Supplementary Material for: Exploration of Hepatoprotective Effect of Gentiopicroside on Alpha-Naphthylisothiocyanate-Induced Cholestatic Liver Injury in Rats by Comprehensive Proteomic and Metabolomic Signatures

Background/Aims: Cholestasis is the major cause of the accumulation of bile acids and results in liver damage, fibrosis, and failure. A growing number of studies have shown that gentiopicroside is a promising prospect that may protect the liver. However, its therapeutic mechanism has not yet been clarified. This study aimed to explore the effect and mechanism of gentiopicroside in cholestasis induced by alpha-naphthylisothiocyanate. Methods: We performed isobaric tags for relative and absolute quantification-based quantitative proteomics and metabolomics using liquid chromatography quadruple time-of-fight mass spectrometry and identified the expression of 73 metabolites and 84 proteins associated with cholestasis-related dysfunctions in the metabolism of bile acids, fatty acids, and glycerophospholipids. Results: Integrated analyses of proteomic and metabonomic studies showed altered pathways in cholestasis-induced liver injury involving increased activity of farnesoid X receptor/retinoid X receptor, bile acid biosynthesis, and peroxisome proliferator-activated receptor-α/retinoid X receptor-α. Gentiopicroside could reverse these metabolite, protein, and blood biochemical indices, as well as alleviate liver damage. The progressive changes in the proteins and genes may be correlated with cholestasis and were confirmed by western blot and quantitative realtime polymerase chain reaction. Conclusion: Gentiopicroside could be used to protect the liver in the presence of cholestasis.