OBJECTIVE The mechanism of action of Tiaozhi Yigan Decoction(TZYGD) in the prevention and treatment of metabolic associated fatty liver disease(MAFLD) was explored using network pharmacology and molecular docking methods, and the results were verified through animal experiments.

METHODS The active components and target proteins of TZYGD were collected and screened from the TCMSP and SwissTargetPrediction databases. The disease-related target proteins of MAFLD/NAFLD were obtained from the GeneCards, DisGeNET, and OMIM databases, and the drug-disease intersection target proteins were determined. The “drug-active component-target” network and protein-protein interaction(PPI) network were constructed using Cytoscape 3.8.2 software, and the core active components and core target proteins were further screened. The Metascape database was used to perform GO and KEGG pathway enrichment analysis on the intersection target proteins, and molecular docking was used to verify the core active components and core target proteins of TZYGD in the treatment of MAFLD. A high-fat diet-induced MAFLD mouse model was established, and the results of network pharmacology were verified through animal experiments.

RESULTS Network pharmacology predicted 164 active components of TZYGD and 321 potential target proteins for the treatment of MAFLD. The core target proteins included IL6, TNF, ESR1, EGFR, AKT1, PPARγ, STAT3, PTGS2, CYP1A1, and PPARα, and the core active components included ethyl linolenate, palmitoyl amyrin, isocryptotanshinone, dihydrotanshinone, and eucommiol A. The results of GO functional enrichment analysis indicated that the regulation of hormone levels, inflammatory response, and small molecule metabolic processes were highly enriched in biological processes; membrane rafts, transcriptional regulatory factor complexes, and low-density lipoproteins were highly enriched in cellular components; protein kinase activity, transcription factor binding activity, and protein tyrosine kinase activity were highly enriched in molecular functions. The results of KEGG pathway enrichment indicated that TZYGD may exert its anti-MAFLD effect by regulating the PI3K-AKT/PPARα signaling pathway. Molecular docking results showed that most of the core active components had good binding activity with the core target proteins. Results from animal experiments indicate that the Lipid-Regulating and Liver-Protecting Decoction reduced serum TG, TC, and LDL-C levels in MAFLD model mice, decreased serum AST and ALT activity, reduced liver MDA, TG, and TC levels, and increased GSH levels, TZYGD effectively alleviated liver steatosis, vacuolar lesions, red lipid droplets, and fibrosis in MAFLD model mice. TZYGD significantly downregulated the protein expression levels of FASN, ACC, LC3II/I, P62, p-mTOR/mTOR, and SREBP1-c in the liver and significantly upregulated the protein expression levels of PPARα and p-AKT/AKT.

CONCLUSION TZYGD can significantly reduce serum transaminases, blood lipids, and lipid accumulation and oxidative stress in MAFLD mice, and improve pathological changes such as liver steatosis. Its mechanism of action may be related to the regulation of the PPARα/AKT/mTOR signaling pathway.