised that this inflammatory milieu exerts a direct detrimental effect on the vascular endothelium but also shifts Trp metabolism toward the KYN pathway through IDO activation, leading to increased generation of neurotoxic metabolites such as 3-HK and QA. In parallel, inflammatory signaling may enhance hepatic FMO3 expression and promotes the oxidation of TMA to TMAO, further amplifying metabolic dysregulation.

At the vascular level, TMAO and Trp-derived metabolites have been reported to jointly promote atherosclerosis and thrombosis. TMAO accelerates plaque formation, foam cell generation, and platelet activation, whereas indole derivatives such as indoxyl sulfate and 5-HT pathway metabolites have been suggested to further potentiate platelet aggregation and endothelial injury. Meanwhile, LPS-mediated activation of NF-κB and MAPK signaling amplifies vascular inflammation, potentially establishing an inflammation-metabolism-thrombosis network.

Importantly, current mechanistic insights into these vascular effects are based primarily on experimental models and findings from related cardiovascular, metabolic, and inflammatory disorders. Direct evidence confirming these mechanisms in human ischemic stroke, however, remains scarce.245

At the BBB level, these metabolites are proposed to exert pronounced synergistic disruptive effects. LPS promotes tight junction protein degradation and increases BBB permeability. TMAO has been reported to aggravate endothelial dysfunction through oxidative stress and NLRP3 inflammasome activation, whereas QA is thought to further disrupts BBB integrity through excitotoxicity and ROS production.

BBB breakdown subsequently facilitates infiltration of inflammatory cells and circulating metabolites into the brain parenchyma, potentially establishing a positive feedback loop that amplifies neuroinflammation.

Within the central nervous system, this metabolic network has been proposed to drive neuronal injury through multiple inflammatory and oxidative mechanisms. TMAO and LPS have been shown to activate microglia and astrocytes and induce NF-κB and NLRP3 signaling, whereas KYN pathway metabolites are thought to promote excitotoxicity, mitochondrial dysfunction, and apoptosis. AhR may serve as a critical molecular hub linking Trp metabolism with immune regulation.

Aberrant AhR activation has been reported to promote Th17 polarization and IL-17 release, further compromising BBB integrity and aggravating cerebral edema and secondary injury.

Importantly, the gut inflammation-metabolism axis is dynamic and bidirectional rather than a strictly unidirectional pathogenic pathway. Certain Trp-derived metabolites, such as KYNA and IPA, have been reported to exert anti-inflammatory and neuroprotective effects, and low-dose LPS preconditioning induces ischemic tolerance under specific conditions. Nevertheless, in the inflammatory milieu of stroke, protective metabolic pathways are frequently suppressed, whereas pro-inflammatory and pro-oxidative mechanisms predominate.

This imbalance may ultimately shifts the metabolic network toward tissue injury and neurological deterioration.

Although direct experimental studies simultaneously targeting LPS, TMAO, and KYN pathway metabolites remain limited, current evidence suggests that these molecules may act synergistically in ischemic injury.246–248 Nevertheless, this conclusion is supported mainly by evidence from experimental studies demonstrating pairwise interactions (such as TMAO-LPS co-exposure and LPS-mediated KYN pathway activation) and by convergent findings from clinical stroke cohorts. Whether these three components interact synergistically has not yet been directly examined in human stroke through simultaneous intervention and quantitative interaction analysis. Future studies integrating multi-omics approaches, germ-free animal models, and quantitative interaction analyses, including combination index analyses and isobologram-based approaches, are needed to clarify the nonlinear synergistic mechanisms among these metabolites and to identify novel therapeutic targets for IS.

Interactive Crosstalk Within the Metabolite Network

Gut-derived metabolites are not merely isolated contributors to the pathophysiology of IS, rather, they operate within an interconnected metabolic network that spans inflammation, oxidative stress, immune regulation, BBB integrity, thrombosis, and lipid metabolism. Under physiological conditions, protective metabolites, such as SCFAs and selected BAs, help sustain immune homeostasis, preserve barrier function, and restrain excessive inflammatory activation. By contrast, TMAO, LPS, and neurotoxic metabolites generated through the KYN pathway are more closely associated with endothelial dysfunction, oxidative damage, thrombotic tendency, and neuroinflammation.

During the transition from stroke susceptibility to acute ischemic injury, gut microbial dysbiosis and host metabolic reprogramming may disrupt this regulatory network, shifting it toward a maladaptive state that amplifies injury-related signaling cascades and contributes to the onset, progression, and poor neurological outcomes of cerebral ischemic injury (Figure 3). Figure 3Mechanistic overview of gut microbiota-associated metabolites in epithelial inflammatory signaling, immune homeostasis, and inter-metabolite crosstalk. Gut microbiota-derived SCFAs (acetate, propionate, butyrate), LPS, and TMAO converge on the epithelial NF-κB signaling axis.

LPS engages LBP and the TLR4/MD-2/CD14 co-receptor complex, initiating MyD88/TIRAP-dependent activation of IKK, phosphorylation and proteasomal degradation of IκBα, and subsequent NF-κB (p65/p50) nuclear translocation to drive transcription of TNF-α, IL-6, pro-IL-1β, and pro-IL-18 and to prime the NLRP3 inflammasome. Circulating SCFAs counter-regulate this cascade: GPR43 engagement activates β-arrestin-2, which suppresses NF-κB, and SCFAs additionally inhibit HDAC to further restrain pro-IL-1β processing. Four magnified sub-modules summarize: (i) BBB integrity—SCFAs upregulate tight junction proteins to protect the BBB, whereas TMAO/LPS-derived signals promote barrier disruption; (ii) microglial polarization—LPS and TMAO drive microglial polarization toward a pro-inflammatory M1-like state, whereas SCFAs suppress the pro-inflammatory activation induced by LPS and TMAO and promote a shift toward an anti-inflammatory, reparative M2-like phenotype; (iii) SCFA-kynurenine axis—butyrate-expanded, IL-10/TGF-β-producing Tregs feedback-inhibit IDO-1, whereas STAT1 signaling promotes IDO-1 expression, jointly modulating downstream KYN/QA production; and (iv) TMAO-bile acid axis—TMAO engages FXR/SHP signaling to suppress CYP7A1 and remodel the bile acid pool, with hepatic FMO3 completing the TMAO-bile acid feedback loop.

Line-style conventions: Solid green arrows indicate pathways directly demonstrated in experimental ischemic stroke models (eg, transient/ permanent middle cerebral artery occlusion). Blue dashed arrows indicate mechanisms supported by in vitro studies or non-stroke disease models (eg, hepatic metabolic models for the FXR-SHP-CYP7A1 loop) but not yet directly confirmed in stroke. Red T-shaped (⊥) lines indicate inhibitory relationships.

The four boxed sub-modules (i–iv) summarize mechanistic crosstalk at the BBB, microglial, tryptophan-kynurenine, and bile acid levels.A) Pathway diagram linking gut microbiota metabolites to epithelial NF-kB signaling. At top left, Gut microbiota connects by a rightward arrow to LPS, TMAO and SCFA. SCFA is labeled Acetate, Propionate and Butyrate and a rightward arrow leads into Blood where Acetate, Propionate and Butyrate are shown again.

LPS connects downward to LBP, then to a membrane complex labeled CD14 and TLR4 or MD-2 complex. Beneath the membrane complex, MYD88 and TIRAP connect downward to an IKK complex with subunits alpha, beta and gamma. A rightward arrow leads to IkBa with two phosphorylation circles labeled P, then to a box labeled proteasomal degradation.

NF-kB p65 or p50 is shown in the cytoplasm and again in the NUCLEUS, with an arrow indicating nuclear entry. In the nucleus, text lists up arrow TNF-alpha, IL-6, pro-IL-1 beta and pro-IL-18. A separate branch shows ROS pointing to NLRP3 inflammasome.

On the right side, SCFA connects downward to a membrane receptor labeled GPR43 with beta-arrestin-2 beneath it. A T shaped inhibitory line from the GPR43 and beta-arrestin-2 area points toward the NF-kB pathway. Another T shaped inhibitory line points to a box labeled HDAC inside the nucleus.

B) BBB integrity submodule. SCFA is shown above an epithelial barrier labeled Tight Junction Proteins, with a downward arrow to BBB protection. A dashed downward arrow from TMAO and LPS points to BBB disruption.

C) Microglial polarization submodule. A microglia labeled M1 with the note pro-inflammatory is shown above and a microglia labeled M2 with the note anti-inflammatory is shown below. A line from SCFA points toward the M2 direction.

A dashed line from TMAO and LPS points toward the M1 direction. D) SCFA KYN axis submodule. TGF-gamma points to IDO-1, then a dashed arrow leads to KYN, followed by an arrow to QA.

STAT1 is shown with an inhibitory line directed toward Butyrate. Tregs are shown with an arrow from Butyrate toward Tregs. Text near Tregs lists IL-10 and TGF-beta with an upward arrow.

An inhibitory line from the Tregs and IL-10 and TGF-beta area points toward IDO-1. E) TMAO bile acid axis submodule. A liver icon labeled FMO3 connects by an arrow to a TMAO icon.

Dashed arrows connect TMAO to FXR, then to SHP. A dashed arrow from SHP points to CYP7A1 and a dashed inhibitory line from SHP points downward toward Bile Acid pool, which is labeled with a downward arrow.Pathway diagram of gut metabolites regulating NF-kB, plus four submodules on BBB, microglia, KYN, bile acids.Abbreviations: SCFAs, short-chain fatty acids; LPS, lipopolysaccharide; TMAO, trimethylamine N-oxide; LBP, LPS-binding protein; TLR4, Toll-like receptor 4; MD-2, lymphocyte antigen 96; CD14, cluster of differentiation 14; MyD88, myeloid differentiation primary response 88; TIRAP, Toll/interleukin-1 receptor domain-containing adaptor protein; IKK, IκB kinase; IκBα, NF-κB inhibitor α; NF-κB, nuclear factor-κB; p65/p50, NF-κB subunit proteins; NLRP3, NOD-like receptor family pyrin domain-containing 3; TNF-α, tumor necrosis factor-α; IL-6, interleukin-6; IL-1β, interleukin-1β; IL-18, interleukin-18; GPR43, G protein-coupled receptor 43; HDAC, histone deacetylase; BBB, blood-brain barrier; M1/M2, classically/alternatively activated microglial phenotypes; IDO-1, indoleamine 2,3-dioxygenase 1; KYN, kynurenine; QA, quinolinic acid; STAT1, signal transducer and activator of transcription 1; Tregs, regulatory T cells; TGF-β, transforming growth factor-β; IL-10, interleukin-10; FMO3, flavin-containing monooxygenase 3; FXR, farnesoid X receptor; SHP, small heterodimer partner; CYP7A1, cholesterol 7α-hydroxylase.

It is imperative to acknowledge from the outset that the evidence supporting this network varies considerably in terms of its strength and scope. The most robust human data relate to associations between individual metabolites and stroke risk or clinical outcomes. Examples of such metabolites include increased circulating TMAO, decreased fecal SCFAs, and altered tryptophan-kynurenine metabolism-and stroke risk or clinical outcomes.

In contrast, the network-level interactions discussed below are supported primarily by experimental studies or inferred from findings in related cardiovascular, metabolic, and inflammatory conditions. In accordance with the aforementioned points, the available evidence is to be categorised into three groups throughout this section. The following three categories of evidence were considered: (i) findings directly demonstrated in human IS or cerebrovascular disease; (ii) evidence obtained from experimental stroke models; and (iii) mechanisms inferred from other disease contexts or converging experimental observations.

Antagonistic Effects Within the Metabolite Network

Antagonistic Effects of SCFAs on TMAO- and LPS-Mediated Injury

Opposing Regulation of NF-κB Signaling by SCFAs, LPS, and TMAO

NF-κB signaling has been identified-largely through experimental studies-as a major downstream effector pathway mediating the pro-inflammatory effects of both TMAO and LPS, whereas SCFAs and several bile acids counteract this pathway and limit inflammatory activation. Therefore, the balance between pro-inflammatory and anti-inflammatory signaling centered on NF-κB provides a mechanistic framework for understanding the antagonistic interactions among gut-derived metabolites.

LPS triggers canonical NF-κB signaling by engaging the TLR4 receptor complex.238 In the circulation, LPS first binds to LBP, which converts LPS aggregates into monomeric forms and presents them to CD14. CD14 subsequently transfers LPS to the TLR4/MD-2 complex, where MD-2 functions as an essential accessory protein associated with the extracellular domain of TLR4. The lipid A moiety of LPS, particularly its hexa-acylated and bisphosphorylated structure, interacts with the hydrophobic pocket of MD-2 and TLR4, inducing dimerization of two TLR4/MD-2 complexes and triggering conformational changes within the intracellular TLR4 domain, thereby initiating downstream inflammatory signaling.

Following TLR4 dimerization, the intracellular Toll/interleukin-1 receptor (TIR) domain recruits the membrane-localized adaptor protein TIRAP/Mal. Through its phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]-binding domain, TIRAP localizes to the plasma membrane and subsequently recruits the core adaptor protein MyD88 to form the TLR4-TIRAP-MyD88 signaling complex. This complex activates downstream kinase cascades involving IL-1 receptor-associated kinases (IRAKs) and tumor necrosis factor receptor-associated factor 6 (TRAF6), ultimately resulting in phosphorylation of the IκB kinase (IKK) complex.

Activated IKK induces phosphorylation and proteasomal degradation of IκBα, thereby releasing NF-κB dimers, mainly p65/p50, which rapidly translocate into the nucleus. Nuclear NF-κB binds to κB sites within target gene promoters and induces transcription of pro-inflammatory mediators, including TNF-α, IL-6, cyclooxygenase-2 (COX-2), and NLRP3 inflammasome-associated precursor proteins such as pro-IL-1β and pro-IL-18. Concurrently, NF-κB may also induce anti-inflammatory mediators, including IL-10, thereby establishing an early feedback mechanism regulating inflammatory intensity.

Despite originating from distinct upstream stimuli, both LPS and TMAO converge on NF-κB-dependent inflammatory signaling. Current evidence, derived predominantly from experimental models, suggests that TMAO promotes NF-κB activation through oxidative stress induction, NLRP3 inflammasome activation, and interactions with TLR-related signaling pathways, thereby amplifying vascular and systemic inflammatory responses.177

Multiple mechanisms, most of which have been characterized in experimental settings, have been proposed to explain the inhibitory effects of SCFAs on NF-κB signaling. One important pathway involves HDAC inhibition, which reshapes the epigenetic landscape of inflammation-related genes. In parallel, SCFAs activate GPR43-dependent signaling and promote β-arrestin-2-mediated stabilization of IκB, thereby limiting NF-κB activation.249,250 Changes in the acetylation status of the NF-κB p65 subunit may further diminish its transcriptional activity.120 Among SCFAs, butyrate appears particularly effective in suppressing NF-κB nuclear translocation and inhibiting the NF-κB/MMP-9 axis.120

Experimental studies further corroborate the antagonistic effects of SCFAs on LPS-induced inflammation. In animal models of IS, SCFA supplementation significantly reduces LPS-induced NF-κB p65 phosphorylation and nuclear translocation in brain tissue, accompanied by decreased expression of TNF-α and IL-6, reduced HDAC activity, and increased histone H3 acetylation.251 These findings indicate that SCFAs inhibit the LPS/NF-κB axis through both classical signaling suppression and epigenetic remodeling of inflammatory responses.

Consistent findings have been reported across different cellular and animal models. Vinolo et al252 demonstrate that SCFAs attenuate LPS-induced inflammatory responses through HDAC inhibition and suppression of NF-κB activation, with propionate and butyrate markedly reducing TNF-α, CINC-2αβ, and NO production in LPS-stimulated neutrophils. Tedelind et al253 further show that acetate, propionate, and butyrate significantly reduce TNF-α release from LPS-stimulated neutrophils, whereas Liu et al254 demonstrate that low concentrations of SCFAs suppress the production of NO, TNF-α, IL-1β, and IL-6 while increasing anti-inflammatory IL-10 expression.

Opposing Regulation of Microglial Phenotypes

Microglia, the resident immune cells of the central nervous system, respond rapidly to cerebral ischemic injury. As representative pro-inflammatory stimuli, LPS and TMAO drive microglia toward a predominantly pro-inflammatory, M1-like state in experimental models of cerebral ischemia and neuroinflammation. This phenotype is associated with increased release of inflammatory mediators, elevated oxidative stress, and enhanced production of neurotoxic molecules, thereby exacerbating secondary neuronal injury.255,256

In contrast, SCFAs suppress LPS- and TMAO-induced pro-inflammatory activation and favor a shift toward an anti-inflammatory, repair-associated M2-like phenotype.257–259 For example, acetate attenuates glial activation in models of LPS-induced neuroinflammation by activating GPR43, reducing IL-1β expression, and inhibiting MAPK phosphorylation.260,261 Together, these findings suggest that SCFAs and TMAO/LPS exert opposing effects on microglial activation and inflammatory polarization, although direct demonstration of this opposing regulation in the human ischemic brain remains limited.

Opposing Regulation of Blood-Brain Barrier Integrity

In experimental models, both TMAO and LPS compromise BBB integrity by reducing the expression of tight junction proteins, including claudin-5, occludin, and ZO-1, in cerebral microvascular endothelial cells. These effects are largely mediated by inflammatory and oxidative stress-related pathways.79,184,262 The resulting increase in barrier permeability promotes BBB leakage and facilitates the entry of peripheral immune cells and inflammatory mediators into the brain parenchyma, thereby amplifying secondary injury after IS.

In experimental studies, SCFAs exert marked protective effects on the BBB. They may act directly on brain endothelial cells to upregulate tight junction proteins through mechanisms involving HDAC inhibition and GPCR activation, thereby preserving endothelial barrier function. In addition, SCFAs may indirectly support BBB integrity by dampening peripheral inflammation and reducing the systemic inflammatory burden.

Regulation of the Tryptophan-Kynurenine Pathway by SCFAs

In experimental models, SCFAs, particularly butyrate, can influence the metabolic fate of Trp and limit its entry into the KYN pathway. Beyond serving as an energy substrate for intestinal epithelial cells, butyrate suppresses the conversion of Trp into KYN metabolites by downregulating IDO-1 expression.263 Mechanistically, butyrate reduces signal transducer and activator of transcription 1 (STAT1) expression and inhibits IFN-γ-dependent STAT1 phosphorylation, thereby attenuating STAT1-mediated transcriptional activation of IDO-1.

In experimental stroke models, peripheral immune activation facilitates the infiltration of multiple lymphocyte subsets into the brain parenchyma, where they contribute to neuroinflammatory responses. Among these cells, Th1 and Th17 cells mainly produce pro-inflammatory cytokines, whereas regulatory Tregs restrain excessive immune activation and support tissue repair.

Experimental studies indicate that SCFAs also shape T-cell differentiation, partly through HDAC inhibition and receptor-mediated signaling involving GPR109A. Through these mechanisms, SCFAs suppress Th1 and Th17 differentiation while promoting Treg generation. Whether these effects are recapitulated in human stroke, however, has not been directly demonstrated.

Relevant evidence from human studies is now beginning to accumulate. In a prospective LC-MS analysis of patients with aneurysmal subarachnoid hemorrhage, circulating SCFAs (such as propionic acid) and tryptophan-related metabolites (including Trp and IPA) were measured at two post-hemorrhage time points. Lower concentrations of these metabolites were independently associated with unfavorable functional outcomes and delayed cerebral ischemia, while serial metabolite profiling provided additional prognostic information beyond conventional clinical predictors.100 Although these findings were obtained in hemorrhagic rather than ischemic stroke populations and do not demonstrate a causal relationship, they provide preliminary support for the clinical relevance of the SCFA-tryptophan metabolic axis in human cerebrovascular disorders and highlight the importance of capturing dynamic temporal changes in metabolite profiles.

Interaction Between TMAO and Bile Acid Metabolism

In experimental hepatic models, TMAO inhibits bile acid synthesis through the hepatic FXR-SHP-CYP7A1 signaling axis. Activation of FXR induces expression of SHP, which in turn suppresses transcription of CYP7A1, the rate-limiting enzyme in bile acid synthesis. This process reduces bile acid production and alters the bile acid profile.164

Such changes may impair cholesterol catabolism and contribute to the progression of atherosclerosis. In addition, reduced bile acid synthesis may decrease production of neuroprotective BAs such as UDCA and TUDCA.

Notably, bile acid-FXR signaling also regulates TMAO production. Bennett et al264 demonstrated that FXR activation induces hepatic FMO3 expression and increases circulating TMAO levels. These findings indicate a reciprocal regulatory loop between TMAO and BA metabolism, although this loop has been characterized largely in hepatic and metabolic models; whether it operates in patients with IS, and how it contributes to stroke risk and outcomes, has not been directly demonstrated.

Furthermore, both arms of this axis are modulated by diet, gut microbial composition, and host metabolic state, underscoring the context-dependent nature of the TMAO-BA interaction.

Consequences of Metabolic Imbalance

Under physiological conditions, the microbiota-associated metabolite network remains in dynamic equilibrium and collectively orchestrates vascular, immune, metabolic, and neural homeostasis. However, risk factors such as aging, unhealthy dietary patterns, hypertension, obesity, and diabetes can shift this balance toward an injury-associated configuration. It is important to acknowledge, however, that the distinction between “protective” and “injurious” network states represents an oversimplification.

Individual metabolites may exert distinct, and sometimes opposing, effects depending on their concentration, biological context, disease stage, and host-related factors, including renal function, dietary influences, and comorbid conditions.

In experimental and observational studies, such metabolic disturbance is associated with sustained inflammatory activation, redox imbalance, increased thrombotic tendency, and disruption of the BBB. As a consequence, susceptibility to IS increases, infarct expansion may be aggravated, and neurological recovery may be compromised.

At the vascular level, TMAO promotes foam cell formation and plaque instability, whereas reduced SCFAs attenuates anti-inflammatory regulation. At the systemic level, gut dysbiosis may diminish the capacity for metabolic buffering. Together, these changes indicate coordinated disruption of the gut-immune-vascular-brain axis.

The clinical literature, however, is not uniformly consistent with this framework: some studies have reported lower circulating TMAO in specific stroke subtypes, higher SCFA levels associated with worse short-term discharge status in patients undergoing thrombectomy, or no independent association between TMAO and recurrent stroke or major adverse cardiovascular events in large prospective cohorts after adjustment for confounders. Such findings caution against overgeneralizing single-direction relationships.

Restoring balance within this metabolic network may therefore represent a promising-but as yet unproven-therapeutic strategy for preventing IS, limiting brain injury, and improving long-term neurological outcomes (Table 3). Table 3Evidence Summary of Metabolites and Metabolite–Metabolite Interactions Within the Microbiota-Associated Metabolite NetworkMetabolite/Metabolite–Metabolite InteractionStudy TypeBiological SampleDisease StageDirection of Metabolite ChangeDirect Evidence vs Indirect InferenceEvidence LevelReferencesI. Single-metabolite evidenceTMAOSystematic review/meta-analysis (15 studies, 40,061 patients); multicenter prospective cohort (1,726 patients); case–control/observational cohortsPlasma/serumAcute stroke through 3-month follow-up (including recurrence and major adverse cardiovascular events); partly covering the pre-stroke risk-factor stageTMAO increased (↑); decreased TMAO reported in the large-artery atherosclerosis stroke subtypeDirect association—elevated TMAO associated with poor 3-month mRS outcome (OR 1.581, subtype-dependent); multicenter cohort showed no independent association with recurrent stroke/MACE after adjustment; causality not establishedHigh (meta-analytic association)/Moderate (independent prognostic value debated)Wang et al,186 Frenger et al,265 Yin et al,15SCFAsProspective cohort (n=140); cross-sectional (thrombectomy patients, n=53)FecesAcute admission through 90-day follow-up; post-thrombectomy to dischargeSCFAs decreased (↓)—reduced acetate independently predicted poor 90-day outcome; higher SCFAs associated with worse discharge status in the thrombectomy cohort (conflicting direction)Direct association (human cohorts); mechanisms inferredModerate (inconsistent direction of association)Tan et al,20 Henry et al,98Tryptophan–kynurenine metabolites (KYN/Trp, L-kynurenine, quinolinic acid)Cohort study (n=149); prospective cognitive assessment (n=41); prospective two-timepoint LC–MS in aSAH (n=80); animal stroke models and cellular experimentsSerum/plasma (human); brain tissue, neurons/glia (experimental)Acute phase (NIHSS, infarct volume) → 3-month/long-term outcome; post-stroke cognitive impairment (PSCI) assessment; aSAH day 1 and day 9KYN/Trp ratio and L-kynurenine increased (↑), quinolinic acid increased (↑); decreased propionic acid, tryptophan and indole-3-propionic acid (↓) predicted poor aSAH outcomeDirect association (human biomarkers); excitotoxicity mechanisms confirmed in animal/cellular models; network-level causality inferredModerate (human association)/Limited–moderate (mechanism)Colpo et al,197Brouns et al,195Gellért et al,198Nagy et al;199Guillemin GJ,200Braidy et al,201Gold et al,196Nagybanyai-Nagy et al,100Bile acids (secondary BAs, UDCA/TUDCA)Predominantly animal/cellular experiments; limited human metabolomicsSerum (human); liver, gut and brain tissue (experimental)Metabolic disease/pre-stroke risk-factor stage; MCAO stroke modelsNeuroprotective BAs (UDCA/TUDCA) decreased (↓, inferred); TMAO-related bile acid pool remodelingIndirect inference—no direct human evidence in strokeLimited–moderateTolhurst et al,149Thomas et al,150Lu et al,151Hylemon et al,156Archer et al,157Koeth et al,163Ding et al,164II.

Evidence for metabolite–metabolite interactionsSCFAs ↔ TMAO/LPS (antagonistic inflammatory signaling)Animal stroke models; in vitro cellular experiments (neutrophils, microglia); human observational cohortsBrain tissue, cell cultures (experimental); feces and serum (human)Acute phase (MCAO models); in vitro LPS stimulationSCFAs suppress NF-κB activation, M1 polarization and BBB disruption (antagonistic); TMAO/LPS enhance NF-κB/NLRP3 signalingAntagonistic mechanisms directly demonstrated experimentally; human evidence based on single-metabolite associations (inferred)Moderate (experimental)/Limited (human)Lee et al,249Caetano-Silva et al,251Vinolo et al;252Tedelind et al,253Liu et al,254Lu et al,255Quan et al,256Fellner et al,257Kim et al,258Schilling et al,259Smith et al,260Kobayashi et al,261SCFAs ↔ tryptophan–kynurenine pathway (butyrate–IDO-1 axis)In vitro immune-cell experiments; experimental models; human metabolomics in aSAH (n=80)Immune cells, intestinal epithelium (in vitro); serum (human)Post-stroke immune-activation phase; aSAH day 1 and day 9Butyrate downregulates IDO-1 and reduces neurotoxic KYN metabolites (↓); low propionic acid/tryptophan/IPA associated with poor outcomeMechanism confirmed in cellular/experimental studies; functional relevance in stroke inferredLimited–moderateMartin-Gallausiaux et al,263Nagybanyai-Nagy et al,100TMAO ↔ bile acid metabolism (FXR–SHP–CYP7A1 / FXR–FMO3 loop)Animal hepatic and metabolic experiments; cardiovascular modelsLiver tissue, serum (animal)Metabolic disease stage; not directly studied in strokeElevated TMAO suppresses bile acid synthesis (CYP7A1↓); FXR activation upregulates FMO3 and TMAO (bidirectional loop)Directly demonstrated experimentally; inferred in stroke patientsModerate (experimental)/Limited (stroke)Koeth et al,163Ding et al,164Bennett et al,264SCFAs ↔ secondary bile acids (barrier and immune synergy)Animal/cellular experiments (GPR43, HDAC, TGR5)Intestinal mucosa, vascular endothelium, cerebral microvascular endothelium (experimental); feces (human, limited)Risk-factor stage → acute phase (barrier disruption window)Cooperative maintenance of gut and BBB integrity, NF-κB/NLRP3 suppression, Treg promotion (protective direction)Mechanisms demonstrated experimentally; human evidence in stroke lacking (inferred)Limited–moderateChen et al,118Wang et al,132Guo et al,135Yanguas-Casás et al,136Haselow et al,137Rodrigues et al,142Zhang et al,143Csecsei et al,101Kynurenine metabolites ↔ neurovascular injury pathwaysHuman biomarker studies; animal stroke models and neuronal/glial experimentsSerum (human); neurons, astrocytes (experimental)Acute phase → long-term follow-upQuinolinic acid increased (↑) → excitotoxicity, oxidative stress ↑, BBB disruption; KYN/Trp ↑ associated with severityDirect human association; network-level causality inferredModerateColpo et al,197Brouns et al,195Gellért et al,198Nagy et al,199Guillemin GJ.200Braidy et al,201Nagybanyai-Nagy et al100Notes: (1) Evidence-level definitions—High: directly demonstrated in human ischemic stroke or cerebrovascular disease cohorts; Moderate: supported by experimental stroke models or multiple convergent mechanistic studies with limited human association data; Limited: inferred from other disease settings, cellular studies, or biological plausibility; Hypothesis-generating: network-level relationships without direct validation. (2) Direction of metabolite change—↑ increase, ↓ decrease; arrows indicate the direction associated with poor outcome or disease progression; “conflicting” indicates opposite directions reported across studies. (3) Disease stages—risk-factor stage (pre-stroke) → acute phase (onset ≤72 h/admission) → recovery phase (3–6-month follow-up); special stages: post-thrombectomy, aSAH day 1/day 9, ruptured vs unruptured intracranial aneurysm.Abbreviations: TMAO, Trimethylamine N-oxide; SCFAs, Short-chain fatty acids; KYN, Kynurenine; Trp, Tryptophan; MCAO, middle cerebral artery occlusion; LPS, Lipopolysaccharide; NF-κB, Nuclear factor kappa B; aSAH, Aneurysmal subarachnoid hemorrhage; FXR, Farnesoid X receptor; SHP, Small heterodimer partner; CYP7A1, Cholesterol 7α-hydroxylase; FMO3, Flavin-containing monooxygenase 3; GPR43, G protein-coupled receptor 43; HDAC, Histone deacetylase; TGR5, Takeda G protein-coupled receptor 5.

Translation and Application

Existing Intervention Strategies and Their Limitations

In recent years, probiotics, prebiotics, and FMT have been explored as representative broad-strategies for modulating the gut microbial ecosystem in various diseases, including IS.31,266–270 Probiotics such as Bifidobacterium, Lactobacillus, and Clostridium butyricum have been shown to attenuate stroke severity and improve outcomes. Bifidobacterium helps maintain a healthy gut microbiota by inhibiting pathogenic bacteria, strengthening the intestinal barrier, and limiting the translocation of toxic substances into the bloodstream, thereby reducing systemic inflammation and conferring neuroprotection.271Lactobacillus supports intestinal health by adhering to the intestinal mucosa, reducing pathogenic bacterial loads, and enhancing mucosal integrity; it also modulates immune activity and alleviates neuroinflammation through the MGBA, promoting neurological recovery and highlighting its potential neuroprotective role.272 Probiotics may also help lower blood pressure by reshaping the gut microbiota and regulating inflammatory and neuroimmune responses.

Prebiotics, including inulin and oligosaccharides, are non-digestible compounds without inherent biological activity; they promote the growth and proliferation of beneficial gut bacteria, thereby potentiating probiotic effects.273 Supplementation with inulin and fructo-oligosaccharides has been shown to promote the growth of Bifidobacterium.274 Another prebiotic, β-glucan, may help lower LDL and total cholesterol levels, stabilize blood glucose, and improve endothelial function-possibly through SCFA production-thereby supporting cardiovascular protection.275 Lactulose, an important prebiotic, increases SCFA levels in the intestine and bloodstream, reduces post-stroke inflammation, and enhances functional recovery after stroke.273 Probiotic-prebiotic combinations (synbiotics) exert synergistic effects, as demonstrated in animal stroke models by improved neurological and cognitive function, reduced infarct size, and attenuated inflammation.276 Collectively, probiotics and prebiotics may support stroke prevention and recovery, and their intake or supplementation may serve as a beneficial adjunct to conventional therapy.

FMT involves the transfer of fecal microbiota from healthy donors into recipients with the aim of alleviating disease-related symptoms. Various delivery approaches have been explored, including rectal administration via endoscopy or enema and oral delivery through frozen capsules. Preclinical studies investigating FMT in stroke models have demonstrated reduced intestinal and cerebral inflammation, potentially mediated by Lactobacillus expansion, enhanced IL-10 production, and increased regulatory T-cell responses.277 In experimental stroke models, FMT restores post-stroke gut dysbiosis, attenuates systemic and neuroinflammation (including microglia-associated inflammatory responses), reduces infarct size, and promotes functional recovery.267,270 Furthermore, FMT has been reported to decrease the expression of pro-apoptotic and cell death-related markers while upregulating Bcl-2, an anti-apoptotic protein, thereby limiting neuronal loss after stroke.270 Importantly, current evidence supporting the therapeutic potential of FMT in ischemic stroke is largely derived from experimental studies, whereas clinical data from stroke patients remain limited.

Despite these encouraging findings, a critical appraisal of the available clinical trials reveals a central and pervasive limitation: the lack of systematic consideration of the metabolite network as an integrative intervention target, which leads to unstable and unpredictable therapeutic effects. Nearly all trials have treated IS patients meeting the inclusion criteria as a homogeneous group. However, the human gut microbiota exhibits enormous interindividual variation-as unique as a fingerprint.

Patients differ widely in pre-stroke microbial architecture, dietary habits, and concomitant medications, and consequently present distinct patterns of post-stroke “metabolite network imbalance”: one patient may display a typical “SCFA-deficient” phenotype, another a “TMAO-excess” phenotype, and still others a combination of both. Existing interventions are inherently broad-spectrum. Whether supplementing one or two common probiotic strains or providing a single prebiotic, the underlying logic is to improve the entire intestinal microenvironment universally by introducing “good bacteria” or providing “good food”; FMT goes further by transplanting an entire “black-box” donor microbiota into the recipient.

These approaches resemble randomly scattering seeds or fertilizer in barren soil in the hope of raising good crops, without analyzing the specific composition and problems of the soil itself.

Importantly, multiple systematic reviews and meta-analyses have concluded that there is currently no conclusive evidence that probiotics significantly improve core neurological functional outcomes in IS patients. Although some studies have observed improvements in gastrointestinal complications, this remains far removed from intervening in the stroke itself. Moreover, current trials are commonly limited by small sample sizes, short study durations, and considerable heterogeneity in strains, dosages, and treatment courses, which hampers cross-study comparison and pooled analysis.

A further limitation is insufficient mechanistic specificity: the colonization efficiency and long-term stability of probiotics are often constrained by host intestinal conditions and ecological competition; the efficacy of prebiotics depends on the pre-existing microbial composition; and FMT, despite its broad-spectrum remodeling capacity, is associated with unresolved challenges including donor selection, long-term safety, microbial stability, and the potential transmission of pathogenic or multidrug-resistant microorganisms.

Furthermore, conventional microbiota-based interventions often lack the precision required to regulate specific pathogenic metabolic pathways. Patients with elevated TMAO, excessive KYN pathway activation, or prominent LPS-associated inflammation may require targeted modulation of specific metabolic outputs rather than general microbiota remodeling. Of note, no prospective randomized trials in human stroke have yet demonstrated that metabolite-guided microbiota interventions improve functional outcomes; accordingly, such precision strategies should currently be regarded as hypotheses requiring prospective validation rather than clinically actionable approaches.

To overcome these obstacles, a fundamental paradigm shift is required: redirecting the focus of intervention from the vague goal of “restoring flora balance” toward the precise “remodeling of the metabolite network”. Precision intervention presupposes precision diagnosis. In the IS field, this entails moving beyond the conventional search for a single “magic biomarker” toward defining “metabolite network signature profiles” that comprehensively reflect individual pathophysiological states.

Such profiles, generated through the combined application of metagenomics and metabolomics, should systematically delineate a multidimensional molecular landscape associated with IS risk, severity, and prognosis, thereby providing a solid foundation for precise patient stratification and prognostic prediction.

Potential Precision Intervention

To address the unstable efficacy and limited mechanistic specificity of current strategies, therapeutic approaches should shift from microbiota composition-based modulation to function-oriented metabolic regulation. This concept is supported by evidence that key pathological processes in IS, including inflammation, vascular injury, BBB disruption, thrombosis, and impaired neurological recovery, are closely associated with dysregulated gut-derived metabolic pathways.

Potential precision intervention requires two key steps. First, metagenomics, metabolomics, and clinical phenotyping should be integrated to establish individualized metabolic profiles and identify dominant metabolic abnormalities. Second, causal links between metabolic disturbances and clinical phenotypes, such as stroke severity, inflammatory status, vascular dysfunction, neurological recovery, and recurrence risk, should be clarified.

These approaches may support a transition from empirical intervention to mechanism-based precision therapy.

Under this framework, intervention strategies should move beyond single-target modulation and adopt coordinated, multi-level regulation.

Multi-Target Metabolic Regulation

Given the highly interconnected nature of gut-derived metabolic pathways, targeting a single metabolite is insufficient to restore systemic balance. Future strategies should enhance protective pathways while suppressing pathogenic ones. Supplementation with beneficial metabolites or their precursors, such as SCFAs or high-fiber substrates, may improve anti-inflammatory signaling, intestinal barrier integrity, and immune homeostasis.

Enrichment of functionally defined bacteria, such as the butyrate-producing Faecalibacterium prausnitzii, further enhances protective metabolite production.47

For patients with elevated TMAO levels, potential strategies include inhibiting microbial TMA production, modulating hepatic FMO3 activity, or reducing dietary intake of precursors such as choline and L-carnitine. The goal is to shift the metabolic environment from a pro-inflammatory, pro-thrombotic, and pro-oxidative state toward one that supports barrier protection, immune regulation, and neural repair.

Personalized Nutritional Intervention

Diet is a major external factor shaping gut-derived metabolite profiles. Personalized nutritional strategies should be guided by microbiota composition, metabolite profiles, and clinical characteristics. For individuals with reduced SCFA levels, increased intake of dietary fiber, resistant starch, and prebiotics may enhance SCFA production.

In patients susceptible to TMAO-associated risk, limiting foods rich in choline and L-carnitine may be beneficial.

Dietary interventions should also consider comorbidities such as diabetes, hypertension, dyslipidemia, obesity, and renal dysfunction to avoid excessive metabolic burden. Given that TMAO is cleared predominantly by the kidneys, dietary adjustments may need to be individualized in patients with impaired renal function, a context-dependent consideration that further argues for personalized rather than generic recommendations. A major challenge remains the absence of robust predictive models linking dietary inputs to metabolite network dynamics.

Integration of multi-omics data with clinical outcomes may facilitate development of computational models enabling precision dietary modulation.

Targeted Delivery Systems

Conventional oral interventions are often limited by degradation, absorption, and metabolic transformation during gastrointestinal transit. Advances in biomaterials, nanotechnology, and synthetic biology provide opportunities to improve delivery specificity.

pH-responsive, enzyme-responsive, and microbiota-responsive delivery systems may allow site-specific release of metabolites, microbial strains, or pathway modulators within defined intestinal regions.278 These systems could improve local bioavailability, reduce off-target exposure, and enhance safety. Therefore, targeted delivery may be an important strategy for improving spatial precision and dose stability in metabolic network regulation.

It should be emphasized that the precision intervention strategies described above remain hypotheses requiring prospective validation rather than clinically actionable approaches. To date, no prospective randomized clinical trials have demonstrated that metabolite-guided microbiota-based interventions improve functional outcomes in patients with IS.

Clinical Trial Design

Clinical trial design should also be optimized within the potential precision intervention framework. Traditional randomized controlled trials evaluate average treatment effects in heterogeneous populations and may fail to identify subgroup-specific benefits.

Future studies should adopt stratified designs based on biological and metabolic characteristics. Patient enrichment according to metabolite network profiles may improve the alignment between intervention targets and enrolled populations.

In addition, multidimensional endpoints should combine mechanistic biomarkers, such as SCFA levels, TMAO concentrations, inflammatory mediators, and KYN metabolites, with clinical outcomes, including modified Rankin Scale scores, NIHSS scores, infarct volume, recurrence, and long-term recovery. This approach would allow simultaneous evaluation of biological activity and clinical efficacy.

Standardization of intervention protocols remains essential, including microbial strain selection, metabolic characteristics, dosage, treatment duration, administration route, and follow-up. With these improvements, clinical trials may shift from population-level evaluation toward stratified validation of interventions targeting specific metabolic phenotypes, thereby strengthening the evidence base for precision metabolite network-based therapies in IS.

Conclusion

IS should be viewed not merely as localized ischemic necrosis in the brain, but as a systemic pathological process involving interactions among the nervous, immune, metabolic, vascular, and peripheral organ systems. Research on the MGBA offers a broader framework for understanding IS onset, progression, and recovery. Stroke-induced brain injury can disrupt the intestinal microenvironment through autonomic, neuroendocrine, and immune-inflammatory pathways, resulting in gut dysbiosis, impaired barrier function, and increased intestinal permeability.

In turn, microbial imbalance and abnormal metabolite profiles may intensify peripheral inflammation, damage the BBB, activate aberrant immune responses, and aggravate neuroinflammation. These reciprocal effects create a feed-forward loop linking brain injury, dysbiosis, metabolic disturbance, inflammation, and neuronal damage.

Clinical and experimental studies show that IS is associated with substantial remodeling of the gut microbiota, including enrichment of pro-inflammatory or opportunistic bacteria, depletion of SCFA-producing and other beneficial commensals, reduced microbial diversity in some-but not all-studies, and impaired metabolic function. Gut microbiota-associated metabolites form an interconnected regulatory network that affects inflammation, oxidative stress, immune polarization, endothelial function, platelet activation, BBB integrity, and neural repair. In general, SCFAs and certain BAs support barrier integrity, immune balance, and inflammatory resolution, whereas TMAO, LPS, and KYN pathway metabolites are more closely linked to inflammation, thrombosis, oxidative injury, and neurotoxicity.

During IS progression, this metabolic network may shift from homeostatic regulation toward pathological amplification.

This review therefore proposes the concept of “gut microbiota-associated metabolite network imbalance” as a conceptual and hypothesis-generating framework, moving beyond the simple classification of metabolites as beneficial or harmful. SCFAs, TMAO, BAs, Trp metabolites, and LPS may interact through synergistic, antagonistic, and feedback mechanisms; however, only a subset of these interactions has been directly demonstrated in IS, and most proposed network relationships remain inferred from studies in other diseases or from convergent downstream pathways. Their biological effects are further influenced by stroke stage, subtype, comorbidities, diet, medication use, organ function, and host microbiota background.

Although TMAO is often associated with adverse cardiovascular and cerebrovascular outcomes, some studies have reported reduced TMAO levels in stroke patients, suggesting stage-dependent and context-specific effects. These findings highlight the need to shift from single-metabolite analysis to dynamic, network-level investigation.

Mechanistically, metabolite network imbalance may contribute to IS through coordinated disruption of multiple pathways. Reduced SCFA levels weaken intestinal and BBB integrity and diminish the inhibition of NF-κB signaling, NLRP3 inflammasome activation, microglial M1 polarization, and Th17-mediated inflammation. Meanwhile, TMAO, LPS, and neurotoxic KYN metabolites promote systemic inflammation, endothelial dysfunction, platelet activation, BBB disruption, and neuronal injury.

Conversely, SCFAs and BAs help maintain metabolic and immune homeostasis through FXR, TGR5, GPR41/GPR43, HDAC, and GLP-1-related signaling. The balance between protective metabolites, such as KYNA and IPA, and harmful metabolites, such as QA, 3-HK, and indoxyl sulfate, may influence the severity of post-stroke neuroinflammation, oxidative stress, and BBB injury.

Future studies should adopt integrative, multi-scale approaches. Large multicenter prospective cohorts combining fecal, blood, neuroimaging, immune, and clinical outcome data across different stroke phases are needed to define the temporal dynamics of microbiota-metabolite interactions. Causal mechanisms should be validated using germ-free models, defined microbial consortia, isotope tracing, metabolite-targeted interventions, and receptor-specific manipulation.

Patient heterogeneity should also be addressed by integrating demographic, clinical, metabolic, and microbiota features to support biologically meaningful subtyping. Recent prospective clinical studies in other acute cerebrovascular conditions support the clinical relevance of such temporal dynamics: gut microbial profiles with distinct butyrate-producing taxa have been associated with intracranial aneurysm rupture status and two-timepoint measurements of circulating SCFAs and tryptophan-derived metabolites after aneurysmal subarachnoid hemorrhage were associated with functional outcome and delayed cerebral ischemia.100,101

From a translational perspective, the metabolite network framework has the potential to improve risk prediction, prognostic assessment, and precision therapy; however, these applications remain largely investigational, and prospective studies are required to determine whether multi-metabolite biomarker panels improve prediction beyond established clinical models and whether metabolite-guided interventions improve clinical outcomes. Multi-metabolite biomarker panels incorporating SCFAs, TMAO, BAs, Trp metabolites, and LPS-related indicators may perform better than single biomarkers in predicting stroke risk, severity, recurrence, and long-term outcomes. Metabolite-based stratification may also guide individualized interventions, including SCFA restoration, TMAO suppression, bile acid modulation, and targeted regulation of KYN and AhR pathways.

Future clinical trials should move beyond average treatment effects and incorporate metabolic phenotyping, mechanistic biomarkers, and clinically meaningful endpoints.

In conclusion, gut microbiota-associated metabolite networks represent a key regulatory interface in IS pathogenesis; however, the proposed network should currently be regarded primarily as a conceptual and hypothesis-generating framework with limited prospective human validation. Clarifying their dynamic interactions will deepen our understanding of stroke as a systemic disease and support the development of mechanism-based precision therapies. With advances in multi-omics, network medicine, and computational modeling, targeting metabolite network imbalance may offer new opportunities for improving IS prevention, treatment, and long-term neurological recovery.