The Effects Of Ursolic Acid On NADPH Oxidase Which Expressed In Hepatic Stellate Cells,Hepatocytes And Kupffer Cells In Vivo

Background:Recent studies indicate that NADPH oxidases(NOX)expressed in hepatic stellate cells(HSCs),hepatic cells(HCs)and kupffer cells(KCs)play an important role in in the pathogenesis of liver fibrosis.Our preliminary studies showed that ursolic acid(UA)had a unique anti-liver frotic effect in vivo.In vitro studies,we found that UA could induce apoptosis of activated HSCs by inhibiting the expression of NOX in HSCs.Besides,UA could protect HCs by decreasing the expression of NOX in HCs.However,in vivo,the effect of UA to NOX in HSCs,HCs and KCs is so far unclear.In this topic,we use fibrosis rats as the research object,dealing with apocynin(AP)sets as positive control,observing the effect of UA intervention(UA prevention and treatment)to reverse liver fibrosis;make sure that the mechanism of UA to anti-liver fibrosis is related to decrease NOX expressed in HSCs,HCs and KCs.Objective:To observe the content of NOX expressed in HSCs,HCs and KCs after the intervention of UA,exploring the mechanism and molecular target of UA to ameliorate liver fibrosis in vivo.Method:1.Sixty male Sprague Dawley(SD)rats were randomly divided into six group: blank control group,liver fibrosis model group,apocynin prevention or treatment group,UA prevention or treatment group.For liver fibrosis model group,we made rats intragastric administration with carbon tetrachloride(CCl4)for eight weeks,2 ml/kg(20% olive oil dilution concentration),twice a week.For rats in prevention groups,after two weeks after gavaging with CCl4,we gavage rat with apocynin or UA(40 mg/kg/d)for the last six weeks at the same time.For rats in treatment groups,we gavage rat with apocynin or UA for another four weeks alonely after eight weeks after gavaging with CCl4.The 3rd day after medication,we obtain blood and liver tissue from rats.2.We used the means of hematoxylin eosin staining(HE)to observate the lobular structure;we utilized the way of sirius red staining to observe the hyperplasia degree of collagen fiber;we made use of colorimetric method to test the hydroxyproline content in serum and liver tissue;we took advantage of western-blotting(WB)to detect the expression of type I collagen,TGF beta 1,MMP-1,TIMP-1 and ?-SMA protein in liver tissue.3.We inspected serum alanine aminotransferase(ALT),albumin(ALB),total bilirubin(TBIL)of rats by automatic biochemical analyzer;we adopted the technology of Immunohistochemistry(IHC)to detect proliferating cell nuclear antigen(PCNA)in liver tissue;we utilized the method of TdT-mediated dUTP Nick-End Labeling(TUNEL)staining to detect the apoptosis of HCs.4.We took advantage of western-blotting to detect t the expression of NOX1,NOX2 and NOX4 protein in liver tissue.5.we took advantage of technique about in situ perfusion and gradient centrifugation to extract primary HSCs,HCs and KCs from rats,detecting the mRNA level of NOXs in those three kinds of cells.Results: 1.The effect of UA intervention to liver fibrosis in vivo.When compared to liver fibrosis group,the fibrous septum and collagen deposition were reduced obviously in liver tissue of UA prevention and treatment group;there were obviously reduction in hydroxyproline in rats serum and liver tissue(P<0.001).The expression of type I collagen,TGF beta 1,TIMP-1 and ?-SMA protein in liver tissue of UA prevention group were declined(P<0.01,P<0.001,P<0.01 and P<0.001 respectively);the expression of type I collagen,TGF beta 1,TIMP-1 and ?-SMA protein in liver tissue of UA treatment group were declined(P<0.001,P<0.001,P<0.01 and P<0.001 respectively);However,the expression of MMP-1 protein was increased in liver tissue of UA prevention and treatment group(P < 0.01).2.The protect effects of UA intervention to liver function and HCs in vivo.When compared to liver fibrosis group,the level of serum alanine aminotransferase(ALT)and total bilirubin(TBIL)appeared significantly lowed in UA prevention and treatment group(all P < 0.001),while the level of serum albumin(ALB)appeared significantly rised in serum of rats(P < 0.001);the proliferation of HCs was increased(P < 0.01),while the apoptosis of HCs was decreased(P < 0.01).3.The effects of UA intervention to the expression of NOX protein in liver tissue in vivo.When compared to liver fibrosis group,the expression of NOX1,NOX2 and NOX4 protein in liver tissue of UA prevention group were decreased(P<0.01,P<0.01 and P<0.001 respectively);the expression of NOX1,NOX2 and NOX4 protein in liver tissue of UA treatment group were decreased,too(P<0.001,P<0.01 and P<0.001 respectively).4.The effects of UA intervention to the expression of NOX mRNA in HSCs,HCs,and KCs in vivo.4.1 The influence of UA intervention to the expression of NOX mRNA in HSCs in vivo.When compared to liver fibrosis group,the expression of NOX1,NOX2 and NOX4 mRNA in HSCs were fallen off in UA prevention group(P<0.01,P<0.01 and P<0.001 respectively);the expression of NOX1,NOX2 and NOX4 mRNA in HSCs were also fallen off in UA treatment group(P<0.001,P<0.01 and P<0.001 respectively).4.2 The influence of UA intervention to the expression of NOX mRNA in HCs in vivo.When compared to liver fibrosis group,the expression of NOX1,NOX2 and NOX4 mRNA in HCs were came down in UA prevention group(P<0.001,P<0.01 and P<0.001 respectively);the expression of NOX2 and NOX4 mRNA in HCs were also came down in UA treatment group(P<0.01 and P<0.001 respectively).There was no significant difference between the data of UA treatment group and liver fibrosis group(P>0.05).4.3 The influence of UA intervention to the expression of NOX mRNA in KCs in vivo.When compared to liver fibrosis group,the expression of NOX1,NOX2 and NOX4 mRNA in KCs were decreased in UA prevention group(all P<0.001);the expression of NOX1,NOX2 and NOX4 mRNA in KCs were decreased in UA treatment group(P<0.01,P<0.05 and P<0.001 respectively).Conclusion:The mechanisms of UA anti-liver fibrosis are related to reduce the expression of NOX in HSCs,HCs,and KCs in vivo.