Lipid-lowering effect of flavanomarein on non-alcoholic fatty liver disease based on network pharmacology

Objective To explore the targets and signaling pathways of flavanomarein in the treatment of non-alcoholic fatty liver disease (NAFLD) by network pharmacology, and to investigate the effect and mechanisms in the treatment. Methods ① The potential active targets of flavanomarein were retrieved in the Swiss Target Prediction database, and the disease-related targets were searched in the DisGeNET database. The prediction targets of lipid metabolism of NAFLD regulated by flavanomarein was obtained through Venn analysis. The Biso Genet database was used to construct the protein-protein interaction (PPI) network diagram, the Metascape database was used to analyze GO functional enrichment and KEGG pathway enrichment, and the Cytoscape software was used to draw the "target-pathway" network diagram. ② NAFLD db/db mice was employed for in vivo verification. Thirty male db/db mice were randomly divided into db/m group, db/db model group, and db/db+flavanomarein group (50 mg/kg). After the administration of 0.5% sodium carboxymethyl cellulose solution and flavanomarein for 12 weeks, the mice were sacrificed, and the liver contents of TG, GSH and GSSG was detected. HE staining and oil red O staining were used to observe the pathological changes of the liver. qRT-PCR and Western blotting were used to detect the mRNA and protein levels of key molecules in the lipid metabolism in the liver. ③HepG2 cells were treated with free fatty acids (FFAs, oleic acid∶palmitic acid =2∶1) and divided into normal group, FFAs treatment group (500 μmol/L), and FFAs+ low, medium and high concentrations of flavanomarein (25, 50, 100 μmol/L) for 24 h. Oil red O staining was used to detect the TG content in HepG2 cells. qRT-PCR and Western blotting were used to detect the mRNA and protein levels of key molecules in the lipid metabolism of HepG2 cells. Results ① A total of 29 core targets of flavanomarein docking NAFLD were screened through Venn analysis, including peroxisome proliferator-activated receptor-gamma (PPARγ), vascular endothelial growth factor (VEGF), and mitogen-activated protein kinase 14 (MAPK14). The GO analysis enriched 6 molecular functions, 20 biological processes and 5 cellular components. KEGG analysis enriched 40 key signaling pathways related to the effect of flavanomarein on NAFLD, which mainly focuses on the AMPK lipid metabolism pathway, insulin resistance (IR) pathway, and apoptosis pathway. ② The results of animal experiments showed that flavanomarein treatment reduced TG level in the NAFLD mice (P < 0.05) and improved the ratio of GSH/GSSG (P < 0.05). HE and oil red O staining showed that flavanomarein could reverse hepatocyte enlargement and reduce lipid drop infiltration of hepatocytes. qRT-PCR results showed that the treatment decreased the mRNA levels of ACC1, SCD1, and FASN for lipid synthesis (P < 0.05), while increased those of ACOX-1 and CPT1α for β-oxidation (P < 0.05). Western blot assay results indicated that the expression levels of SREBP and FASN were obviously lower (P < 0.05), and those of PPARγ and PPARα were increased (P < 0.05) in the flavanomarein group than the model group. ③ In vitro cell experiments showed that flavanomarein reduced the TG content of HepG2 cells (P < 0.05) and the expression of SREBP, SCD1 and ACC1, but increased the levels of CPT1α and ACOX-1 mRNA (P < 0.05). And, the protein expression of SREBP and FASN was also significantly reduced (P < 0.05). Conclusion Flavanomarein may play a therapeutic role in NAFLD by improving oxidative stress, up-regulating fatty acid β-oxidation, reducing lipid synthesis through regulating the PPARγ/SREBP/FASN signaling pathway.