Analysis of the biological mechanism of Gurigumu-13 in the treatment of non-alcoholic fatty liver disease based on network pharmacology.

contextGurigumu-13 (GR) was a classical Mongolian medicinal compound widely used in clinical practice for the treatment of non-alcoholic fatty liver disease (NAFLD).

objectivesThis study aimed to investigate the underlying mechanism by which GR exerted its therapeutic effects against NAFLD. MATERIALS AND

methodsThe main active components of GR were identified using high-resolution mass spectrometry. Using network pharmacology approaches, key targets and potential pathways of GR against NAFLD were predicted and preliminarily validated through molecular docking. Furthermore, transcriptomic sequencing analysis, combined with in vivo and in vitro experiments was performed to elucidate the efficacy and mechanism of GR in alleviating NAFLD. In vivo, a rat model of NAFLD was established by feeding Wistar rats a methionine-choline deficient (MCD) diet. Serum levels of AST, ALT, TC, TG, MDA, GSH, SOD, HDL-C, and LDL-C were measured. Liver tissues were collected for histopathological examination via H&E staining. In vitro, HepG2 cells were treated with oleic acid and palmitic acid to induce lipid accumulation. Intracellular TG content, as well as MDA and SOD levels, were assessed. Lipid deposition was visualized by Oil Red O staining. The expression of relevant genes and proteins was further analyzed by Western blotting and quantitative real-time PCR (qRT-PCR).

resultsMass spectrometry analysis identified a total of 151 major bioactive compounds in GR. Through network pharmacology, 406 effective active ingredients and 1,012 potential targets of GR were screened. After intersecting with NAFLD-related genes (1,929 genes collected from Genecards and OMIM databases), 329 common targets were obtained. Protein-protein interaction network analysis indicated that STAT3, AKT, and others served as core targets. GO and KEGG enrichment analyses revealed that these genes are primarily involved in biological processes such as oxidative stress and lipid metabolism, as well as signaling pathways including PI3K/AKT and insulin resistance. In vivo, after 8 weeks of MCD diet feeding, rats in the model group exhibited significant hepatocellular fat accumulation, along with increases in body weight (1.82 times of the control group) and liver index (p < 0.05). GR intervention ameliorated these pathological changes, reduced serum levels of AST, ALT, TC, TG, MDA, and LDL-C (p < 0.05), and elevated levels of GSH, SOD, and HDL-C (p < 0.05). In vitro, GR significantly decreased triglyceride content in lipid-loaded HepG2 cells, alleviated intracellular lipid deposition (p < 0.05), and reduced the elevated glucose concentration in the culture medium of the model group. Additionally, GR reversed the increase in ROS and MDA contents and the decrease in SOD activity induced by the lipid-accumulation model. Western blotting results demonstrated that GR upregulated the phosphorylation levels of PI3K, AKT, and mTOR, and reduced STAT3 protein expression (p < 0.05). These changes were further supported by qPCR results (p < 0.05).

conclusionsThe improvement of NAFLD by GR may be primarily achieved through its active components (such as ellagic acid and terchebin) via modulation of key targets (such as STAT3) and signaling pathways (such as PI3K/AKT/mTOR), thereby regulating lipid metabolism, alleviating oxidative stress and insulin resistance, and exerting therapeutic effects.