| Objective:To investigate the hepatoprotective effect of dioscin, a natural product, against CCl4-induced acute liver damage in mice, and to study the mechanisms of its effect and possible drug-targets prediction.Methods:In this study, dioscin (25,50,100 mg/kg) was pretreated to the CCl4 exposed mice for 5 days, and silymarin (200 mg/kg) was used as a positive drug. Hematoxylin and eosin (H&E) staining and DNA laddering assay were deployed to evaluate liver necrosis and apoptosis. The levels of ALT, AST, ALP, LDH, MDA, SOD, GSH and liver index were detected to investigate the effect of dioscin on CCl4-induced hepatotoxicity. By radio-immunity assay (RIA) and reverse transcription-polymerase chain reaction (RT-PCR), the levels of TNF-αand IL-6 were investigated. The caspase-3 and-8 activities were detected by kits. Western blotting assay and immunohistochemistry (IHC) were used to evaluate proteins expression levels involved in the mechanisms of dioscin's hepato-protective effect, including Fas, FasL, Bax, Bcl-2, cytochrome c, as well as ICAM-1, vimentin, prohibitin, HGF and c-Met. In addition,1-D gel electro-phoresis coupled with nanoRPLC-ESI-MS/MS/MS was used to search possible biomarkers, and then the distributions of protein functions were analyzed by Gene Ontology (GO) enrichment analysis. Meantime, the possible targets of dioscin for hepatoprotective effect to CCl4-induced heaptotoxicities in mice were found through a bioinformatics approach, INVDOCK program.Results:The hepatoprotective effect of dioscin at the dose of 100mg/kg is equal to the effect produced by silymarin at the dose of 200mg/kg, which is widely used in various kinds of liver injury as a famous hepatoprotective drug. Pretreatment with dioscin prior to administration of CCl4 significantly inhibited CCl4-induced elevations in ALT (4488±568.33 vs.1265±238.41 U/L), AST (2691±332.98 vs.831±229.60 U/L), ALP (24.88±6.89 vs.15.63±3.06 U/100 mL) and LDH (3648.89±508.97 vs. 2010.96±209.46 U/L) serum levels, reversed CCl4-induced increases of hepatic MDA content (1.71±0.13 vs.0.69±0.09 nmol/mg protein) and decreases of SOD (27.23±0.74 vs.46.81±3.42 U/mg protein), GSH activities (0.24±0.03 vs.0.54±0.02 mg GSH/g protein) in liver homogenates and liver index (5.35±0.30 vs.4.11±0.24%). The concentrations and mRNA expression level of TNF-a and IL-6 were also down-regulated by dioscin. Liver histopathologic study and DNA laddering assay indicated that dioscin protected hepatocyte from CCl4-induced apoptosis and necrosis. Deeply study indicated that dioscin markedly decreased Fas/FasL protein expressions, increased Bcl-2/Bax ratio, and inhibited release of cytochrome c from mitochondrion to cytosol. Further-more, dioscin in turn attenuated CCl4-induced caspase-3,-8 activities. Additionally, the levels of phosphorylation of mitogen-activated protein kinases (MAPKs) were also regulated by dioscin, as well as the expressions of ICAM-1, vimentin, prohibitin, HGF and c-Met. Then,71 proteins from total protein were identified by proteomic method, in which more than 50% of them are associated with the functions of response to stimulus and stress, regulation of apoptosis, oxidation reduction and so on. Six differentially expressed proteins in them, HSPA5 and ANXA6 expressions were down-regulated by dioscin, while IVD, RPS6, cytoglobin and NDPK-A expressi-ons were up-regulated. At last, bioinformatic technique, inverse dock prog-ram (INVDOCK), was used to forecast the possible targets of dioscin for the hepatoprotective effect from mouse protein data base, and two proteins, iNOS and GSTA1, were found.Conclusion:The present study is the first time to discover the significant hepatoprotective effect of dioscin against liver injury, which might be developed into a new hepatoprotective drug. The mechanism investigation and drug-target finding are benefit for the therapy of liver damage in the future.
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