Exploring The Mechanism Of Danshen-Huzhang Herb Pair On The Treatment Of Non-alcoholic Steatohepatitis Based On Network Pharmacology And Experimental Verification

Objective1.To investigate the mechanism of action of Danshen-Huzhang herb pair in the treatment of non-alcoholic fatty liver disease(NAFLD)based on network pharmacology and molecular docking techniques.2.Validation of the therapeutic effects of the major active ingredients of Danshen-Huzhang herb pair on NAFLD through enrichment of the major signaling pathways based on in vitro experiments.Methods1.The TCMSP platform was used to screen drug compound targets.GeneCards database and OMIM database were used to screen disease targets of NAFLD.The intersection targets of drugs and NAFLD were extracted to obtain potential action targets.And then PPI and drug-active ingredient-target-disease network diagram were constructed.Accumulation analysis of GO and KEGG pathways at intersecting targets was performed using DAVIAD database.Molecular docking verification of key compounds and core targets was performed,and the results were visualized by Pymol software.2.The HepG2 fatty liver cell model was constructed using sodium palmitate.Luteolin was use to interfere with fatty liver cell.The intracellular lipid deposition was determined by oil red O staining and triglyceride content detection,which was inhibited by AKT activator SC79.The therapeutic effect of luteolin is inhibited by AKT activator SC79.The enriched key signal pathway(PI3K-AKT pathway)was verified in vitro by Western Blot experiment.Results1.Network pharmacology prediction showed that there were 59 active components and a total of 89 target points in the treatment of non-alcoholic fatty liver with Danshen-Huzhang herb pair.The core compounds obtained are quercetin,luteolin,tanshinone iia,beta-sitosterol,cryptotanshinone,etc.The core targets are AKT1,TP53,MYC,ESR1,CASP3,etc.A total of 100 entries were enriched for molecular function,430 entries were enriched for biological process,and 56 entries were enriched for cellular component.The KEGG pathway was enriched to a total of 122 signaling pathways,including PI3K-AKT signaling pathway,AGE-RAGE signaling pathway in diabetic complications,MAPK signaling pathway,HIF-1 signaling pathway,p53 signaling pathway and TNF signaling pathway.The results of molecular docking showed that the binding energies of five key effective components to five key target proteins were all less than-5 kcal/mol,which indicated that the binding activity was good.2.Luteolin,the core active compound of Danshen-Huzhang herb pair,and PI3K-AKT signaling pathway,the key signaling pathway were selected for experimental verification.It was confirmed that luteolin could effectively reduce the fat accumulation and intracellular triglyceride content in HepG2 fatty liver cell model.Luteolin inhibits the mTOR signaling pathway by inhibiting the phosphorylation of PI3K-AKT signaling pathway,thus activating autophagy to relieve NAFLD.Conclusion1.The results of network pharmacology in this study predicted and validated the therapeutic effects of multiple compounds in Danshen-Huzhang herb pair on NAFLD possibly through the synergistic cascade of multiple targets and signaling pathways2.The in vitro experiment further confirmed that luteolin,the core compound of Danshen-Huzhang herb pair,exerted the pharmacological effect of alleviating NAFLD by acting on autophagy induced by PI3K-AKT-mTOR signaling pathway.