Network Pharmacology, Molecular Docking, Molecular Dynamics Simulation, and in vitro Experiments to Explore the Mechanism of Asiatic Acid Inhibiting Acetaldehyde-Induced Activation of Hepatic Stellate Cells

Abstract

Purpose: This study utilized network pharmacology, molecular docking, molecular dynamics simulation, and in vitro experimental verification to explore the mechanisms of action by which asiatic acid (AA) inhibits acetaldehyde-induced activation of hepatic stellate cells (HSCs). Methods: Databases were screened to identify intersections between AA and alcoholic liver fibrosis. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) Enrichment analysis were performed, followed by molecular docking and molecular dynamics simulation to predict further the interactions between AA and cross-targets. Mechanisms of action of AA were assessed experimentally in HSC-T6 rat HSCs. Results: Screening of the relevant databases using web pharmacology identified several genes, including those encoding signal transducer and activator of transcription 3, nuclear factor kappa-B (NF-κB)-P65 (RELA), tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), and interleukin 1 beta (IL-1β), that were closely associated with alcoholic liver fibrosis. GO and KEGG enrichment analysis showed that the alcoholic liver disease pathway was the most relevant one, and molecular docking showed critical binding activity of RELA, TNF-α, IL-6, and IL-1β with AA. Molecular dynamics simulation showed that the binding between RELA and AA was stable. AA at concentrations of 0 to 30 μM were determined to be nontoxic to HSC-T6 cells. Real-time quantitative polymerase chain reaction and Western blotting showed that the levels of expression of α-SMA and type 1 collagen were higher in acetaldehyde-treated than untreated HSC-T6 cells, with AA reducing their expression dose-dependently in acetaldehyde-treated cells. Western blotting also showed that AA could upregulate the expression of the apoptotic proteins BCL2 associated X and cleaved caspase 3 and could inhibit the phosphorylation of RELA and IKB-α. Conclusion: AA inhibited acetaldehyde-induced HSC-T6 cell proliferation and suppressed the secretion of fibrillar factors by inhibiting RELA phosphorylation.