The Role Of Nrf2 And Oleanolic Acid In Alcoholic Liver Fibrosis Of Mice

The NF-E2-related Factor 2 (Nrf2) plays an crucial role in protecting hepatic cell against oxidative stress (OS) and toxin-induced tissue damage through its capacity to induce the expression of antioxidant proteins and phaseⅡdetoxifying enzymes. Relationship is demonstrated between alcohol-induced OS and the development of liver pathology.Ethanol-induced OS is the result of the combined impairment of antioxidant defences and the production of ROS by the mitochondrial electron transport chain, the alcohol-inducible CYP2E1 and activated phagocytes. Furthermore, hydroxyethyl free radicals (HER) are also generated during ethanol metabolism by CYP2E1. OS can contribute to liver fibrosis by triggering the release of pro-fibrotic cytokines and collagen gene expression in HSC. Humoral and cellular immune(Kupffer cells) responses stimulated by reactions of HER and LPO might contributes to the perpetuation of chronic hepatic inflammation which can explain progressive fibrosis rationally after alcohol abstinence. As a traditonal liver-protecting drug whose major effective ingredient is a pentacyclic triterpenoid, oleanolic acid (OA) can be extracted from many kinds of Chinese herbs and.induce cytoproctective genes probably through Nrf2-keap pathway.Nevertheless, molecular biological mechanism of oleanolic acid OA is still unkown.Therefore, it is necessary to invesitigate the expression of Nrf2 in alcoholic liver fibrosis(ALF) and elucidate molecular biological mechanism against fibrosis of OA.The present study may offer a novel strategy to prevent and ameliorate ALF.Objective.1.To made an ALF model by alcohol gastric infusion for kunming mice;To detect serum level of ALT, AST and TBIL; To summerize pathological features of ALF.2.To observe Nrf2 expression of hepatocytes in normal mice, drinking mice, normal mice treated with OA and drinking mice treated with OA.3.To investigate pretreatment with OA would be able to reduce inflammatory response, improve hepatocyte injury, prevent or slowed fibrosis and elucidate molecular biological mechanism of OA against liver fibrosis.4.To study influence on hepatocyte proliferation and apoptosis of OA in normal mice and drinking mice.5.To explore the role of alcohol and OA in activations of mononuclear macrophages, in order to explore the impact of macrophages on liver fibrosis procession in future.MethodsAll animal work was performed according to protocols approved by the Moral and Ethnic Committee of Shandong Univesity.Thirty female Kunming mice were randomly divided into four groups:A1 control group (normal saline ig.,5 mice), A2control group (OA ig.,5 mice), model group (alcohol ig.,10 mice) and treatment group(alcohol and OA ig.,10 mice). All mice were free to drink, eat and feed in coops in animal experimental center,where temperature was 20～25℃, light and dark were 12 hours alternately. At the end of 12 weeks,2 hours before sacrifice, mice were injected i.p. with 250mg/kg BrdU to label proliferating cells. Mice (fasting during the previous night) were all sacrificed by celio-injection of 10% chloral hydrate solution(0.3ml/100g.bw).Blood sample was taken by heart punctiuation of mice Following blood coagulation, serum was collected after centrifugation and preserved under temperature 80℃below zero.The liver samples were picked off quickly, then 3/4 part of each sample was fixed by 10% formalin solution,embedded in paraffinfor and sectioned for histological evaluation,1/4 part of each sample was frozen rapidly by liquid nitrogen and preserved under temperature 80℃below zero in freezer preparing for mRNA suspension. Detections will be done in batches as follow:1.Ievels of ALT, AST and TBIL in blood serum sample were determineded by automatic biochemistry analyser in clinical biochemistry lab using standard procedures.2. Histopathology abnomal was detected by HE stain,such as inflammatory cell infiltration and parenchymal damage graded numerically according to the scoring system devised by Thompson microscopically. Fibrosis was observed by MT stain and quantified by professional image quantitative analysis system.3. Immunofluorescence and immunohistochemistry The expression of Nrf2 and extracellular matrix protein FN in liver tissue were detected individually by immunohistochemistry stain in each group. Immunofluorescence stain with an antibody against ER-MP23(a kind of macrophage-specific lectin) was used to determine the number of macrophages. Terminal dUTP nick-end labeling (TUNEL) assays were performed to evaluate apoptosis of hepatocytes. We analyzed proliferatiion of liver Cells by incorporation of 5-bromo-2-deoxyuridine (BrdU,nucleotide analogon).4.COL1α,TGF-β1,α-SMA mRNA of frozen liver samples were detected by the highly sensitive technique of quantitative RT-PCR in molecular biological lab.Results1. Serum concentrations of ALT,AST and TBIL were increased in model mice obviously compared with control mice indicating an injury in liver function. Serum concentrations of ALT,AST and TBIL in mice pretreating with OA were decreasd more than twofold compared with model mice indicating an improvement in liver function.2. Histology manifestion paralleled to biochemical findings. In the control group, constitution of hepatic lobules was normal,central veins was big and had thin paries;.Hepatic cells were polygon which were arranged in hepatic cord, which were distributed as radial around central veins. There were only a few inflammatory cells infiltrating in portal regions in control mice. Abnormal hepatic lobules featuring by severe fatty degeneration, focal and regional necrosis of hepatic cells, inflammatory cells infiltrate in quantity, mesenchyma fibrosis could be seen in model mice. Histological grading performed by two independent experienced liver histopathologists revealed more severe inflammation and liver parenchyma damage in model mice compared with control mice. Compared with model mice, the parenchymal injury of the hepatic lobules and inflammatory response were improved in mice pretreating with OA statistically.Fibrosis was not observed and only a small area around blood vessels was stained with aniline blue(MT stain) in control mice. The percentage of fibrotic area was significantly lower in treatment mice compared with model mice. This result was confirmed by staining of the sections with MT stain (1.86±1.23%VS3.55±1.37%, p=0.02)3.There was more extended deposition of FN detected by immunohistochemistry stain in model mice in accord with fibrosis findings. The percentage of deposition area was significantly lower in treatment mice compared with model mice(2.32±1.13%VS5.17±2.05%, p=0.01).4.Low expression of Nrf2 was observed within cytoplasm of only a few hepatic cells in Al and A2 control mice. Ethanol induced an obvious up-regulation of Nrf2 expression in liver tissue probably through OS.The expression of Nrf2 in model mice was higher compared with control mice(69.31±12.18 VS2.85±0.72, p=0.00); The expression of Nrf2 in treatment mice was higher compared with model mice(92.31±15.45 VS 69.31±12.18, p=0.03).OA was capable of up-regulating the expression of Nrf2 in liver tissue.There were few macrophages(ER-MP23 positive liver cells) infiltrating in A1 and A2 control mice.The number of macrophages in treatment mice was statistically lower than model mice (178±35 VS 113±29/mm2, p<0.05)Only very few proliferating cells were seen in the liver of A1 and A2 control mice (1.09±1.03% VS 1.15±1.24%, p>0.01). The difference of the percentage of BrdU positive cells was not statistically differnt in model mice and treatment mice (2.35±1.21% VS 2.75±1.06%,p>0.05)Normal saline and OA did not induce apoptosis in control mice. Hepatocytes could undergo apoptosis in response to alcohol ingestion, but there was no statistical difference in the percentage of TUNEL-positive cells between model mice and treatment mice (232±45 VS209±27/mm2,p>0.05)5. Compared with A1 control group, the expression of Nrf2,FN,collagenⅠ,TGF-β1,α-SMA mRNA in liver tissue up-regulated(p<0.05);Compared with model group, the expression of FN. collagenⅠ,TGF-β1,α-SMA mRNA in liver tissue down-regulated(p<0.05).Conlusion1. ALF model in mice was established successfuly by ethanol ig. at the end of 12th week; This is an effective method which is characteristic of high success rate and conform to the human drinking.2.The expression of Nrf2 was increased in ALF model in mice. It might be a protective reaction to oxidative stress induced by alcohol injury. OA could up-regulate Nrf2 expression of hepatocytes in drinking mouse and had no influence on normal mice.3.OA could significantly decrease serum level of ALT,AST and TBIL and reduce the degeneration and necrosis of liver cells in drinking mouse. Infiltration of inflammatory cells and lobular fibrosis were also improved. Pretreatment with oleanolic acid can prevent macrophages infiltration and increase the level of Nrf2 expression and up-regulates mRNA expression of CollagenⅠ,TGF-β1,α-SMA, it may protect the liver from alcohol-induced fibrosis by inhibiting activation of HSC and decrease the synthesis and depositon of extracellular marix.4.Oleanolic acid had no influence on haptocyte proliferation and apoptosis in normal mice. To a certain degree, it could promote proliferation and lower apoptosis rate of liver cells in drinking mouse but without statistical difference.5 Macrophages(Kupffer cells) might be activated by oxidative stress which was triggered by HER and lipid peroxidation products and mediated immune response favouring the progression of liver damage towards fibrosis. Activated macrophages could secrete anti-inflammatory cytokines(TGF-β1 and IL-10) and express profibrosis factors (TGF-β1,angiotensionⅡand others).Then, the fibrogenic activation of HSC was initiated. Hence, perpetual inflammatory response and progressive fibrosis occurred.