Effects of Cyclocarya paliurus Flavonoid Extract in Non-alcoholic Steatohepatitis Mice: Intermeshing Network Pharmacology and in vivo Pharmacological Evaluation

Objectives: Non-alcoholic steatohepatitis (NASH) is the second most common cause of the hepatitis. In this study, we aimed to investigate the effects and mechanisms of Cyclocarya paliurus (CPF) flavonoids extract in the treatment of NASH. To achieve this, we performed compound target prediction and network analysis based on the method of network pharmacology. We investigated the effect of CPF extract on NASH and the underlying molecular mechanisms using the in vivo pharmacological evaluation technique. Materials and Methods: First, NASH was induced in mice with a high‑fat diet. Furthermore, experimental validation was carried out with a high‑fat feed‑induced NASH mouse model. Chemical compounds and human target proteins of CPF, as well as NASH‑related human genes, were obtained from TCMSP, PubChem, and GenBank Database, respectively. Subsequently, molecular networks and typical pathways presumably involved in the treatment of CPF on NASH were performed by Metoscape and String software. Then, the network was constructed by using Cytoscape v3.7.2. The Kyoto Encyclopedia of Genes and Genomes pathway enrichment was performed by using Database for Annotation. Then, the possible underlying mechanisms were studied using the quantitative reverse transcription polymerase chain reaction and Western blot analysis. Results: The network pharmacology research showed that the CPF, which contained 26 compounds and 27 genes, regulated the most signaling pathways related to NASH. Moreover, the protein–protein interaction network based on the targets of CPF on NASH revealed that the core compounds were luteolin, quercetin, kaempferol, ellagic acid, isoquercitrin, gallic acid, inositol, and myricetin, which were regulated by more than 4 compounds, remarkable related with peroxisome proliferator-activated receptor (PPAR) signaling pathway, cholesterol metabolism, bile secretion, type II diabetes mellitus, adenosine 5′-monophosphate-activated protein kinase signaling pathway, and insulin signaling pathway. In addition, carnitine palmitoyltransferase 2, cytochrome P450 family 7 subfamily A member 1 (CYP7A1), cytochrome P450 family 27 subfamily A member 1 (CYP27A1), and fatty acid‑binding protein 1 were treated as target genes of CPF in NASH. In vivo experiments indicated that CPF promoted Peroxisome proliferator‑activated receptor alpha (PPAR α)/ Peroxisome proliferator‑activated receptor gamma (PPAR γ) signaling pathway in NASH mouse model. Conclusion: CPF played a role in inhibiting lipid response in NASH, which was closely associated with the modulation effect of CPF on PPAR α/PPAR γ signaling pathway.