The Regulation Mechanism Of ACE2in The Hepatocytes Epithelial Mesenchymal Transition Induced By Angiotensin Ⅱ

Background and ObjectionChina is a big country of liver disease with the higher incidence of chronic liver disease, and liver fibrosis is essential pathological changes for a variety of chronic liver disease to liver cirrhosis. Liver fibrosis is reversible, so as early as possible to block and delay the occurrence and development of liver fibrosis, to some extent, can effectively control the formation of liver cirrhosis.Explore the occurrence of liver fibrosis development mechanism and find effective anti liver fibrosis drugs is an important subject between human life and health, and profound clinical significance and social value.Hepaticfibrosis (HF) refers to the necrosis of hepatocytes, activation of increase in the number of fibroblasts or muscle fibroblasts, collagen and other extracellular matrix in the liver (ECM) hyperplasia and degradation of imbalances, bring about fiber of connective tissue in the liver of pathological changes of the deposit. Liver fibrosis is a variety of cytokines and growth factors, oxidative stress and a series of complex. Studies have found that in the process of liver damage, liver is three kinds of cells within the hepatocytes, bile duct epithelial cells and static rate of hepatic stellate cell(Q-HSC) that is affected by various regulatory phenotypic changes of cytokines, gain myofibroblast(MFb) characteristics, showed that hepatic fibrosis associated with epithelial-mesenchymal transition in the process. Epithelial mesenchymal transition refers to the epithelial cells in morphology, structure, adhesion and migration ability obtain mesenchymal cells characteristic of process. Its main performance is:the epithelial phenotype mucin genes such as E-cadherin express decreased; Mesenchymal cells phenotype gene such as vimentin expression increased; Mesenchymal cells functional genes such as collagen Ⅰ expression enhance.Renin angiotensin system (RAS) is another research focuses on liver fibrosis during recent years. Studies have shown that the intrahepatic ACE-AngⅡ-AT1R axis is closely linked to liver fibrosis. Angiotensin Ⅱ (AngⅡ) is an important effector molecule of RAS that can activation of hepatic stellate cells, the portal area myofibroblast cells and bone marrow-derived stromal cells and many other induced liver fibrosis potential cells, contributing to the occurrence of liver fibrosis development. Our previous found that AngⅡ can induce BRL-3A epithelial mesenchymal transition. Accordingly, in this study will further observe whether AngⅡ can also induce primary rat hepatocytes epithelial mesenchymal transition in vitro experiments; while observing the AngⅡ can change the cell migration ability of BRL-3A.With the deepening understanding of the RAS, and gradually found a new component of the RAS, such as angiotensin-converting enzyme2(ACE2), angiotensin1-7(Ang (1-7)), Ang (1-7) receptor Mas, which constitute the ACE2-Ang-(1-7)-MAS receptor axis become the RAS another important branch, having a significant inhibitory effect on ACE-AngⅡ-AT1R axis. Ang (1-7) in vitro and in vivo in conjunction with the AT1R competitive antagonist activity of AngⅡ play the role of anti-fibrosis. Therefore, in this study will observe whether the ACE2-Ang-(1-7)-Mas receptor axis can inhibition AngⅡ in vivo and in vitro experiments, inhibition hepatocytes epithelial mesenchymal transition and then inhibition hepatic fibrosis.MethodsThe regulation mechanism of ACE2in the hepatocytes epithelial mesenchymal transition induced by angiotensin Ⅱ in vitro 1. AngⅡ induced rat hepatocytes epithelial mesenchymal transitionRat hepatocytes were stimulated with different concentrations of AngⅡ for48hours, then the protein expression of collagen, vimentin and E-cadherin was detected by Western Blot. It would be divided into control group,10-9mol/L AngⅡ group and10-7mol/L AngⅡ group. The experiment is repeated three times.Rat hepatocytes were stimulated with10-7mol/LAngⅡ for different time period, and then the protein expression of collagen, vimentin and E-cadherin was detected by Western Blot. It would be divided into24hours control group,24hours AngⅡ group,48hours control group,48hours AngⅡ group,72hours control group and72hours AngⅡ group.The experiment is repeated three times.AngⅡ stimulation in BRL-3A after48hours, transwell invasion experiment to observe changes in hepatocytes migration ability; it would be divided into control group and AngⅡ stimulation group.The experiment is repeated three times.2. Angiotensinl-7inhibit rat hepatocytes epithelial mesenchymal transition induced by angiotensin ⅡRat hepatocytes were stimulated with different stimuli, then the protein expression of vimentin and E-cadherin was detected by Western Blot. It would be divided into control group, AngⅡ group, Ang (1-7) group, AngⅡ+Ang (1-7) group and AngⅡ+Ang (1-7)+A779group.The experiment is repeated three times.3. Angiotensin converting enzyme2inhibit rat hepatocytes epithelial mesenchymal transition induced by angiotensin ⅡBuild lentiACE2slow virus vector and transfection into the rat hepatocytes and then the protein expression of collagen, vimentin, albumin and E-cadherin was detected by Western Blot. It would be divided into control group, lentiGFP group, lentiACE2group and lentiACE2+A779group.The experiment is repeated three times.Build lentiACE2slow virus vector and transfection into the rat hepatocytes and then stimulated with different stimuli, the protein expression of vimentin and albumin was detected by Western Blot. It would be divided into control group, lentiGFP group, lentiACE2group, lentiGFP+AngⅡ group, lentiACE2+AngⅡ group, lentiACE2+AngII+A779group and AngⅡ group.The experiment is repeated three times.Build lentiACE2slow virus vector and transfection into the rat hepatocytes and then stimulated with different stimuli, transwell invasion experiment to observe changes in hepatocytes migration ability; it would be divided into control group, lentiGFP group, lentiACE2group, lentiGFP+AngⅡ group, lentiACE2+AngⅡ group, lentiACE2+AngⅡ+A779group and AngⅡ group.The experiment is repeated three times.The regulation mechanism of ACE2in the hepatocytes epithelial mesenchymal transition induced by angiotensin Ⅱ in vivo1.ISHAK and METAVIR fibrosis score were evaluated by Masson staining was detected in liver tissue among sham operation model (Sham)、double bile duct ligation rat model (BDL) and the treatment model (BDL+Ang-(1-7)).2. The frozen section in the liver tissue collagen albumin and immunofluorescence double dye; it would be divided into control group, liver fibrosis group, to observation whether people’s hepatocyte are epithelial mesenchymal transition.ResultsThe regulation mechanism of ACE2in the hepatocytes epithelial mesenchymal transition induced by angiotensin Ⅱ in vitro1. AngⅡ can be induced rat hepatocytes epithelial mesenchymal transitionRat hepatocytes were stimulated with10-9mmol/L and10-7mmol/L AngⅡ for48hours. Compared with control group,10-9mmol/L AngⅡ group and10-7mmol/L Ang II group E-cadherin protein expression in hepatocytes decreased (P<0.05), collagen, vimentin protein expression increased (P<0.05); And compared with10-9mmol/L AngⅡ group,10-7mmol/L AngⅡ group E-cadherin protein expression in hepatocytes decreased (P<0.05); Collagen, vimentin protein expression increased (P<0.05).To treatment of rat hepatocytes with AngⅡ for24,48and72hours, at the dose of10-7mmol/L,compared with control group, the rat hepatocytes AngⅡ group collagen, vimentin protein increase is not obvious, no statistical significance, E-cadherin protein expression decreased (P<0.05), the difference was statistically significant; Cultivation of48hours, AngⅡ group compared with control group, collagen, vimentin protein increased significantly(P<0.05), the difference was statistically significant, E-cadherin protein expression decreased (P<0.05), the difference was statistically significant;72hours training, AngⅡ group compared with control group, collagen protein increase is not obvious, no statistical significance, vimentin protein increased significantly (P<0.05), the difference was statistically significant, E-cadherin protein expression decreased (P<0.05), the difference was statistically significant.AngⅡ stimulation in BRL-3A after48hours, AngⅡ group transfer cell number was significantly increased (P<0.05).The difference was statistically significant.2. Angiotensin1-7can be inhibited rat hepatocytes epithelial mesenchymal transition induced by angiotensin ⅡOriginal cultivation generation in rat hepatocytes after48h, compared with control group, AngⅡ group E-cadherin protein expression decreased (P<0.05), vimentin protein expression increased (P<0.05), the difference was statistically significant; Compared with AngⅡ group, AngII+Ang(1-7) group E-cadherin protein expression increased (P<0.05), vimentin protein expression decreased (P<0.05), the difference was statistically significant; Compared with AngII+Ang(1-7) group, AngⅡ+Ang(1-7)+A779group E-cadherin protein expression decreased (P<0.05), vimentin protein expression increased (P<0.05), the difference was statistically significant.3. Angiotensin converting enzyme2can be inhibited rat hepatocytes epithelial mesenchymal transition induced by angiotensin ⅡThis experiment has successful build lentiACE2slow virus vector, and transfection into BRL-3A. Compared with control group, the transfection of ACE2slow virus albumin in BRL-3A and E-cadherin protein expression significantly increased (P<0.05), collagen, vimentin protein expression decreased (P<0.05), the difference was statistically significant; AngⅡ intervention lentivirus ACE2BRL-3A transfection, factorial analysis results:virus transfection and AngⅡ interaction effect between two factors, ACE2transfection group can significantly inhibit AngⅡ albumin expression caused by falling, vimentin expression. The regulation mechanism of ACE2in the hepatocytes epithelial mesenchymal transition induced by angiotensin Ⅱ in vivo1. METAVIR&ISHAK fibrosis score demenstrated that BDL group was significantly higher than that of Sham group (P=0.000), and Ang (1-7) group significantly reduced compared with BDL group (P=0.000), but both higher than the Sham group.2.Human liver tissue frozen section of collagen and albumin co-immunofluorescence showed that in the normal liver tissue albumin (green)+collagen (red), few positive cells, in liver fibrosis albumin decreased significantly, collagen increase, positive cells increased markedly.Conclusion1. AngⅡ can induce hepatocytes epithelial mesenchymal transition.2. ACE2-Ang (1-7)-Mas axis can inhibit AngⅡ, inhibiting hepatocytes epithelial mesenchymal transition and having the function of anti-fibrosis.