Evaluating the mechanism of action of Si Ni San in improving stress-induced gastric ulcers based on the TLR4/NF-κB signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Sinisan (SNS) is a classic traditional Chinese herbal formula originating from Treatise on Cold Damage Disorders, which has been traditionally used to harmonize the liver and spleen, regulate qi dynamics, and relieve abdominal pain and gastrointestinal discomfort. Clinically, SNS has long been applied for disorders characterized by liver-spleen disharmony, including functional and stress-related gastrointestinal conditions. However, its protective effects and underlying mechanisms in stress-induced gastric ulcer (SGU) remain unclear.

OBJECTIVE

To evaluate the protective effects of SNS on SGU in mice and to elucidate the underlying molecular mechanisms.

METHOD: Mice were pretreated with SNS at doses of 2.4, 4.8, and 9.6 g/kg by oral gavage for seven consecutive days prior to induction of stress gastric ulcer using the water-immersion restraint method. Gastric mucosal injury was evaluated by macroscopic observation and histopathological examination (H&E and PAS staining). Oxidative stress parameters, including ROS, MDA, SOD, and GSH-Px, were measured using commercial assay kits. Protein expression levels of TLR4, MyD88, and NF-κB in gastric tissues were determined by Western blot analysis, and serum inflammatory cytokines (TNF-α, IL-1β, and IL-6) were quantified by ELISA. LC-MS/MS was performed to identify absorbed constituents, followed by network pharmacology and molecular docking analysis.

RESULTS

Study showed that SNS protected the gastric mucosal structure, alleviated inflammation, effectively preserved the epithelial layer, and reduced oxidative stress in gastric tissues. It inhibited the expression of TLR4, NF-κB, and MyD88 proteins in gastric tissues, thereby reducing the inflammatory response. ROS and MDA levels were decreased, whereas SOD and GSH-Px activities were increased in gastric tissues compared with the model group. Enrichment analysis suggested that SNS may exert anti-SGU effects through multiple pathways, including the Toll-like receptor signaling pathway, TNF signaling pathway, and IL-17 signaling pathway. Potential active compounds contributing to the protective effects of SNS on the gastric mucosa were identified using LC-MS combined with bioinformatics analysis.

CONCLUSION

SNS pretreatment exerts protective effects against stress-induced gastric ulcer by modulating the TLR4/NF-κB signaling pathway, thereby attenuating inflammatory responses and gastric mucosal injury.