Pioglitazone Ameliorates Nutritional Hepatic Fibrosis By Inhibiting Toll-like Receptor4/Nuclear Factor κB Signaling Pathway In Mice

Objective: Non-alcoholic fatty liver disease (NAFLD) has become themost common liver disease wildworld. The disease spectrum of NAFLDincludes simple steatosis, non-alcoholic steatohepatitis (NASH),NASH-related hepatic fibrosis and cirrhosis, hepatocellular carcinoma.Appromate twenty to forty percent of NASH progress into cirrhosis and causeliver failure. Understanding hepatic fibrogenesis in NAFLD is an importantresearch area and will be valuable for identifying potential therapeutic targetsto prevent the progression of fibrotic steatohepatitis to more severe disease.There is increasing evidence that toll-like receptors (TLRs), especially TLR4,play a pivotal role in the pathogenesis and progression of many liver diaease.The binding of specific TLR ligands leads to activation of adaptor molecules.This results in stimulation of inhibitor of nuclear factor-κB kinase-β (IKK-β)and translocation of nuclear factor κB (NF-κB), and then leads to theexpression of pro-inflammatory as well as anti-inflammatory cytokines, suchas tumor necrosis factor α (TNF-α), interleukin-1(IL-1), IL-6. Peroxisomeproliferator-activated receptorγ (PPARγ) agonists, pioglitazone, has beenshown to alleviate liver steatosis, inflammation and fibrogenesis in murineNASH models. However, molecular mechanisms of these effects are yet to befully defined. The aim of this study is to elucidate the effect and the molecularmechanism of PPARγ on liver fibrosis by TLR4/NF-κB signaling pathway inMCD-diet induced non-alcoholic fatty liver disease in mice.Methods: Eight-week-old male C57BL/6J mice were randomly dividedinto four groups of six: Control group, MCD diet supplemented with cholinebitartate (2g/kg) and DL-methionine (3g/kg), as control;2) MCD group,methionine-choline deficient diet;3) MCD+PIO group, MCD diet plus pioglitazone (50mg/kg/d, chow);4) MCD+GW group, MCD diet plus2-chloro-5-nitrobenzani-liden (GW9662;1mg/kg, three times/week, i.p.). Thebody-weight and diet consumption were recorded during the experiment of8weeks. After fasting and water deprivation for more than12hours, all of theanimals were anesthetized and weighted. Blood samples were collected fromfemoral artery for biochemical analysis. Livers were weighted and partly fixedin10%formalin for histological analysis or snap-frozen in lipid nitrogen.Liver injury was estimated by serum alanine aminotransferase (ALT) andaspartate aminotransferase (AST) levels and histological examination. ThemRNA and protein levels of PPARγ, TLR4, IKK-β and NF-κB were analyzedby real-time PCR and western blot.Results:1The mice of MCD group showed less activity than the Controlgroup, sikness and dim hair.2The serum alanine aminotransferase (ALT) andaspartate aminotransferase (AST) levels were increased by the MCD diet(431.12±35.17vs.32.97±3.31and423.85±28.02U/L vs.33.82±3.68U/L,P＜0.05) compared to the controls, and were amelioration by theadministration of pioglitazone (321.98±39.69U/L vs.431.12±35.17U/Land330.30±43.99U/L vs.423.85±28.02U/L，P＜0.05).3The MCD dietresulted in steatosis, inflammatory and fibrosis in mice liver section.4Thehepatic mRNA expressions of PPARγ, TLR4, IKK-β and NF-κB: The hepaticmRNA expressions of PPARγ were downregulated in the mice fed MCD diet,whereas the levels of TLR4, IKK-β and NF-κB were increased compared withthe controls (0.54±0.06copies vs.0.96±0.05copies,4.60±0.02copies vs.1.01±0.06copies,3.59±0.54copies vs.1.01±0.03copies and4.62±0.28copies vs.0.99±0.01copies, P＜0.05). Pioglitazone ameliorated hepaticsteatosis, inflammatory and fibrosis as we observed in histological section.With the induction of PPARγ by pioglitazone, the severity of liver injury wassignificantly attenuated, and the hepatic mRNA expressions of TLR4, IKK-βand NF-κB were depressed (0.74±0.02copies,2.42±0.10copies,1.34±0.51copies and2.42±0.50copies, P＜0.05). These effects were contrary inmice treated with GW9662.5The hepatic protein expressions of PPARγ, TLR4, IKK-β and NF-κB: The hepatic protein expressions of PPARγ weredownregulated in the mice fed MCD diet, whereas the levels of TLR4, IKK-βand NF-κB were increased compared with the controls (0.81±0.04vs.1.58±0.09,1.17±0.04vs.0.44±0.02,1.06±0.07vs.0.43±0.05and1.04±0.02vs.0.44±0.05, P＜0.05). Pioglitazone ameliorated hepatic steatosis,inflammatory and fibrosis as we observed in histological section. With theinduction of PPARγ by pioglitazone, the severity of liver injury wassignificantly attenuated, and the hepatic protein expressions of TLR4, IKK-βand NF-κB were depressed (1.07±0.09,0.76±0.01,0.71±0.08and0.70±0.05, P＜0.05). These effects were contrary in mice treated with GW9662.Conclusion:1PPARγ agonist, pioglitazone, could ameliorateinflammation and fibrogenesis in MCD induced hepatic fibrosis in mice.2TLR4/NF-κB signaling pathway was involved in the inhibitory effects ofpioglitazone on the progression of nutritional hepatic fibrosis in mice.