Effects And Mechanisms Of Dioscin On BDL-and DMN-induced Hepatic Fibrosis And Liver Regeneration

Objective:To investigate the effects and potential mechanisms of dioscin on BDLand DMN-induced hepatic fibrosis and liver regeneration.Methods:(1)In the study of dioscin against BDL-and DMN-induced hepatic fibrosis,in vitro experiments,HSC-T6 and LX2 were pretreated with different concentrations of dioscin(0.6,1.2 or 2.4 μg/mL)under different treatment times(6,12 and 24 h).The effects of dioscin on HSCs activation were assayed by MTT,flow cytometry,immunofluorescence and real-time PCR assays.In vitro,one experimental hepatic fibrosis model was induced by BDL,and at the begaining of test,the rats were daily administered wth dioscin at the doses of 60,40 or 20 mg/kg,and the positive drug UDCA(25 mg/kg)was used for 4 weeks.Each group contained 10 animals.In addition,another experimental hepatic fibrosis model was induced by intraperitoneal(i.p.)DMN(1%)treatment three times per week for 6 weeks.At the begaining of the test,the rats were daily administered with dioscin at the doses of 60,40 and20 mg/kg,and the positive drug silymarin(100 mg/kg)was also used.Each group contained 10 animals.In this study,the effects of diosin against hepatic fibrosis were evaluated by the changes of the body weights,the levels of AST,ALT,TBIL,MDA,SOD,GSH and GSH-Px,the histopathological changes and immunofluorescence assays.The methods of immuno-histochemical,immunofluorescence,real-time PCR and western blotting assays were used to detect the expression levels of protein/gene releated to Sirt1/Nrf2 signaling pathway.Furthermore,the abrogation of p38 MAPK by SB-203580(a p38 MAPK inhibitor),and Sirt1,Nrf2 siRNA were applied for underlying the molecular mechanisms(2)In the study of dioscin to promote liver regeneration,the in vitro experiments,the rat primary hepatocytes and AML 12 cells were pretreated with different concentra-tions of dioscin under different treatment times(6,12 and 24 h).The effects of dioscin on cell proliferation were detected by MTT,Brdu and PCNA immunofluorescence assays.In vivo,70%PH models were induced in C57BL/6 mice and SD rats.The C57BL/6 mice were randomly divided into four groups:the animals in sham and PH(model)groups were treated with vehicle(0.5%CMC-Na),and the animals in dioscin+PH groups were given dioscin at the doses of 40 mg/kg and 80 mg/kg.Each group contained 12 animals.The rats were also randomly divided into four groups:the animals in sham and PH(model)groups were treated with vehicle(0.5%CMC-Na),and the rats in dioscin+PH groups were given dioscin at the doses of 30 mg/kg and 60 mg/kg.Each group contained 12 animals.Dioscin was administered intragastrically(i.g.)once daily for seven consecutive days.70%PH was performed on the 4th,5th,6th or 7th day,and the sham operation was only laparotomy without liver resection.Two hours before sacrifice,all animals were injected intraperitoneally with Brdu(100 mg/kg).At different times after PH or sham-operation,all animals were sacrificed.Then the blood and liver tissue were collected and stored for further assay.The liver to body weight ratios,serum ALT/AST levels,Brdu and PCNA immunohistoche-mical stainings were investigated.For molecular mechanism investigation,the methods of ELISA,immunofluorescence,flow cytometry and western blotting assays were used to detect the effects of dioscin on the expression levels of protein/gene releated to Notchl/Jaggedl signaling pathway.In addition,Notchl siRNA and DAPT(γ-secretase inhibitor)were applied.Results:(1)In the study of protecting BDL-and DMN-induced hepatic fibrosis,dioscin markedly inhibited the cell viabilities of HSC-T6 and LX-2 cells.Furthermore,dioscin markedly decreased the levels of ROS and significantly reduced the mRNA levels of COL1A1,COL3A1,α-SMA and fibronectin.Notably,dioscin inhibited HSCs activation in vitro.In vivo.dioscin obviously improved body weights,decreased the levels of ALT,AST,TBIL and MDA,markedly increased the levels of SOD,GSH and GSH-Px,reversed the histopathological changes and reduced the mRNA levels of COL1A1,COL3A1,α-SMA and fibronectin.In the study of molecular mechanisms,dioscin markedly up-regulated the levels of Sirtl,HO-1,GST,GCLC and GCLM via increasing the nuclear translocation of Nrf2,which in turn inhibited p38 MAPK phosphorylation and reduced the levels of COL1A1,COL3A1,α-SMA and fibronectin.These results were further validated by knockdown of Sirtl and Nrf2 using siRNAs silencing,and the abrogation of p38 MAPK by SB-203580 in HSC-T6 and LX-2 cells.Collectively,our findings confirmed the potent effects of dioscin against BDL-and DMN-induced hepatic fibrosis via Sirtl/Nrf2-mediated inhibition of p38 MAPK pathway.(2)In the study of promoting liver regeneration,in vitro,compared with control groups,dioscin(300,600 ng/mL)clearly increased cell viability and proliferation on the rat primary hepatocytes,and clearly increased cell viability and proliferation on AML 12 cells at the concentions of 150,300 ng/mL.In addition,dioscin significantly increased the level of PCNA based on immunofluorescent staining in AML12 cells.In vivo,compared with PH groups,dioscin obviously increased the ratios of liver/body weight,decreased the serum ALT and AST levels,and stimulated cell proliferation.In mechanism study,dioscin dramatically enhanced y-secretase activity via up-regulation of PS1.In addition,dioscin up-regulated the levels of Notchl,Jagged 1 and the translocation of NICD1 to nucleus,increased the levels of Heyl,Hesland then promoted cell cycle progression and hepatocyte proliferation.In addition,the results of Notchl siRNA and DAPT inhibition further confirmed that diosicn showed potent effect on liver regeneration via activating Notchl/Jaggedl signaling pathway.Conclusion:(1)Dioscin alleviates BDL-and DMN-induced hepatic fibrosis via Sirtl/Nrf2-mediated inhibition of p38 MAPK pathway.(2)Our data also suggested that dioscin promotes liver regeneration by regulating Notchl/Jaggedl signaling pathway.In short,our results provide useful experimental data for prevention of hepatic fibrosis and promotion of liver regeneration of dioscin,which should be considered as one potent new drug in the future.