The Role And Mechanism Of UPA/PAI-1in Non-alcoholic Fibrosing Steatohepatitis

Objective: Non-alcoholic fatty liver disease (NAFLD) is an acquiredmetabolic stress-related liver disorder that was assumed to be present byheredity. With changes of dietary pattern and life style, NAFLD is increasingaround the world. NAFLD includes a spectrum of simple steatosis, non-alcoholic steatohepatitis/fibrosis, cirrhosis and hepatocellular carcinoma(HCC). Non-alcoholic fibrosing steatohepatitis which correlated with patientprognosis is the crucial pathological stage for progression to liver cirrhosis,HCC and liver failure. Unfortunately, today there is no specific and effectiveantifibrotic therapy available, therefore it is important to diagnose liverfibrosis in early stages and search for new treatment method to preventprogressive liver fibrosis. It has been proven that Radix Salvia Miltiorrhizae,Semen Persicae, Gynostemma Pentaphyllammak could significantly reversehepatic fibrosis. Fuzhenghuayu recipe (FZHY), a compound Chinese herbalmedicine consists mainly of the three traditional Chinese medicines. It wasreported that FZHY could significantly improve liver function and reversehepatic fibrosis in patients with hepatitis B virus infection. However, the effectand molecular mechanism of FZHY against nutritional fibrosing steato-hepatitis is still unclear. In this study, experimental non-alcoholic fibrosingsteatohepatitis models were established by feeding male C57BL/6J mice withmethionine and choline deficient (MCD) diet for8weeks, and interventedwith FZHY or/and heme oxygenase-1(HO-1) chemical inducer (Hemin) orinhibitor (zinc protoporphyrin Ⅸ, ZnPP-Ⅸ). The aim of this study was toinvestigate the potential effect and molecular mechanism of uPA/PAI-1innon-alcoholic fibrosing steatohepatitis, the mechanism of FZHY and HO-1chemical inducer (Hemin) on ameliorating uPA/PAI-1expression.Methods: Eight-week-old male C57BL/6J mice (Experimental Animal Center of Chinese Academy of Medical Sciences) were fed with MCD diet for8weeks to induce non-alcoholic fibrosing steatohepatitis and methionine andcholine supplemented diet for Control group. FZHY and/or Hemin or ZnPP-Ⅸwere administered to the mice, respectively. All of the animals were sacrificedat the end of experiments. Blood samples were collected for biochemicalanalysis. Parts of the livers were fixed in4%formalin for histological analysisor snap-frozen in lipid nitrogen followed by storage at-80oC freezer untilrequired.1The common state of mice including mental status and hair was observedbefore sacrificed. Liver indexes were calculated by liver weight (g)/bodyweight (g)×100％.2Serum alanine aminotransferase (ALT) and aspartate aminotransferase(AST) levels were tested by enzymatic kinetic method with an automaticbiochemical analyzer (Olympus AU5400, Japan) according to themanufacturer’s instructions.3The grades of hepatic steatosis, inflammation and fibrosis were observedwith haematoxylin and eosin and Masson-trichrome stained liver sections.4Hepatic mRNA and protein expression of HO-1, plasminogen activatorinhibitor-1(PAI-1), urokinase type plasminogen activator (uPA), transforminggrowth factor beta1(TGF-β1), matrix metalloproteinase-9(MMP-9), tissueinhibitor of matrix metalloproteinase-1(TIMP-1) were detected by reversetranscription polymerase chain reaction (RT-PCR), Western blot andimmunohistochemistry, respectivly.5All data were presented as mean±SD (standard deviation). Statisticalanalysis was performed by one-way analysis of variance (ANOVA) andStudent-Newman-Keuls test for evaluating differences between groups usingSPSS13.0software (v.13.0; SPSS Inc., Chicago, IL). A P-value of less than0.05was considered statistical significance.Results:1The common state of mice: Control mice were active, with bright hair,and their body weight and liver weight increased gradually with time going. The body weight and liver weight in model group and intervention groupsdecreased remarkably compared with Control group (P<0.05). However, theliver indexes were no significant difference among the seven groups (P>0.05).2Serum ALT and AST levels: Compared with Control group, the serumlevels of ALT and AST increased remarkably in model group. FZHY or Hemincould lower serum of ALT and AST levels. FZHY combined with Hemincould futher decrease the serum ALT and AST levels. Contrarily, the serumlevels of ALT and AST showed a increasing tendency by using ZnPP-Ⅸ(P<0.05).3Routine pathologic examination: In control mice, the liver sectionspresented almost normal histopathology. The liver sections from mice fed anMCD diet alone exhibited disordered lobule structure, severe macrosteatosis,spot or focal hepatocyte necrosis and inflammatory infiltration, portal fibrosisand fibrous septum. However, mice treated with FZHY in the presence orabsence of hemin could notably ameliorate hepatic steatosis, necroticinflammation and improved liver fibrosis. Co-administration of FZHY andhemin had a further improved effect on hepatic inflammation and fibrosis.4mRNA and protein expression of fibrotic related genes: Hepatic steatosis,inflammatory infiltration and fibrosis were induced by feeding mice withMCD diet, which was associated with enhanced expression of hepatic HO-1,PAI-1, uPA, TGF-β1, MMP-9and TIMP-1mRNAs and proteins. FZHY couldameliorate the liver injury by down-regulation of PAI-1, TGF-β1, TIMP-1expression and up-regulation of uPA, MMP-9expression. An additive effectwas observed in mice fed MCD supplemented with FZHY or/and Hemin.However, liver injury and related gene expression showed a contrary tendencyby using ZnPP-Ⅸ(P<0.05).Conclusions:1PAI-1, uPA and related genes TGF-β1, MMP-9and TIMP-1played acrucial role in development of non-alcoholic fibrosing steatohepatitis inducedby feeding mice with methionine-choline deficient diet for8weeks.2FZHY recipe could prevent non-acoholic fibrosing steatohepatitis through suppressing oxidative stress, hepatic stellate cells activation andmodulating the gene expression of proinflammation and fibrogenesis inexperimental nutritional steatohepatitis.3FZHY combined with HO-1chemical inducer played an ameliorate effecton ameliorating hepatic inflammatory response and fibrogenesis which wasassociated with up-regulation of anti-fibrotic related genes and down-regulation of fibrotic related genes.