Integrated network pharmacology analysis and in vitro cell experiments to reveal the mechanisms of Ligusticum chuanxiong Hort. in the treatment of non-alcoholic fatty liver disease

Abstract

Background: Non-alcoholic fatty liver disease (NAFLD) is a liver disease of unknown etiology. A traditional Chinese medicine Ligusticum chuanxiong Hort. (CX), it has been used about 2,000 years. Until now, the mechanism of action of CX on NAFLD remains unclear. Method: We first tested the toxicity of CX to AML12 cells with CCK-8. In vitro cell models of NAFLD were made using free fatty acid, and used Oil Red O staining tested lipid droplets. Then the active compounds of CX were collected from TCMSP and literatures, and SwissTargetPrediction, Search Tool for Interacting Chemicals, Encyclopedia of Traditional Chinese Medicin, Bioinformatics Analysis Tool for Molecular mechANism of Traditional Chinese Medicine database were used to predict the targets of the compounds. DRUGBANK, Online Mendelian Inheritance in Man, Therapeutic Target Database, DisGeNET and GeneCards database were used to predict the targets of NAFLD. Use Venn diagram to obtained the intersection targets by, and analyzed the protein-protein intersection network. Use Kyoto Encyclopedia of Genes and Genomes and Gene Ontology to forecast the function of intersection genes. Molecular docking was used to evaluate the interaction between hub gene and active ingredients. Finally, use western blotting to determine the effects of CX on PPARA, PPARG, IL1B and TNF proteins. Result: CX can reduce the production of AML12 cell lipid droplets. A total of 15 chemical components were identified from CX. Folic acid, chrysophanol and sitosterol were the main components of CX against NAFLD. ALB, TNF, PPARG and PPARA proteins were the main targets of CX in the treatment of NAFLD. PPAR signaling pathway and fatty acid degradation were closely related to anti-NAFLD. Molecular docking results shows that folic acid was the main active ingredient of CX for NAFLD treatment, and TNF is the main potential target. The cellular NAFLD model showed that CX up-regulated the expression of PPARA and PPARG protein and down-regulated inflammatory factor IL-1B and TNF expression. Conclusion: Our study suggests that CX has a therapeutic effect on NAFLDA, which may be related to the PPAR pathway and the reduction of inflammatory cytokines.