Introduction
Gastric ulcer (GU) is a common gastrointestinal disorder characterized involving disruption to the gastric mucosal barrier, leading to epithelial damage, bleeding, and, in severe cases, perforation (Arshad et al., 2025; Lanas and Chan, 2017). Despite advances in clinical management, GU remains a significant global health burden. Its pathogenesis is multifactorial, involving Helicobacter pylori infection, non-steroidal anti-inflammatory drug use, and excessive alcohol consumption, contribute to its pathogenesis (Arshad et al., 2025).
Ethanol-induced gastric injury is a widely used as a classical experimental model, as it rapidly induces mucosal damage through oxidative stress, epithelial barrier disruption, and inflammatory responses (Rahman et al., 2020). Accumulating evidence suggests that inflammation plays a central role in the development of gastric mucosal injury (Yan et al., 2025; White et al., 2015). Ethanol exposure triggers the release of pro-inflammatory cytokines, such as interleukin (IL)-1β, IL-6, and tumor necrosis factor-α (TNF-α), which leads to the amplification of inflammatory cascades and tissue damage (Wang et al., 2010).
The Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway is a key regulator of inflammatory signaling, and its activation has been associated with gastric mucosal injury and ulcer progression (Agashe et al., 2022; Hu et al., 2021). However, the upstream mechanisms that regulate inflammatory signaling in GU remain incompletely understood.
Recent studies have emphasized the critical role of gut microbiota in gastrointestinal diseases. Beyond its established involvement in intestinal disorders, emerging evidence indicates that gut microbiota also influences gastric mucosal homeostasis and inflammatory responses via the gut-stomach axis (Shabani et al., 2025; Aldriwesh et al., 2026). Specifically, substantial evidence suggests that gastric ulceration is frequently accompanied by gut microbiota dysbiosis, with a marked reduction in both microbial diversity and abundance in patients with gastric ulcers, especially, Firmicutes, Actinobacteria and Lactobacillus (Chen et al., 2018; Zhang et al., 2022).
Lactobacillus is well recognized for its immunomodulatory properties in the gastrointestinal tract, acting in part through the inhibition of NF-κB signaling to facilitate the healing of gastric ulcerative lesions (Shen et al., 2018; Yang et al., 2021; Li et al., 2026). Mechanistically, this protective effect is thought to be associated with lactic acid secretion, which enhances the permeability of H. pylori cell walls and may thus exert direct antimicrobial activity (Westerik et al., 2018). Moreover, microbiota-targeted interventions have shown promise in modulating host inflammation by enhancing superoxide dismutase (SOD), glutathione (GSH) and CAT activities and protecting against mucosal injury (Hitch et al., 2022).
Nevertheless, whether gut microbiota plays a causal role in mediating inflammatory signaling pathways, such as JAK/STAT, in GU remains to be clarified.
Traditional Chinese medicine has long been used to manage the management of gastrointestinal disorders and is attracting increasing attention due to its multi-component and multi-target characteristics (Tian et al., 2025). Chaihuang weikuining (CHWKN) is a classical herbal formula comprising multiple medicinal plants, including Astragalus membranaceus and Bupleurum species, and has been clinically applied for the treatment of gastric disorders in China (Weng et al., 2023; Guo et al., 2024a; Zheng et al., 2025; Zhang et al., 2025). Astragalus membranaceus, a representative herbal medicine and its bioactive constituents have been reported to modulate immune cells and improve gut microbiota composition (Guo et al., 2024a; Weng et al., 2023).
The homogeneous astragalus polysaccharide and cycloastragenol, which are isolated from Astragalus membranaceus, have been shown to reduce ulcer area and improve mucosal integrity (Zhang et al., 2025; Zheng et al., 2025). Polygonum cuspidatum has been reported to exert protective effects in various animal models of acute gastric ulcer. Its antioxidant properties effectively increase the levels of SOD, GSH, and prostaglandin E2, thereby promoting ulcer healing (Kim et al., 2020).
Previous studies have suggested that individual components of these herbs possess anti-inflammatory and microbiota-modulating properties (Kim et al., 2020; Zhang et al., 2025; Zheng et al., 2025). However, the therapeutic effects of CHWKN on gastric ulcer and, more importantly, its underlying mechanisms, particularly with respect to gut microbiota, remain largely unexplored.
In this study, we aimed to investigate the protective effects of CHWKN against ethanol-induced GU and to elucidate its underlying mechanisms. Specifically, we hypothesized that CHWKN exerts its gastroprotective effects by modulating gut microbiota, thereby suppressing inflammatory signaling pathways such as JAK1/STAT3. To test this hypothesis, we combined antibiotic-mediated microbiota depletion, fecal microbiota transplantation (FMT), and 16S rDNA sequencing to systematically evaluate the role of gut microbiota in CHWKN-mediated protection.
Our findings provide new insights into the microbiota-dependent mechanisms of CHWKN and highlight its potential as a therapeutic strategy for GU.
Material and methods
Preparation and quantitative evaluation of the CHWKN
Astragalus membranaceus, Bupleurum chinense, Polygonum cuspidatum, Cyperus rotundus, Codonopsis pilosula, Paeonia, Glycyrrhiza uralensis, Cinnamomum cassia, Zingiber officinale, Ziziphus jujuba, maltose were purchased from the outpatient department of Zhejiang Chinese University (Zhejiang, China). CHWKN was extracted in 100 mL of distilled water at 100 °C for 1 hour and concentrated (4.8 g/L) via rotary evaporation. The chemical components were analyzed by the UHPLC-QExactive system (Thermo Fisher Scientific, USA).
The data were processed by Progenesis QI software (Waters Corporation, Milford, MA, USA). Feature peaks were then annotated by matching both MS and MS/MS spectra against the Majorbio Traditional Chinese Medicine-specific metabolite database (Majorbio, Shanghai, China).
Animal
Male SD rats were purchased from the Shanghai Family Planning Research Institute (Shanghai, China). All animal experiments were approved by the Institutional Animal Care and Use Committee of Zhejiang Chinese Medical University (IACUC-20210726-05). The rats were housed in a specific pathogen-free (SPF) barrier facility under controlled conditions: temperature was maintained at 23 ± 1 °C, and relative humidity at 50%−65%, with free access to food and water.
Gastric ulcer models
Following a 1-week of acclimatization period, the rats were randomly divided into five groups: control group (n = 8), model group (n = 8), low-dose CHWKN group (n = 8), high-dose CHWKN group (n = 8), and omeprazole group (n = 8). As described previous study (Shu et al., 2026), rats except those in the control group were administered 5 mL/kg absolute ethanol by gavage once before the first administration of CHWKN or other treatments, while the control group received an equal volume of normal saline. Meanwhile, based on clinical equivalent dose conversion and according to the body surface area conversion principle, the low- and high-dose CHWKN groups were treated with 7.5 and 15 g/kg of CHWKN, respectively, and the omeprazole group received 20 mg/kg of omeprazole.
After 14 days of treatment, all rats were sacrificed, and blood and gastric tissue were collected.
ABX rat models
Rats were randomly assigned to the model group (n = 8), the CHWKN group (n = 8), ABX group (n = 20) and the CHWKN + ABX group (n = 8). Rats in the model and CHWKN groups received sterile deionized drinking water. Rats in the ABX and CHWKN + ABX groups had ad libitum access to drinking water containing vancomycin (0.5 g/L), ampicillin (1 g/L), neomycin (0.5 g/L), and metronidazole (1 g/L) for 2 weeks to establish the ABX model (Vicentini et al., 2021; Rakoff-Nahoum et al., 2004).
All antibiotics were purchased from MCE (Shanghai, China).
Fecal samples were collected on day 10 of antibiotic treatment. Bacterial load was assessed by quantitative PCR targeting the bacterial 16S rRNA gene to evaluate the reduction in gut bacteria following ABX treatment (Supplementary Figure 1A). After completion of antibiotic treatment, the 20 rats initially assigned to the ABX group were randomly allocated to the ABX group (n = 8), ABX + ethanol + FMT-ethanol group (n = 6), and ABX + ethanol + FMT-ethanol-CHWKN group (n = 6).
FMT was initiated 24 h after discontinuation of antibiotics, as described in Section 2.5.
FMT rat models
Fecal donors comprised ethanol-treated rats (n = 6) and rats treated with ethanol and CHWKN (n = 6). Both donor groups received their respective treatments for 14 days as described above. Fresh fecal samples were collected from donor rats using sterile saline, at a concentration of 10 mg/mL, soaked for 1 min, and homogenized for 10 min.
The homogenate was then centrifuged at 6000 rpm for 15 min, and the supernatant was collected for transplantation. The prepared fecal suspensions were administered to recipient rats by oral gavage at 1 mL per 100 g body weight.
FMT was initiated 24 h after discontinuation of antibiotic treatment and administered once every 2 days for 14 days. The ABX + ethanol + FMT-ethanol group (n = 6) received fecal suspension from ethanol-treated donors, whereas the ABX + ethanol + FMT-ethanol-CHWKN group (n = 6) received fecal suspension from donors treated with ethanol and CHWKN. The efficiency of gut microbiota restoration was assessed by q-PCR after 10 days of antibiotic treatment (Supplementary Figure 1B).
Biochemical analysis
The serum levels of pepsinogen I (PGI) (ELK Biotechnology, Shanghai, China) and pepsinogen II (PGII)(ELK Biotechnology, Shanghai, China), IL-10 (ELK Biotechnology, Shanghai, China), IL-6 (ELK Biotechnology, Shanghai, China), IL-1β (ELK Biotechnology, Shanghai, China), TNFα (ELK Biotechnology, Shanghai, China), MPO(Nanjing Jiancheng Bioengineering Institute, Nanjing, China), and LPS (Beyotime, Shanghai, China), according to the manufacturer's recommended protocol, were measured by multimode reader (Molecular Devices, USA).
Histopathological analysis
The gastric tissue was fixed in a 4% paraformaldehyde solution, embedded in paraffin wax, and then cut into 4-μm-thick sections. Hematoxylin and eosin staining (H&E) and periodic acid-Schiff (PAS) staining were performed on the sections to evaluate gastric injury and damage to the gastric mucosal barrier.
Real-time PCR
Total RNA was extracted from tissues using the FastPure Complex Tissue/Cell Total RNA Isolation Kit (Vazyme, Nanjing, China), followed by cDNA synthesis using HiScript III RT SuperMix for qPCR (+gDNA wiper) (Vazyme, Nanjing, China) both according to the manufacturer's instructions. Real-time quantitative PCR (qPCR) was performed using a QuantStudio 6 Pro (Thermo Fisher Scientific, USA). The primer sequences are listed in the Table 1.
16S rRNA microbiome analysis
We collected feces from rats in the control group (n = 6), model group (n = 6), and CHWKN-H group (n = 6) for 16S rRNA gene sequencing. 16S rRNA gene sequencing was performed by LC-Bio (Hangzhou, China), and the microbiota data were analyzed via the Majorbio Cloud Platform (Shanghai, China). For alpha diversity indices, the Kruskal–Wallis rank-sum test was used for multi-group comparisons, followed by False Discovery Rate correction to control for multiple testing.
Beta diversity was evaluated based on Bray–Curtis distances, and group separation was assessed using ANOSIM with 999 permutations. Differential taxa were identified using LEfSe with an all-against-all comparison strategy.
Statistical analysis
All data are expressed as mean ± standard deviation (SD). Statistical analyses were performed using GraphPad Prism 8.0 software (GraphPad Software, San Diego, CA, USA). Differences among multiple groups were evaluated using one-way analysis of variance (ANOVA) followed by Tukey's post hoc test for multiple comparisons.
Normality and homogeneity of variance were assessed prior to analysis. A value of P < 0.05 was considered statistically significant.
Results
Chemical characterization of CHWKN
To elucidate the chemical basis underlying the pharmacological effects of CHWKN, LC-MS/MS analysis was performed, and representative total ion chromatograms are shown in Figure 1A. As illustrated in Figure 1B, carbohydrates and their derivatives (25%) and amino acids and derivatives (15%) were the predominant components, followed by flavonoids (11%), terpenoids (9%), and phenolic acids (4.5%). Notably, these bioactive compounds are widely reported to possess antioxidant, anti-inflammatory, and microbiota-modulating properties.
Since carbohydrates and amino acid derivatives can serve as substrates for gut microbiota (Mora-Flores et al., 2023), while flavonoids and phenolic compounds exert immunomodulatory effects (Al-Khayri et al., 2022), the chemical composition of CHWKN suggests a potential dual role in regulating host inflammation and gut microbial homeostasis. Detailed information on the major constituents is summarized in Table 2.
CHWKN protects against ethanol-induced gastric mucosal injury
To evaluate the gastroprotective effect of CHWKN, as detailed in a previously published study (Huang et al., 2024), an ethanol-induced GU model was established (Figure 2A). Ethanol administration resulted in severe gastric mucosal injury, characterized by hemorrhage, epithelial disruption, and inflammatory cell infiltration. In contrast, CHWKN treatment markedly alleviated these pathological changes, as demonstrated by improved mucosal integrity in H&E and PAS staining (Figure 2B).
Quantitative analysis further showed that CHWKN significantly reduced the ulcer index and ameliorated mucosal damage compared with the model group (Figure 2C). Additionally, CHWKN increased serum pepsinogen I level and the PGI/PGII ratio (Figure 2D), indicating improved gastric secretory function and reduced mucosal atrophy. Notably, these protective effects were comparable to those observed in the omeprazole (OME) group, a standard anti-ulcer agent, supporting the therapeutic potential of CHWKN.
CHWKN suppresses gastric inflammation via inhibition of JAK/STAT3 signaling
To investigate the anti-inflammatory effects of CHWKN, circulating inflammatory mediators were assessed. Exposure to ethanol exposure significantly increased the level of pro-inflammatory cytokines, including IL-1β, IL-6, TNF-α, MPO, and LPS, while decreasing the level of the anti-inflammatory cytokine IL-10 (Figures 3A–F). CHWKN treatment markedly reversed these changes, indicating a robust anti-inflammatory effect.
Notably, similar trends were observed in the OME group, which served as a positive control. Given the central role of the JAK/STAT pathway in inflammatory responses (Agashe et al., 2022), we further examined its activation. qRT-PCR analysis showed that ethanol markedly upregulated Jak1 and Stat3 expression, whereas both CHWKN and OME treatment significantly suppressed their expression (Figures 3G, H). These findings suggest that CHWKN alleviates ethanol-induced gastric inflammation, at least in part, through inhibition of JAK1/STAT3 signaling.
Given the emerging role of gut microbiota in regulating gastric inflammation, we next investigated whether the protective effects of CHWKN depend on microbiota.
Gut microbiota is required for the gastroprotective effects of CHWKN
To determine whether gut microbiota contributes to the protective effects of CHWKN, antibiotic-treated (ABX) rats were used to deplete gut microbiota (Figure 4A). Histological analysis revealed that depletion of the microbiota markedly attenuated the protective effects of CHWKN, as evidenced by aggravated mucosal injury, increased epithelial erosion, and enhanced inflammatory infiltration in the ABX+CHWKN group compared with the CHWKN group (Figures 4B, C). Consistently, the beneficial effects of CHWKN on gastric function, as indicated by increased PGI levels and the PGI/PGII ratio, were significantly diminished following microbiota depletion (Figure 4D).
Gut microbiota mediates the anti-inflammatory effects of CHWKN
We next examined whether microbiota depletion affects CHWKN-mediated anti-inflammatory responses. Compared with the CHWKN group, the ABX+CHWKN group exhibited significantly elevated levels of IL-1β, IL-6, TNF-α, MPO, and LPS, accompanied by a reduction in IL-10 levels (Figures 5A–F). Furthermore, the inhibitory effects of CHWKN on Jak1 and Stat3 expression were abolished following microbiota depletion (Figures 5G, H).
These findings indicate that gut microbiota is essential for the anti-inflammatory effects of CHWKN and its regulation of JAK1/STAT3 signaling.
CHWKN-associated microbiota is sufficient to confer protection against gastric injury
To further establish the causal role of gut microbiota, FMT was performed (Figure 6A). Rats receiving microbiota from CHWKN-treated donors (the donor rats treated with ethanol and CHWKN, CHWKN-FMT) exhibited significantly improved gastric mucosal integrity compared with those receiving microbiota from model donors, as demonstrated by reduced epithelial damage and inflammatory cell infiltration (Figures 6B, C). In addition, CHWKN-FMT restored gastric secretory function, as evidenced by increased serum PGI levels and PGI/PGII ratio (Figure 6D).
Consistently, CHWKN-FMT significantly reduced pro-inflammatory cytokines (IL-1β, IL-6, TNF-α, MPO, and LPS) while increasing IL-10 levels (Figures 7A–F). Moreover, Jak1 and Stat3 expression were markedly suppressed in the CHWKN-FMT group (Figures 7G, H). These findings demonstrate that CHWKN-associated microbiota is sufficient to confer protection against gastric injury and inflammation, highlighting a causal role of gut microbiota in mediating the therapeutic effects of CHWKN.
CHWKN remodels gut microbiota composition in GU
To characterize microbiota alterations, 16S rDNA sequencing was performed. Alpha diversity analysis revealed no significant differences in Chao, ACE, Shannon, and Simpson indices among groups (Figure 8A), suggesting that CHWKN does not significantly impact microbial richness. However, β-diversity analysis (PCoA) showed a clear separation between the model and CHWKN groups on ASV level (Figure 8B), indicating that CHWKN reshapes the structure of the microbial community.
LEfSe analysis further exhibited the representative altered by ethanol and CHWKN treatment. The g_Lachnospiraceae_NK4A136 group, g_Christensenellaceae_R-7 group, g_Allobaculum, g_Rothia were enriched in control group. The genus g_unclassified_f_Anaerovoracaceae, g_unclassified_f_Lachnospiraceae, g_unclassified_f_Oscillospiraceae, g_unclassified_f_Ruminococcaceae, g_norank_f_Desulfovibrionaceae were enriched in model group.
G_unclassified_f_Prevotellaceae, g_norank_f_Prevotellaceae, g_Dubosiella, g_Faecalibaculum were enriched in CHWKN group (Figure 8C). Notably, CHWKN treatment increased potentially beneficial bacteria such as Lactobacillus, while reducing taxa enriched in the model group. At the genus level, CHWKN partially reversed ethanol-induced dysbiosis, characterized by reduced abundance of norank_f_Muribaculaceae and increased Lactobacillus (Figure 8D).
These results suggest that CHWKN alleviates GU injury, at least in part, by remodeling gut microbiota composition.
Discussion
In the present study, we demonstrate that CHWKN exerts significant protective effects against ethanol-induced GU. Importantly, our findings reveal that these effects are mediated via a gut microbiota-associated mechanism involving the suppression of inflammatory responses and inhibition of JAK1/STAT3 signaling. By integrating antibiotic-mediated microbiota depletion, FMT, and microbiome profiling, provides evidence supporting a causal role for gut microbiota in CHWKN-mediated gastroprotection (Figure 9).
Ethanol-induced gastric injury is characterized by disruption of the mucosal barrier disruption, oxidative stress, and excessive inflammatory responses (Zou et al., 2021; Guo et al., 2024b). Consistent with previous reports, we observed that ethanol exposure resulted in severe mucosal damage, accompanied by increased levels of pro-inflammatory cytokines, including IL-1β, IL-6, TNF-α, MPO, and LPS (Guo et al., 2024b; Zhao et al., 2024; Li et al., 2018). These mediators are known to amplify inflammatory cascades and contribute to epithelial injury.
Treatment with significantly alleviated these pathological changes, indicating its potent gastroprotective and anti-inflammatory properties. Notably, the effects of CHWKN were comparable to those of omeprazole (Oosterhuis and Jonkman, 1989), a clinically established anti-ulcer agent, further supporting its therapeutic potential. Inflammation plays a central role in GU progression, and the JAK/STAT signaling pathway is a key regulator of inflammatory responses (Agashe et al., 2022).
Activation of JAK1/STAT3 has been reported to promote cytokine production and sustain inflammatory signaling in gastrointestinal disorders (Sarapultsev et al., 2023). In this study, CHWKN markedly suppressed the expression of Jak1 and Stat3, suggesting that inhibition of JAK/STAT3 signaling contributes to its anti-inflammatory effects. These findings are consistent with the notion that targeting inflammatory signaling pathways represents an effective strategy for mitigating gastric mucosal injury.
A major finding of this study is the identification of gut microbiota as a critical mediator of the action of CHWKN. Increasing evidence indicates that gut microbiota plays an essential role in maintaining gastrointestinal homeostasis and regulating host immune responses (Yoo et al., 2020; Wang et al., 2024). Dysbiosis has been associated with enhanced inflammatory signaling and impaired mucosal repair (Santana et al., 2022).
In the present study, antibiotic-mediated depletion of gut microbiota abolished both the protective and anti-inflammatory effects of CHWKN, demonstrating that microbiota is required for its therapeutic action. More importantly, fecal microbiota transplantation experiments further established the causal role of microbiota. Transfer of microbiota from CHWKN-treated donors was sufficient to confer protection against gastric injury and suppress inflammatory responses in recipient rats.
These results provide strong evidence that CHWKN does not merely act on host tissues directly, but rather exerts its effects, at least in part, through modulation of gut microbiota. Such microbiota-associated mechanisms have been increasingly recognized in the pharmacological actions of herbal medicines.
Consistent with these functional findings, 16S rDNA sequencing revealed that CHWKN reshaped gut microbial composition. Although microbial richness was not significantly altered, CHWKN induced clear changes in microbial community structure and increased the abundance of beneficial taxa, such as Lactobacillus. We acknowledged only reliance on 16S rRNA sequencing, does not make sure definitively pinpoint the functional bacterial taxa and metabolites mediating the protective effects against ethanol induced gastric injury.
Nevertheless, previous study reported Lactobacillus rhamnosus GG facilitates gastric ulcer healing by reducing the apoptosis to proliferation ratio and promoting angiogenesis (Lam et al., 2007). In addition, L. rhamnosus SHA113 and its conditioned medium have been reported to exert anti-inflammatory activity and attenuate ethanol induced mucosal damage (Yang et al., 2021). These data collectively suggest that Lactobacillus species may participate in the protective effects to gastric ulcer.
However, definitive attribution of causality requires further experimentation, including targeted bacterial depletion or reconstitution studies. Lipopolysaccharide (LPS), an integral component of the outer membrane of Gram-negative bacteria, is a well-recognized pro-inflammatory endotoxin and serves as a reliable indicator of low-grade systemic inflammation (Di Vincenzo et al., 2024). We found serum LPS levels were significantly reduced following CHWKN administration.
Notably, this effect was largely abolished by gut microbiota depletion and restored by fecal microbiota transplantation, suggesting that the impact of CHWKN on systemic inflammatory status is mediated, through the gut microbiota. The JAK1/STAT3 signaling pathway is a classical pathway through which LPS exerts its pro-inflammatory effects (Ji et al., 2025). Our findings also suggest a potential link between gut microbiota and JAK1/STAT3 signaling.
Therefore, microbiota remodeling induced by CHWKN may contribute to suppression of inflammatory signaling and protection of the gastric mucosa. Our findings also suggest a potential link between gut microbiota and JAK1/STAT3 signaling. Although the precise mechanisms remain to be fully elucidated, it is plausible that microbiota-derived metabolites or microbial-host interactions may regulate inflammatory pathways, thereby influencing JAK1/STAT3 activation.
This microbiota-inflammation signaling axis may represent a key mechanism underlying the therapeutic effects of CHWKN.
Several limitations of this study should be acknowledged. First, although we demonstrated a causal role of microbiota using ABX and FMT approaches, the specific microbial taxa and metabolites responsible for the observed effects remain unclear. Second, the molecular mechanisms linking microbiota alterations to JAK1/STAT3 signaling require further investigation.
Future studies incorporating metabolomics and mechanistic analyses will help to clarify these pathways.
Conclusion
This study demonstrates that in contrast to single compound therapies, CHWKN exerts concurrent modulation of gut microbiota and suppression of the JAK1/STAT3 inflammatory pathway, offering a dual mechanism that addresses both microbial dysbiosis and mucosal inflammation. These findings not only provide mechanistic insight into the therapeutic effects of CHWKN but also highlight the potential of targeting gut microbiota as a strategy for GU management, although further evidence from pharmacokinetic, toxicological, and clinical studies is required to validate this potential. Collectively, these findings position CHWKN as a promising multi-targeted therapeutic candidate for gastric ulcer disease, with potential to serve as an adjunct or alternative to antibiotics and proton pump inhibitors.