Integrating network pharmacology and transcriptomics reveals the mechanism by which AS-IV prevents ethanol-induced acute gastric injury in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Gastric ulcer (GU), characterized by complex and multifactorial etiology, remains a prevalent gastrointestinal disease globally. The traditional Chinese medicine Astragalus membranaceus (Fisch.) Bunge is recorded in numerous ancient books such as "Shennong Bencao Jing", "Bencao Gangmu", "Yao Lei Fa Xiang", and "Zhenzhu Nang" to have the functions of tonifying qi, strengthening the spleen, harmonizing the stomach, and promoting ulcer healing. Astragalus membranaceus, a classical Chinese medicinal plant embodying the concept of "medicine-food homology," prominently features astragaloside IV (AS-IV) among its active pharmacological constituents. Accumulating evidence suggests AS-IV confers gastroprotective benefits in various experimental gastric injury models through mechanisms likely involving antioxidant, and anti-apoptotic effects. However, the specific molecular nodes through which AS-IV modulates its gastroprotective effects have not been fully defined.

AIM OF THE STUDY: This experiment combined network pharmacology and transcriptomics to elucidate the underlying mechanisms of AS-IV in alleviating acute ethanol-induced GU in a rat and cell model.

MATERIALS AND METHODS: This investigation employed Sprague-Dawley (SD) rats subjected to ethanol-induced gastric injury and human gastric epithelial GES-1 cells to establish robust in vivo and in vitro GU models, respectively. Initially, we comprehensively assessed the protective efficacy of AS-IV on gastric mucosal lesions using diverse analytical techniques such as Hematoxylin and Eosin (H&E) staining, Alcian Blue-Periodic Acid-Schiff (AB-PAS) staining, Enzyme-Linked Immunosorbent Assays (ELISA), real-time quantitative PCR (RT-qPCR), immunohistochemical (IHC) analysis, and immunofluorescence (IF). Next, we identified essential molecular targets and signaling cascades influenced by AS-IV through integrated analyses encompassing network pharmacology predictions and transcriptomic profiling. Furthermore, the transcriptome further revealed five key molecular nodes (Ereg, Tnfsf11, Nr1d1, Socs3, IL-6), from which we proposed the "triple imbalance - triple repair" model. Finally, in vitro and in vivo experiments verified the inhibitory effect of AS-IV on the EGFR-mediated PI3K/Akt/NF-κB signaling axis.

RESULTS: Our in vivo findings revealed that AS-IV substantially ameliorated pathological manifestations in gastric tissues, markedly reduced inflammatory markers, alleviate oxidative stress, and decreased apoptotic cell death. Network pharmacology and molecular docking predicted EGFR as the key target. MD simulation and CETSA confirmed that there was a stable direct binding between AS-IV and EGFR. Transcriptomics analysis further identified five key molecular nodes on the EGFR/PI3K/Akt/NF-κB signaling axis - Ereg, Tnfsf11, Nr1d1, Socs3 and IL-6. Both in vitro and in vivo experiments confirmed that AS-IV exerted a protective effect against ethanol-induced gastric injury by inhibiting the abnormal activation of the PI3K/Akt/NF-κB signaling pathway mediated by EGFR.

CONCLUSIONS: AS-IV has a significant gastric protective effect against acute gastric injury induced by ethanol. Transcriptomics screening identified five key molecular nodes on the EGFR/PI3K/Akt/NF-κB signaling axis - Ereg, Tnfsf11, Nr1d1, Socs3 and IL-6. Based on this, the "triple imbalance - triple repair" model was proposed. Experimental verification confirmed that AS-IV exerts anti-inflammatory and anti-apoptotic effects by inhibiting the EGFR-driven PI3K/Akt/NF-κB pathway. These findings provide valuable scientific basis for the development of new therapeutic strategies for gastric mucosal protection.