The Effects Of Angiotensin Ⅱ And Aldosterone On Rat Hepatic Stellate Cells Contraction Mediated By Rhoa-rock Pathway

BackgroundIt was known to all.various kinds of chronic liver disease could develop to hepatic cirrhosis from hepatic fibrosis,portal hypertension was the serious complication of hepatic cirrhosis.Increasing for the vascular resistance intrahepatic was the important factor under the state of hepatic cirrhosis.Hepatic stellate cells was the major cells of extracellular matrix,its activation played key role in the trail process of the occurrence and development for hepatic fibrosis as well as the form for portal hypertension.The activated HSC could convert to myofibroblast and fibroblast proliferate and gain contractility which resulted in the diminution for the anteroposterior diameter of sinus hepaticus,then,the vascular resistance intrahepatic would increase:besides,it caused the hepatic tissue cicatrices to contract,and increased the vascular resistance intrahepatic progressly,and resulted in portal hypertension finally.So,the regulative mechanism for the contraction of HSC was the important component element for the research to the mechanism of portal hypertension.Recently,renin- angiotensin- aldosterone system intrahepatic was the hot spot of investigation for hepatic fibrosis,angiotensinⅡ(AngⅡ) was the important effector molecule,it could induce HSCs to activate and proliferate,and cause the hepatic fibrosis to form.ACE-AngⅡ-AT1R was relate with the form of portal hypertension intimately.The researchs also indicated that aldosterone(Aldo) could paly some functions resembly as AngⅡ.But,the researchs about the mechanisms for the contraction of HSC induced by Ang and Aldo was rare,their mechanism of action was unknown.The activated HSC could convert to myofibroblast,and it had the characters as the same as smooth muscle celI(SMC),so the molecule mechanism of HSC's contraction was resemble as the SMC.Both Ca²⁺ -dependent and -independent pathways were involved in HSC contraction.The regulative mechanisms for the contraction of HSC mediated by the RhoA-Rock pathway which was one of the Ca²⁺ -independent pathways was the hot spot for research recently.So,our objective was to illuminate the mechanisms of rat HSC contraction induced by AngⅡand Aldo through investigating the effects of AngⅡand Aldo on rat HSCs contraction mediated by Rho-Rock pathway.Objective]To investigate the effects of AngiotensinⅡand Aldosterone on rat hepatic stellate cells contraction mediated by Rho-Rock pathway.MethodsHSC-T6 cell line was under pre-disposal treatment with AngⅡand Aldo 1μmol/L,respectively,The cell contraction was detected by silicone-rubber-membrane cultivation directly.The concentration variation of intracellular free calcium in rat HSC was observated by laser confocal microscopy.Besides.HSC-T6 cell line was under pre-disposal treatment with AngⅡand Aldo 10μmol/L respectively.And the protein level of MLC and phosphorylation MLC were detected by Western blotting.Then.the present study observated the difference of phosphorylation MLC protein level after using the blocking agent of AngⅡ1-receptor(AT-1 receptor) -irbesartan.Aldo receptor blocking agent-antisterone,protein kinase C(PKC)special blocking agent-Stauro,Rho kinase blocking agent-Y27632. and MLCK special blocking agent-ML-7,respectively.RT-PCR was used to detect the expression of Rock2,RhoAGTP and RhoGEF in Ca²⁺ - independent pathways mediated by Rho-kinase,and that of PKC.ResultsBoth AngⅡand Aldo could induce HSCs contraction;The concentration of intracellular free calcium in rat HSC was higher after it was under pre-disposal treatment with AngⅡ,but it had no change after HSC was under pre-disposal treatment with Aldo.The diversity of levels of phosphorylation MLC protein induced by AngⅡand Aldo showed time-dependent manner.It would be decreased after peaking in 15 minutes.The levels of phosphorylation MLC protein of the irbesartan + AngⅡgroup and Y27632 + AngⅡgroup were significantly lower than that of the AngⅡgroup.The level of phosphorylation MLC protein of the ML-7+ Stauro+AngⅡgroup was higher than that of the Y27632 + AngⅡgroup(P =0.003).The level of phosphorylation MLC protein of the Y27632 +ML-7+Stauro+ AngⅡgroup was significantly lower than that of the AngⅡgroup(P=0.000).The levels of phosphorylation MLC protein of the Aldo group were significantly higher than that of the control group(P = 0.001).Antisterone and Y27632 could inhibit the effective(P = 0.004,P = 0.042,respectively).The level of phosphorylation MLC protein of the ML-7+Stauro+ Aldogroup was lower than that of the Y27632 + Aldo group(P =0. 022).The level of phosphorylation MLC protein of the Y27632 +ML-7+Stauro+ Aldo group was also significantly lower than that of the Aldo group(P = 0.001 ).The mRNA expression of Rock2,RhoAGTP and RhoGEF increased significantly after the treatment of AngⅡ.while decreased in irbesartan + AngⅡgroup.The mRNA expression of Rock2 and RhoGEF was lower,but it was higher for RhoAGTP in Y27632 + AngⅡgroups.The mRNA expression of the three elementums of both the ML-7+Stauro+ AngⅡgroup and the Y27632 +ML-7+ Stauro+ AngⅡgroup were higher.The mRNA expression of Rock2,RhoAGTP,RhoGEF and PKC increased significantly after the treatment of Aldo,while inhibited by antisterone.The mRNA expression of the four elements was lower than that of Aldo group,but it was higher in ML-7+Stauro + Aldo groups than control group.In the Y27632 +ML-7+ Stauro+Aldo group,the mRNA expression of RhoGEF was higher than that of the ML-7+Stauro+ Aldo group,but the mRNA expression of PKC was lower.ConclusionBoth AngⅡand Aldo could induce HSCs contraction;The concentration of intracellular free calcium in rat HSC was higher after it was under pre-disposal treatment with AngⅡ,but it had no change after HSC was under pre-disposal treatment with Aldo.AngⅡinduced HSCs contraction in Ca²⁺ independent pathways mediated by Rho-kinase mainly.Aldo induced HSCs contraction in RhoA-Rock pathways.