The Protective Effect Of Sedum Sarmentosum Bunge Against DMN-induced Liver Fibrosis Via Sirt1-AMPK-LXR Signaling Pathway

Objective:Liver fibrosis caused by a variety of pathofenic factors intrahepatic connective tissue dysplasia is considered to be a pathological process.The process of fibrosis will continue to develop long-term cirrhosis.In the process of hepatic fibrosis,it is characterized by activation of hepatic stellate cells and other extracellular matrix cells.In this study,we established the chronic hepatic fibrosis model and activated hepatic stellate cells by using the valuable dimethylnitrosamine to explore the effects of Sedum sarmentosum Bunge(SS)on hepatic fibrosis,lipid deposition,apoptosis and the improvement of inflammatory factors and regulatory mechanisms.Methods:In vivo,the rats were divided into five groups of six rats each group,such as:normal group,DMN group,DMN+SS100 group,DMN+SS300 group and DMN+FZHY group.Liver fibrosis was induced by intraperitonea linjection of 1%DMN(2ml/kg body weight in saline)on three consecutive days per week for up to five weeks.The gavage was performed one hour earlier than intraperitonea linjection in the early morning.The SS groups rats were daily gaveged 100 or 300mg/kg the extract of SS extract,and FZHY group rats were administered with 2g/kg by oral gavage every day for five weeks.At the end of the stipulated period,the animals were euthanized.Blood and liver tissues were collected.The levels of alanine aminotransferase(ALT)and aspartate aminotransferase(AST)in serum were measured by the kit.The effects of SS on liver histomorphology were observed by H&E,Masson staining and immunohistochemical staining.The expression of a-SMA,Collagen I,TIMP-1,SREBP-1,FASN,ACCa,Caspase3,Caspase9,FLIP,Bax,Bcl-2,Bid,Caspase1,TNF-?,Sirtl,AMPK?,p-AMPK?,LXR?,LXR? protein in liver were detected by Western blotting and PCR.In vitro,HSC-T6 and LX-2 cells were cultured in Dulbecco's modified eagle medium(DMEM)containing 10%fetal bovine serum(FBS),100 units/ml penicillinG and 100 mg/ml streptomycin in a 37?,5%CO2 and saturated humidity environment.For the experiment,the cells were plated in 6-well plate a density of 5×105scells/well,cells were exposed to TGF-?(10ng/ml)2h before treatment with SS 6h.The expression of ?-SMA,CollagenI,Sirtl,AMPK,p-AMPK,LXRa and LXR? protein were detected by Western blotting,PCR and immunofluorescence analysis.In addition,TGF-?(10ng/ml)stimulated HSC-T6 and LX-2 cells for 2h,and then treated with SS extract,AICAR(AMPK agonist)and GW3965(LXR agonist)for 6 hours,then collecting cells to extract total protein and nuclear protein for detection.The effects of SS on the expression of Sirtl,AMPKa,p-AMPKa,LXRa and LXR? proteins in HSC-T6 and LX2 cells were detected by Western blotting and immunofluorescence.Results:In vivo experiment,the results showed DMN-induced rats liver injury by that the activities of ALT and AST in serum were significantly inhibited.H&E staining and Masson staining showed that SS extract could significantly improve the liver tissue lesion induced by dimethylnitrosamine in a dose-dependent.The expression of a-SMA,Collagen I,TIMP-1 protein and mRNA in liver tissue was inhibited,and the results were consistent with a-SMA immunohistochemical results.The results of RT-PCR showed that the expression of SREBP1,ACCa and FASN was significantly decreased.The expression of Caspasel and TNFa in the liver can be inhibited,and SS extract reduce the expression of FLIPL,inhibition of Bid,resulting in a increasing expression of cleaved-Bid,then Bax expression was inhibited,thereby increasing the level of Bcl-2.The expression levels of SIRT1,p-AMPKa,LXRa and LXRP were significantly increased in SS group.In vitro,our results showed that SS extract could significantly reduce the expression of a-SMA and Collagenl in hepatic stellate cells to inhibit the activation of hepatic stellate cells.The expression of ?-SMA were more obvious in immunofluorescence,and SS significantly inhibited the expression of a-SMA protein.In addition,SS extract significantly promote the activation of Sirtl,LXRa,LXR? and AMPK phosphorylation in hepatic stellate cells in a dose-dependent manner.Conclusion:SS can inhibit the fibrosis and reduce the accumulation of fat,inhibit inflammation and apoptosis,and the activation of Sirtl-AMPK-LXR signaling pathway significantly inhibits dimethylnitrosamine-induced hepatic fibrosis and activation of hepatic stellate cells.