Renqingchangjue ameliorates MNNG-induced chronic atrophic gastritis by inhibiting the TNF/NF-κB/Caspase-3 axis

Chronic atrophic gastritis (CAG) is a pivotal premalignant stage in the Correa cascade, characterized by progressive and largely irreversible loss of gastric glands and an elevated risk of gastric cancer. Renqingchangjue (RQCJ), a classical Tibetan multi-herb formula, has demonstrated clinical and pharmacological benefits in gastritis, yet its mechanisms of action in CAG remain inadequately defined.

This study aims to clarify the therapeutic efficacy and mechanistic basis of RQCJ in CAG.

We established a network pharmacology-bioinformatics workflow to predict potential RQCJ targets in CAG, integrating targets obtained from TCMSP/SwissTargetPrediction with CAG-related genes to construct a STRING protein-protein interaction (PPI) network and perform GO/KEGG enrichment analyses. Constituents were characterized by UHPLC-HRMS/MS. An MNNG-induced CAG mouse model (control group, model group, Weifuchun positive control group, RQCJ low-dose group and RQCJ high-dose group) was validated by histopathology (H&E), immunohistochemistry (IHC), immunofluorescence (IF), TUNEL, ELISA, and Western blot (WB). In vitro, MNNG-injured GES-1 cells were evaluated using CCK-8, scratch wound-healing, and Annexin V-FITC/PI flow cytometry. Mechanistically, RT-qPCR, WB, and reference-based transcriptome sequencing of gastric tissue were employed, and compound druggability was assessed by AutoDock Vina docking.

RQCJ markedly ameliorated MNNG-induced chronic atrophic gastritis in vivo and in vitro. UHPLC-HRMS/MS profiling identified 43 constituents, of which 31 were detected as circulating prototypes. Network pharmacology first predicted 154 putative RQCJ-CAG targets enriched in TNF/NF-κB signaling; integrating serum-absorbed component targets with CAG-related genes refined this to 140 high-confidence targets with consistent TNF/NF-κB enrichment. Functionally, RQCJ (20-40 μg/mL) improved GES-1 cell viability and migration while suppressing apoptosis, and in mice dose-dependently repaired gastric mucosal architecture, lowered TNF-α, IL-1β and IL-6, and normalized gastrin and pepsinogen. RQCJ also reduced IL-8, CCL2 and CXCL1 mRNA and increased IL-10 in both models. Mechanistically, it inhibited phosphorylation of IKKβ, IκBα and NF-κB p65 in a dose- and time-dependent manner, decreased cleaved caspase-8/3, restored the Bax/Bcl-2 ratio. Transcriptomics confirmed enrichment of NF-κB and apoptosis pathways, and intersecting differentially expressed genes with the 140 serum-based targets yielded 99 core genes converging on TNF/NF-κB-mediated apoptosis. Molecular docking further supported target engagement, with a chromen-7-ol derivative showing strong predicted affinity for MMP9.

Together, these multi-level data indicate that RQCJ exerts clinically relevant protection against CAG via multi-component suppression of TNF-NF-κB/Caspase-3 axis, broad rebalancing of pro-/anti-inflammatory cytokines, and restoration of gastric function.