Study On The Mechanism Of PPARα Induction In Ameliorating Ethanol Induced Liver Injury In Mice

Alcoholic liver injury is a progressive process encompassing hepaticsteatosis, steatohepatitis, fibrosis, cirrhosis and even hepatocellular carcinoma.Fatty liver develops in about90%of individuals who drink excessive ethanolfor a long time, and5-15%of patients progress to fibrosis and cirrhosisdespite abstinence. Continued alcohol use increases the risk of progression tofibrosis or cirrhosis to30-37%. Chronic ethanol exposure results intriglyceride accumulation in the liver, and also contributes the initiation ofoxidative stress and the development of inflammatory response, whichactivates hepatic stellate cell (HSC) and induces liver fibrosis.Pharmacological treatment for patient with alcoholic liver disease (ALD) isstill not available. There is compelling need to identify agent to protect liveragainst ethanol related injury.Peroxisome proliferator activated receptor alpha (PPARα), interacts withthe retinoid X receptor to function as a transcription factor to induce theexpression of a series of genes involved in fatty acid transport and oxidation,as well as inflammatory responses. However, the role and the mechanism ofPPARα in pathogenesis of ALD remain largely unknown.In the present study, we aim to establish alcoholic steatohepatitis andalcoholic liver fibrosis mice model, and to elucidate the pathogenesis of ALDas well as the effect and the molecular basis of PPARα in ethanol inducedhepatic injury in mice.Part1Establishment of alcoholic steatohepatitis/liver fibrosis model andhepatic expression of PPARα in miceObjective: To establish alcoholic steatohepatitis and liver fibrosis modeland explore the effect of PPARα in alcoholic liver injury in mice.Methods: C57BL/6J mice were fed with4%ethanol-containing Lieber-DeCarli liquid diet for12weeks to induce alcoholic steatohepatitis,and fed with ethanol liquid diet alone in the first4weeks and combined withmicroamount of carbon tetrachloride intraperitoneal injection in the following4weeks to induce alcoholic liver fibrosis. Serum alanine aminotransferase(ALT) and aspartic transaminase (AST) levels were measured by enzymaticmethod using an automatic biochemical analyzer. Haematoxylin and eosin(HE) stain and Masson stain were performed for observation of hepaticsteatosis, inflammation and fibrosis in the liver sections. Ultrastructuraldamage of hepatocytes was evaluated by transmission electron microscopy.The expression of α-smooth muscle actin (α-SMA) was detected byimmunohistochemistry strain. The mRNA and protein expression levels ofPPARα were detected by quantitative real-time PCR and Western blot.Results:1Serum ALT, AST levels, histological changes, and hepatic PPARαexpression of alcoholic steatohepatitis model1.1Serum levels of ALT and AST in miceMice fed with ethanol liquid diet for12weeks showed significantlyhigher serum ALT (150.33±20.68vs58.83±15.79U/L, P <0.001) and AST(259.67±28.13vs105.17±13.83U/L, P <0.001) levels compared with thecontrol mice.1.2Histological changes in miceMice in control group showed normal liver histology. The liver sectionsfrom mice fed with ethanol-containing liquid diet exhibited disordered lobulestructure, hepatocyte ballooning, moderate steatosis, inflammatory infiltrationand mild hepatocyte necrosis.1.3Ultrastructural damages of hepatocytes in miceRich organelles including mitochondria, endoplasmic reticulum andribosomes are observed in hepatocytes of normal control livers under electronmicroscopy. Whilst, in the liver sections of ethanol feeding mice,mitochondrial damage in hepatocytes with broken cristae, rupturedmembranes and merged cristae/membranes are observed. Granule fusion and degranulation phenomenons are also found in rough endoplasmic reticulum.1.4Hepatic expression of PPARα in miceHepatic expression of PPARα mRNA (0.72±0.03vs0.97±0.07, P <0.05)and protein (0.48±0.12vs0.83±0.13, P <0.05) was down-regulated byethanol.2Serum ALT, AST levels, histological changes, and hepatic PPARαexpression of alcoholic liver fibrosis model2.1Serum ALT and AST levels in miceSerum ALT level of mice treated with carbon tetrachloride(107.00±20.77U/L, P <0.01) or feeding with ethanol (76.00±5.76U/L, P <0.05) was higher than that of control mice (63.50±7.82U/L). Serum AST levelof mice feeding with ethanol was higher than that of control mice(300.50±34.16vs103.17±16.15U/L, P <0.01). Serum ALT (1072.50±109.19U/L) and AST (838.17±153.73U/L) levels of mice treated with ethanol pluscarbon tetrachloride were significantly higher than that of the mice in othergroups (P <0.01) and increased with the progression of the liver injury, theserum ALT levels were73.00±10.33,66.83±6.27,159.90±19.75,199.00±17.60,176.17±15.63,1072.50±109.19U/L, and serum AST levelswere98.17±12.62,252.50±23.84,480.00±17.40,544.50±72.88,491.67±13.94,838.17±153.73U/L, at zero, forth, fifth, sixth, seventh and eighth weeks,respectively.2.2Histological changes in miceAt eighth week, the control mice showed normal liver histology. Theliver sections of mice treated with carbon tetrachloride showed mildinflammatory infiltration, while mice fed with ethanol liquid diet exhibitedmoderate hepatocyte steatosis and mild inflammatory infiltration. Miceadministrated with ethanol plus carbon tetrachloride developed mild tomoderate hepatic steatosis at forth week, and progressive necroinflammationas well as perisinusoidal and portal fibrosis from fifth to eighth week, andpiecemeal necrosis and bridging fibrosis formed at eighth week.2.3Hepatic expression of α-SMA in mice Few α-SMA was deposited in liver of control mice and mice treated withcarbon tetrachloride or ethanol. In liver of ethanol plus carbon tetrachloridetreated mice, increasing α-SMA expression in the activated HSC and fibroticareas with the progression of the liver injury was observed.2.4Hepatic expression of PPARα in miceHepatic PPARα mRNA expression was reduced by carbon tetrachloridetreatment (P <0.01), while mRNA and protein expression was reduced byethanol treatment (P <0.05and P <0.01) in mice. Hepatic mRNA and proteinexpression of PPARα was further down-regulated by ethanol plus carbontetrachloride treatment, compared with carbon tetrachloride (P <0.01and P <0.01) or ethanol treatment (P <0.01and P <0.05).Conclusion:1Alcoholic steatohepatitis model could be established by feeding mice withLieber-DeCarli ethanol liquid diet for12weeks, while alcoholic liver fibrosismodel could be established by feeding mice with ethanol liquid diet for the4weeks and combined with microamount of carbon tetrachloride intraperitonealinjection for the following4weeks.2Hepatic expression of PPARα were suppressed by ethanol and ethanol pluscarbon tetrachloride treatment, which might play important roles in theprogression of ethanol induced steatohepatitis and fibrosis.Part2Activation of peroxisome PPARα ameliorates ethanol inducedsteatohepatitis in miceObjective: To explore the pathogenesis of alcoholic steatohepatitis andto elucidate the effect and the molecular basis of PPARα in ethanol inducedhepatic steatosis and inflammation in mice.Methods: C57BL/6J mice were fed with ethanol liquid diet for12weeks,and supplemented with PPARα agonist WY14643or antagonist GW6471inthe last2weeks. Serum ALT and AST levels were measured by enzymaticmethod using an automatic biochemical analyzer. HE stain was performed forobservation of hepatic steatosis and inflammation in the liver sections.Ultrastructural damage of hepatocytes was evaluated by transmission electron microscopy. The expression levels of PPARα, PPARα target genescytochrome P4504A10(CYP4A10), CYP4A14, lipid metabolism relatedgenes fibroblast growth factor21(FGF21), sirtuin1(SIRT1), PPAR-gammacoactivator-α (PGC-1α), fatty acid synthase (FAS), pro-inflammatorycytokines phosphatidylinositol3-kinase (PI3K), osteopontin (OPN),cyclooxygenase2(COX-2), as well as anti-inflammatory cytokinesadiponectin and heme oxygenase-1(HO-1) were detected by quantitativereal-time PCR and Western blot.Results:1Serum levels of ALT and AST in miceA significant reduction of serum ALT (84±25.81vs150.33±20.68U/L, P<0.001) and AST (180.33±16.48vs259.67±28.13U/L, P <0.001) levels wasnoticed after WY14643treatment in ethanol feeding mice. However, GW6471treatment further elevated ALT level (186±20.86vs150.33±20.68U/L, P <0.01).2Histological changes in miceThe liver sections from mice fed with ethanol liquid diet exhibitedmoderate steatosis, inflammatory infiltration and mild hepatocyte necrosis.WY14643significantly ameliorated ethanol induced hepatic steatosis andinflammation.3Ultrastructural damages of hepatocytes in miceBroken cristae, ruptured membranes and merged cristae/membranes wereobserved in hepatocellular mitochondria of ethanol feeding mice, as well asgranule fusion and degranulation phenomenons were found in roughendoplasmic reticulum. These ultrastructural damages in hepatocytes weresignificantly improved by WY14643administration.4Hepatic expressions of PPARα and PPARα-responsive genes in miceHepatic expression of PPARα-responsive genes CYP4A10and CYP4A14was down-regulated by ethanol. WY14643administration increased theexpression levels of PPARα mRNA (P <0.05) and protein (P <0.01), CYP4A10(P <0.001) and CYP4A14(P <0.001) mRNA in ethanol feeding mice. However, GW6471furhter reduced hepatic PPARα (P <0.05) and CYP4A14(P <0.001) mRNA expression.5Hepatic expression of lipid metabolism related genes in miceRelative to control mice, hepatic expression of SIRT1mRNA and protein(P <0.001) as well as PGC-1α protein (P <0.01) was reduced, whileexpression of FAS mRNA and protein (P <0.001) was enhanced in ethanolfeeding mice. Administration of WY14643increased mRNA and proteinexpressions of FGF21(P <0.001and P <0.01), SIRT1(P <0.05and P <0.01), PGC-1α (P <0.001and P <0.001), and reduced FAS expression (P <0.001and P <0.05) as compared with mice fed ethanol liquid diet only. While,GW6471decreased the FGF21mRNA (P <0.001) and protein (P <0.05)expression, and increased FAS mRNA expression (P <0.01).6Hepatic expression of pro-inflammatory factors in miceEthanol increased hepatic expression of pro-inflammatory factors PI3K,OPN and COX-2, but decreased expression of anti-inflammatory factoradiponectin. WY14643treatment significantly reduced hepatic mRNA andprotein expression of PI3K (P <0.001and P <0.01), OPN (P <0.001and P <0.001) and COX-2(P <0.01and P <0.05), and increased expression ofadionectin (P <0.001and P <0.01) and HO-1(P <0.001and P <0.001) inethanol feeding mice. On the other hand, GW6471further increased PI3Kprotein (P <0.05) and COX-2mRNA (P <0.05) expression.Conclusions:1PPARα showed a protective role in ethanol induced liver injury, asevidenced by diminished hepatic steatosis, inflammation, and improvedhepatocyte ultrastructure.2Activation of PPARα by WY14643ameliorated hepatic steatosis throughincreasing lipids oxidation promoting genes expression, and suppressing fattyacid synthesis promoting genes expression.3Induction of PPARα attenuated liver inflammatory response by repressingexpression of pro-inflammatory cytokines, as well as enhancing expression ofanti-inflammatory factors. Part3Activation of PPARα ameliorates alcoholic liver fibrosis in miceObjective: To explore the pathogenesis of alcoholic liver fibrosis and toelucidate the effect and molecular basis of PPARα in alcoholic liver fibrosis inmice.Methods: C57BL/6J mice were fed with ethanol liquid diet alone in thefirst4weeks and combined with microamount of carbon tetrachlorideintraperitoneal injection in the following4weeks to induce alcoholic liverfibrosis, and supplemented with PPARα agonist WY14643or antagonistGW6471in the last2weeks. Serum ALT and AST levels were measured byenzymatic method using an automatic biochemical analyzer. HE stain andMasson stain were performed for observation of hepatic steatosis,inflammation and fibrosis in the liver sections. The mRNA and proteinexpressions of PPARα, inflammatory factors tumor necrosis factor-alpha(TNF-α), Interleukin-10(IL-10), pro-fibrotic factors α-SMA, OPN,transforming growth factor-β1(TGF-β1), visfatin, PI3K, matrixmetallopeptidase-2(MMP-9), MMP-2, and anti-fibrotic factors adiponectinand HO-1were detected by real-time PCR, Western blot assay andimmunohistochemistry strain, respectively.Results:1Serum levels of ALT and AST in miceWY14643significantly reduced serum ALT (131.17±48.11vs1072.50±109.19U/L) and AST (202.83±31.73vs838.17±153.73U/L) levelsin mice treated with ethanol plus carbon tetrachloride (P <0.01), whileGW6471further increased the AST level (1000.33±85.54U/L)(P <0.05).2Histological changes in miceMice administrated with ethanol plus carbon tetrachloride developedmild hepatic steatosis, progressive necroinflammation, piecemeal necrosis andbridging fibrosis. WY14643prominently alleviated the liver injury induced byethanol plus carbon tetrachloride, merely mild inflammation and few fibersdeposition were found in the liver sections.3Hepatic expression of α-SMA in mice WY14643treatment reduced hepatic expression of α-SMA in ethanolplus carbon tetrachloride treated mice, which mainly expressed in centralveins, vessels of portal areas and few fiber deposition areas.4Hepatic expression of PPARα in miceWY14643administration significantly increased hepatic PPARαexpression in ethanol plus carbon tetrachloride treated mice (P <0.01and P <0.001). However, GW6471further reduced hepatic PPARα mRNA expression(P <0.01).5Hepatic expression of inflammatory factors in miceCarbon tetrachloride or ethanol treatment increased hepatic TNF-αmRNA expression (P <0.01), and reduced IL-10mRNA expression (P <0.01).While ethanol plus carbon tetrachloride treatment further increased TNF-αmRNA expression (P <0.01), and reduced IL-10mRNA expression (P <0.01),as compared with carbon tetrachloride or ethanol treatment. WY14643suppressed TNF-α mRNA expression (P <0.01) and promoted IL-10mRNAexpression (P <0.01) in ethanol plus carbon tetrachloride treated mice.6Hepatic expression of pro-fibrotic factors in miceCarbon tetrachloride or ethanol treatment increased hepatic OPN,TGF-β1, visfatin, PI3K, MMP-2and MMP-9expression (P <0.05), whileethanol plus carbon tetrachloride treatment further increased the expression ofthese genes (P <0.05). WY14643suppressed OPN (P <0.01and P <0.001),TGF-β1(P <0.01and P <0.001), visfatin (P <0.01and P <0.001), PI3K (P<0.01and P <0.01), MMP-2(P <0.01and P <0.001), MMP-9(P <0.01andP <0.01) mRNA and protein expression in ethanol plus carbon tetrachloridetreated mice.7Hepatic expression of anti-fibrotic factors in miceCarbon tetrachloride or ethanol treatment reduced adiponectin mRNAand protein expression (P <0.01), and increased HO-1expression (P <0.05).Ethanol plus carbon tetrachloride treatment further decreased adiponectin andincreased HO-1expression (P <0.05). WY14643up-regulated adiponectin (P<0.01and P <0.001) and HO-1(P <0.01and P <0.001) mRNA and protein expression in ethanol plus carbon tetrachloride treated mice.Conclusions:1PPARα showed a protective role in alcoholic liver fibrosis, as evidenced bydiminished hepatic steatosis, inflammation and fibrosis.2Activation of PPARα by WY14643ameliorated hepatic injury throughsuppressing hepatic expression of pro-inflammatory factors and pro-fibroticfactors, while increasing expression of anti-inflammatory factors andanti-fibrotic factors.