Network pharmacology analysis to explore mechanism of Three Flower Tea against nonalcoholic fatty liver disease with experimental support using high-fat diet-induced rats

Objective:Nonalcoholic fatty liver disease(NAFLD)has become a common chronic liver disease that is harmful to human health.Moreover,there is currently no FDA-approved first-line drug for the treatment of nonalcoholic steatohepatitis(NASH)or NAFLD.Traditional Chinese medicine(TCM)is widely used to ameliorate liver diseases,such as the traditional ancient recipe called Three Flower Tea(TFT),which consists of double rose(Rosa rugosa),white chrysanthemum(Chrysanthemum morifolium),and Daidaihua(Citrus aurantium).However,the mechanisms of the action of TFT are not clear.Therefore,this study aimed to elucidate the mechanisms of TFT against NAFLD in high-fat diet(HFD)-induced rats.Methods:This study utilized bioinformatics and network pharmacology to establish the active and potential ingredient-target networks of TFT.Furthermore,a protein–protein interaction(PPI)network was constructed,and enrichment analysis was performed to determine the key targets of TFT against NAFLD.Furthermore,an animal experiment was conducted to evaluate the therapeutic effect and confirm the key targets of TFT against NAFLD.Results:A total of 576 NAFLD-related genes were searched in Gene Cards,and under the screening criteria of oral bioavailability(OB)≥30%and drug-likeness(DL)≥0.18,a total of 19 active ingredients and 210 targets were identified in TFT.Network pharmacology analysis suggested that 55 matching targets in PPIs were closely associated with roles for NAFLD treatment.Through the evaluation of network topology parameters,four key central genes,PPARγ,SREBP,AKT,and RELA,were identified.Furthermore,animal experiments indicated that TFT could reduce plasma lipid profiles,hepatic lipid profiles and hepatic fat accumulation,improve liver function,suppress inflammatory factors,and reduce oxidative stress.Through immunoblotting and immunofluorescence analysis,PPARγ,SREBP,AKT,and RELA were confirmed as targets of TFT in HFD-induced rats.Conclusion:In summary,our results indicate that TFT can prevent and treat NAFLD via multiple targets,including lipid accumulation,antioxidation,insulin sensitivity,and inflammation.