The Mechanism Of Angiotensin Ⅱ Progressing Alcoholic Liver Fibrosis

With the elevating of living condition and the prevailing of HBV vaccine,ALD has become the major desease that threatening our Chinese people.ALD includes alcoholic fatty liver,alcoholic hepatits and alcoholic liver cirrosis.The stage of fibrosis is reversible,so considerable attention has been paid to genesis of liver fibrosis and how to reverse it.The RAAS is an important regulator of cardiovascular homeostasis and systemic blood pressure.Recent reseaches indicated AngⅡhad a number of blood-pressure independent actions including upregulating cytokines and mitogenic and trophic effects on cell growth.The RAAS is important in cardiac and renal fibrosis,so studies are focused on its effect in liver fibrosis.Some studies show there are non-blood AngⅡexisting in liver,and the serum AngⅡand ACE are higher in patient with liver cirrosis. This indicates the importance of the RAAS in the genesis of liver fibrosis.The HSC is the principal effector of hepatic fibrosis.Recent researches indicate HSC can secrete AngⅡand express the major receptor AT1R of AngⅡ.This indicates AngⅡpromoting fibrosis through the activation and proliferation of HSC,and TGF-β1 and MAPK has been shown to contribute to this process.To study the function of AngⅡin the genesis of liver fibrosis,we make the experimental rat model of alcoholic fibrosis by alcoho intragastric infusion,observe the dynamic changes of serum AngⅡin the process of fibrosis and the effects of the ACEI drugs(Captopril) in the genesis of fibrosis.Furthermore,observe the effect of AngⅡon proliferation and collagen secretion and the expression of TGF-β1,p38 MAPK of the HSC by isolation and culture the primary HSC,to confirm the effect of AngⅡin alcoholic fibrosis and find a new suitable agent for clinical treatment. Materials and methods1,Animal model of rat alcoholic diseaesWistar male rats devide into 3 groups randomly,intragastric infused with alcohol and Captopril.2,Histological detectionsThe liver sections were fixed in formaldehyde solution,embedded in paraffin,and used HE and van Giesion staining,to observe the histological changes.3,Determination of serum HA,LNThe experiments were performed using radioimmunoassay method according to instruction.4,Detection of serum and tissue levels of AngⅡThe experiments were performed using radioimmunoassay method according to instruction.5,Immunohistochemistry staining of typeⅠ,ⅣcollagenApply SABC method.For each specimen,10 more positive staining areas were selected,and analyzed the strength and area of the staining.6,Isolation and culture of the HSCFollowed the method by Song shao gang et al to isolate the HSC and cultured in DEME7,Identification of the HSCApply SP immunohistochemiscal staining techniques by using anti-Desmin antibody.8,Detection of cell proliferationApply the MTT chromometry method.9,Immunohistochemiscal staining of HSCα-SMABy SP immunohistochemiscal staining techniques.10,Detection of typeα1(Ⅰ),α1(Ⅳ) collagen by RT-PCR(real time quantitative PCR) SYBR-Green-based real-time PCR was used to measure the expression of typeα1 (Ⅰ),α1(Ⅳ) collagen mRNA.11,Detection of TGF-β1By ELISA method.12,Determination of p38 MAPKApply Western blot analysis.13,The effect on proliferation and expression of TGF-β1,α1(Ⅰ),α1(Ⅳ) collagen of p38 MAPKinhibitor SB203580Apply Western blot analysis.Results1,General observationThe rats of alcohol group were dark-colored,dull reponsed,and with poor appetite,and lost weight.However,the rats of ACEI group were active,light-colored, and fat.The death rate was highest of alcohol group.2,Histological detectionsHE and van Giesion staining showed the no degeneration or necrosis or inflammatory cell infiltration or fibro proliferation in control group.At the end of 4w in alcohol group,there was local small fatty degeneration,dotted necrosisof hepatocyte and inflammatory cll infiltration.At the end of 12w,there was mixed fatty degeneration, and with apprant proliferation of fiber tissue.But in ACEIgroup,at the end of 8w,there was slightly local fatty degeneration,ang no apparent necrosis of hepatocyte or inflammatory cell infiltration,and at the end of 12w there was only slightly increased fatty degeneration without fiber proliferation.3,Determination of serum HA,LNAt the end of 8 w,the serum LN level of alcohol group began elevate,and at the end of 12 w the LN level continuely elevated,apprantly higher than control and ACEI group.The LN level of ACEI group had no diffrence with control. At the end of 12 w the serum HA level began elevate,apprantly higher than control and ACEI group(P＜0.0005).4,Detection of serum and tissue levels of AngⅡAt the end of 8 w,the serum AngⅡlevel of ACEI1 group began elevate,and incontinuely elevated but remained a high level.At the end of 4 w,the serum AngⅡlevel of alcohol group was apprantly higher than group(t=7.51,P＜0.0005),and continuely elevated with time prolonged.5,Immunohistochemistry staining of typeⅠ,ⅣcollagenIn control group,typeⅠcollagen was mainly existed in portal area with filament staining.In alcohol group,typeⅠcollagen was mainly existed in central vein area with large band staining,the positive area percent was apprantly higher than normal group (P＜0.0005).The result of ACEI group had no diffenrance with control group (P=0.054)In control group,typeⅣcollagen was existed in vesicle wall of central wein.In alcohol group,there were large quantity typeⅣcollagendeposit with stong positive staining.The result of ACEI group was similar to control group.The positive area percent of alcohol group was apprantly higher than that of control group or ACEI group(P＜0.0005),the result ACEI group has no difference with control group (P=0.055).6,AngⅡpromoting theα-SMA expression in HSCOnly few cultured HSC withα-SMA positive staining in plasma without AngⅡstimulation.The percent of positive cells increased when stimulating with 10^-8moL/L AngⅡfor 24h,and the percent still increased when AngⅡconcentrationincreased, (P＜0.0005),and became highest when stimulating with 10^-6moL/L AngⅡ.7,AngⅡpromoting the HSC proliferation10^-8moL/L AngⅡcould promote the HSC proliferation(P=0.015),the promotion was apprant when added higher concentration of AngⅡ,and the peak was at 10^-6moL/L(P＜0.0005).This indicates AngⅡcould promote the HSC proliferation,and the effect was dose-dependent. 8,AngⅡpromoting the expression ofα1(Ⅰ),α1(Ⅳ) Col mRNAWhen stimulating with differentconcentration AngⅡfor 24h,α1(Ⅰ) Col mRNA were increased than control group respectively(P＜0.0005).The expression ofα1(Ⅳ) Col mRNA was similar withα1(Ⅰ)Col mRNA.These results indicate AngⅡupregulate the expression of HSCα1(Ⅰ),α1(Ⅳ) Col mRNA.9,AngⅡpromotiong secretion TGF-β1 of HSCIn normal conditions,HSCcan secrete foe concentration TGF-β1,but when stimulating with 10^-8,10^-7,10^-6moL/L AngⅡ,the concentration of TGF-β1 was appantly higher than that of control group(P＜0.0005).10,AngⅡpromoting p38 MAPK expression of HSCWestern blot results showed p38MAPK with a specific protein band at 38 kDa.When stimulating with 10^-8,10^-7,10^-6moL/L AngⅡfor 24h,the protein content of p38 MAPKwere higher than that of normal control(P＜0.0005).11,p38 MAPK inhibitor SB203580 blockade AngⅡon the expression of TGF-β1The TGF-β1 secretion of HSC was increased when 10^-6moL/L AngⅡstimulating for 24h(P＜0.0005).But when formerly added SB203580 to blockade p38 MAPK pathway,the secretion decreased(P＜0.0005).This indicated p38 MAPKmaybe the upstream signal of TGF-β1.12,p38 MAPK inhibitor SB203580 blockade AngⅡon the HSC proliferationThe HSC proliferation was promoted with 10^-6moL/L AngⅡstimulating for 24h. But when formerly added SB203580 to blockade p38 MAPK pathway,the HSC proliferation decreased(P＜0.0005).This indicated AngⅡpromting HSC proliferation mediated by p38 MAPK13,The effect on proliferation and expression of TGF-β1,α1(Ⅰ),α1(Ⅳ) collagen of p38 MAPKinhibitor SB203580When stimulating with 10^-6moL/L AngⅡfor 24h,α1(Ⅰ) Col mRNA was apprantly higher than normal control(P＜0.0005).When formerly added SB203580 to inhibit the p38 MAPK activity,α1(Ⅰ) Col mRNA was apprantly lower than no SB203580 group (P＜0.0005)。Expression ofα1(Ⅳ) Col mRNAwas similar withα1(Ⅰ) Col.These results indicate AngⅡupregulating theα1(Ⅰ),α1(Ⅳ) Col mRNA expression of HSC through p38 MAPK pathway.Conclusion1,AngⅡincreases both in plasma ang also in liver tissue,and in latter AngⅡis positively related to the level of liver fibrosis.2,ACEI can inhibit the formation of liver fibrsis,decrease the level of HA and LN in plasma,reduce the expression of collagen typeⅠandⅣin liver.Thus AngⅡmay promote the alcoholic liver fibrosis.3,AngⅡpromote the activation of the primarily cultured HSC.4,AngⅡpromote the multiplication of the primarily cultured HSC.5,AngⅡup regulate the expression ofα1(Ⅰ),α1(Ⅳ) Col mRNA in of the primarily cultured HSC.6,AngⅡcan promote the cell sythesis of HSC and the secretion of TGF-β1.7,AngⅡcan promote the expression of p38 MAPK protein in HSC.8,p38 MAPK mediate AngⅡin HSCmultiplication,and increase the expressionof collagen typeα1(Ⅰ),α1(Ⅳ) mRNA.9,p38 MAPK mediate the promomotion of AngⅡin the secretion of TGF-β1 in HSC.10,p38 MAPK mediate the proliferation of HSC promoted by AngⅡ.