Integrative pharmacological analysis of modified Zuojin formula: Inhibiting the HIF-1α-mediated glycolytic pathway in chronic atrophic gastritis

Zuojin formula (ZJF) is a well-known herbal medicine in Pharmacopoeia of China, which is widely used for gastritis. Modified Zuojin formula (MZJF) was adapted based on traditional Chinese medicine (TCM) theories concerning gastric atrophy and dysplasia, along with extensive clinical experience, has been clinically employed to treat chronic atrophic gastritis (CAG). However, the underlying mechanisms by which MZJF intervenes in CAG remain to be fully elucidated. The aim of this study was to evaluate the effects of MZJF intervention in CAG and explore its potential mechanisms. Four induction factors were used to establish a CAG rat model. HE and AB-PAS staining was utilized to assess the effects of MZJF in the intervention of CAG. The stomach weight index and gastric acid pH was used to assess the overall state of stomach. ELISA was used to assess the gastric mucosal inflammatory response. Using transmission electron microscopy to observe chief cells and parietal cells, we evaluated the improvement of ultrastructure by MZJF. Through network pharmacology analysis, the possible regulatory mechanism of MZJF in CAG was preliminarily explored. Binding interactions between MZJF components and predicted targets were explored using molecular docking. Subsequently, quantitative real-time PCR (qRT-PCR), Western blot, biochemical analysis and TUNEL staining were applied to validate the effect of MZJF on predicted pathway. MZJF treatment ameliorated gastric mucosal pathology, inflammation, cellular ultrastructural damage and PG levels, halted the exacerbation of CAG in rats, along with a reduction in stomach weight index and gastric acid pH. A total of 79 compounds in MZJF targeting 203 CAG-related molecules were identified through network pharmacology. Enrichment analysis of the core targets was focused on the hypoxia inducible factor-1α (HIF-1α) signaling pathway. Molecular docking results identified HIF-1α as stable binding targets for MZJF primary components. Subsequently, PCR, WB, and biochemical results showed that MZJF suppressed the gene and protein expression levels of HIF-1α and its downstream molecules including glycolytic enzymes and transporters, modulated glucose, pyruvic acid and lactate levels in gastric mucosal tissue. Moreover, MZJF induced apoptosis of gastric epithelial cells, as evidenced by the upregulation of cleaved caspase-3, Bax, Bax/Bcl-2 and TUNEL positive cells ratio. MZJF suppressed the HIF-1α-mediated glycolytic pathway, and promoted cell