Molecular Mechanism Of Simvastatin Acting On The Progression Of Non-alcoholic Fatty Liver Fibrosis

Objective: Non-alcoholic fatty liver disease (NAFLD) is characterized by excessive deposition of free fatty acids and triglycerides in the liver. It comprises a disease spectrum which includes simple steatosis, non-alcoholic steatohepatitis (NASH), and cirrhosis. In China, NAFLD is increasingly diagnosed and became common chronic liver disease. Adiponectin is secreted by adipocytes and is considered to possess anti-inflammatory effects, to improve hepatic and peripheral insulin sensitivity, and to decrease triglyceride accumulation in the hepatocytes. Recent studies suggest that adiponectin have anti-fibrotic property. It can inhibit the activation, proliferation, and migration of hepatic stellate cells (HSC), and induce apoptosis of activated HSC, but the pathogenesis is still unclear. Nitric oxide (NO) is generated from L-arginine by NO synthase (NOS), which include endothelial NOS (eNOS), inducible NOS (iNOS), and neuronal NOS (nNOS). Previous studies have demonstrated that adiponectin could increase eNOS activity, and thus restricted the contraction of endothelial cells. But it is still indistinct whether adiponectin inhibit the activation of HSC by mediating NOS. Simvastatin, an inhibitor of 3-hydroxy-3-methyglutaryl-coenzyme A reductase, has been proven to be effective on dyslipidemia. In addition, it has pleiotropic effects such as anti-inflammatory and antioxidant properties, inhibition of cell proliferation, and so on. Some studies have shown that simvastatin could reduce the circulating low-density lipoprotein and improved the hepatic steatosis of patients with NAFLD. However, it is controversial that whether simvastatin increase the contents of adiponectin in serum and/or hepatic tissue and improve hepatic fibrosis. Therefore, the rat models of non-alcoholic fatty liver fibrosis were established by high-fat diet administration. We examined the levels of serum adiponectin and detected the mRNA expression and protein contents of adiponectin and AMPK in the hepatic tissues to investigate their effects on the pathogenesis of non-alcoholic fatty liver fibrosis. In vitro, we detected the expressions of AMPK, iNOS, and eNOS of human HSC line LX-2 CELLS treated with recombinant adiponectin to explore its anti-fibrosis mechanisms. In addition, we detected the expressions of adiponectin and foregoing cytokines in hepatic tissues of model rats and cultured LX-2 CELLS treated with simvastatin both in vivo and in vitro to investigate the therapeutic and preventive effects of simvastatin on non-alcoholic fatty liver fibrosis from macroscopic, microscopic, cellular, and molecular levels, and to provide rational data and therapeutic targets for using simvastatin to treat patients with non-alcoholic fatty liver fibrosis.Methods:1 Expression and significance of adiponectin in hepatic tissues of rat models with non-alcoholic fatty liver fibrosisForty clean wistar rats weighting from 140 grams to 160 grams were purchased from the experimental animal center of Hebei medical university. After one week of acclimatization, rats were randomly divided into two groups. The rats in normal control group (n=10) were fed with standard diet and those in model group (n=30) were fed with high-fat diet (standard diet+10% lard+2% cholesterol+5% corn oil). Then six rats in model groups were respectively sacrificed at 8th, 12th, 16th, 20th, and 24th week by bloodletting at femoral vein, and the serum and liver specimens were collected. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), cholesterol (TC), and triglyceride (TG) were measured using an auto-biochemical analyzer, and serum adiponectin was examined by enzyme-linked immunosorbent assay (ELISA). Some of liver tissues were made of frozen sections and were stained by Sudan IV for hepatic steatosis. Another part of liver tissues were fixed with 10% formalin and then made of paraffin sections. Hematoxylin-eosin (HE) staining was used for observing the general hepatic pathological changes and Masson staining for fibrosis. The rest livers were stored in -80℃refrigerator to detect the expressions of adiponectin, AMPK and Collagen ? in both mRNA and protein levels by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot, respectively.2 Molecular mechanism of adiponectin inhibiting the activation of hepatic stellate cellsHuman HSC line LX-2 cells from the cell bank of Chinese Academy of Sciences were cultured in DMEM containing 10% fatal bovine serum (FBS), penicillin (100 U/ml), and streptomycin (100μg/ml) at 37℃with 5% CO2. Cells were seeded in six well plates at a density of 5×105 cells per well, and treated with the adipogenic differentiation mixture (ADM) containing 0.5 mM isobytylmethylxanthine, 1μM dexamethazone, and 1μM insulin for 72 hours. After that, ADM was changed to DMEM with 0.2% FBS, penicillin, and streptomycin for 24 hours to achieve cell synchronization. For experiments with adiponectin, cells were treated with solvent (the control group) or transforming growth factorβ1 (TGF-β1, 100 pM), adiponectin (5μg/ml), TGF-β1+adiponectin, L-NAME (NOS inhibitor, 100μM), and L-NAME+adiponectin, respectively. Then cells were harvested at one hour to assess AMPK phosphorylation or at 24 hours to determine iNOS, eNOS,α-smooth muscle actin (α-SMA), and Collagen ?.3 Molecular mechanism of simvastatin ameliorating the progression of non-alcoholic fatty liver fibrosisIn vivo, 18 clean wistar rats weighting from 140 grams to 160 grams were randomly divided into two groups after one week of acclimatization. The rats in normal control group (n=6) were fed with standard diet and those in model group (n=18) were fed with high-fat diet (standard diet+10% lard+2% cholesterol+5% corn oil). At the end of 16th week, six rats randomly separated from the model group were treated with simvastatin intragastrically besides the high-fat diet. The other model rats were still fed with high-fat diet, and meanwhile with normal saline intragastrically. All rats were sacrificed at 24th week and the serum and liver specimens were collected. Serum ALT, AST, TC, and TG were measured using an auto-biochemical analyzer, and serum adiponectin was examined by ELISA. Sudan IV staining was used for hepatic steatosis, HE staining for general hepatic pathological changes, and Masson staining for fibrosis. RT-PCR and Western blot were used for analyzing the adiponectin, eNOS, and Collagen ? mRNA and protein expressions.In vitro, LX-2 cells were prepared for examinations as methods mentioned above. For experiments with simvastatin, cells were treated with solvent (the control group) or TGF-β1 (100 pM), simvastatin (10μM), TGF-β1+simvastatin, L-NAME (100μM), L-NAME+simvastatin, respectively. Then cells were harvested at 24 hours to determine the expressions of eNOS,α-SMA, and Collagen ?. To investigate the role of simvastatin of the expression of adiponectin in HSCs, LX-2 cells were treated with simvastatin for different doses (1μM, 2.5μM, 5μM, 10μM) and different times (1 h, 1.5 h, 3 h, 6 h, 12 h, 24 h). After that, cells were harvested to determine adiponectin expression.Results:1 Expression and significance of adiponecitn in hepatic tissues of rat models with non-alcoholic fatty liver fibrosis1.1 Changes of serum levels of ALT, AST, TC, TG, and adiponectin in model rats:Compared with that of normal rats in control group, the serum levels of ALT, AST, TC, and TG in model rats were all increased with the consumption of high-fat diet except adiponectin, which decrease dramatically, all P<0.05 or P<0.01. The level of serum adiponectin was negatively correlated with that of ALT, AST, TC, and TG (r=-0.58, r=-0.52, r=-0.55, r=-0.45; all P<0.01).1.2 Pathological changes of hepatic tissue: Fed with high-fat diet, the model rats gradually showed steatosis, inflammation, necrosis, and fibrosis in their hepatic tissues. Slightly sinusoidal fibrosis appeared only in part of model rats at 16th week. Until 24^th week, all model rats showed hepatic fibrosis in sinusoids, partly in portal area.1.3 The mRNA expression and protein contents of adiponectin, AMPK, CollagenⅠin the hepatic tissues of normal and model rats: Compared with that in normal control group, the expressions of adiponectin and AMPK both in mRNA and protein levels were gradually decreased in livers of model rats, while the expressions of CollagenⅠwere gradually increased (P<0.05 or P<0.01). Adiponectin mRNA expression was positively correlated with that of AMPK (r=0.81,P<0.01), and both of them were negatively correlated with CollagenⅠmRNA expression (r=-0.88, r=-0.87,all P<0.01). And the same results were obtained from the comparison of their protein contents.2 Molecular mechanism of adiponectin inhibiting the activation of hepatic stellate cells2.1 TGF-β1 induced the activation of quiescent LX-2 cells by reducing the activity of AMPK, decreasing the expression of eNOS, and increasing the expression of iNOSLX-2 cells approximately acquired quiescent phenotypes mediated by ADM for three days. These cells were used for investigating the molecular mechanism of activation and inhibition of HSC. TGF-β1 activated quiescent LX-2 cells to generate excessive extracellular matrix, the mRNA and protein expressions ofα-SMA (0.55±0.05 vs. 0.12±0.02; 0.58±0.07 vs. 0.13±0.03, all P<0.05) and CollagenⅠ(0.46±0.10 vs. 0.09±0.02; 0.43±0.07 vs. 0.08±0.03, all P<0.05) were all increased compared with the control group, as well as that of iNOS (0.53±0.07 vs. 0.37±0.04; 0.55±0.07 vs. 0.39±0.05, P<0.05). On the contrary, TGF-β1 inhibited AMPK phosphorylation and reduced the activity of AMPK activity (0.24±0.04 vs. 0.43±0.07, P<0.05), followed by lowering eNOS mRNA and protein expressions (0.30±0.10 vs. 0.44±0.08; 0.30±0.09 vs. 0.46±0.07; P<0.05).2.2 Adiponectin maintained the quiescent phenotype of LX-2 cells by enhancing the activity of AMPK, increasing the expression of eNOS, and decreasing that of iNOSQuiescent LX-2 cells were directly treated with recombinant adiponectin, and theirα-SMA and Collagen ? were detected. The results showed no significant differences compared with that of the control group. In addition, AMPK's activity (0.53±0.06 vs. 0.43±0.07, P<0.05) and eNOS mRNA and protein expression (0.56±0.10 vs. 0.44±0.08; 0.55±0.05 vs. 0.46±0.07, P<0.05) were all increased, while iNOS were decreased (0.30±0.02 vs. 0.37±0.04; 0.32±0.03 vs. 0.39±0.05, P<0.05).2.3 Adiponectin inhibited the activation of quiescent LX-2 cells induced by TGF-β1 via increasing AMPK's activity and eNOS expression and decreasing iNOS expressionQuiescent LX-2 cells were pre-treated with TGF-β1, and then were used for investigating the molecular mechanism of adiponectin inhibiting the activation of HSC. The results were similar to that above. Compared with the control group, the mRNA and protein expressions ofα-SMA (0.32±0.08 vs. 0.55±0.05; 0.32±0.04 vs. 0.58±0.07, P<0.05), Collagen ? (0.29±0.05 vs. 0.46±0.10; 0.32±0.04 vs. 0.43±0.07; P<0.05), and iNOS (0.44±0.05 vs. 0.53±0.07; 0.46±0.07 vs.0.55±0.07, P<0.05) were reduced, that of eNOS (0.43±0.08 vs. 0.30±0.10; 0.42±0.07 vs. 0.30±0.09, P<0.05) were increased, as well as AMPK's activity (0.43±0.07 vs. 0.24±0.04, P<0.05) increased.2.4 Adiponectin inhibited the activation of quiescent LX-2 cells due to L-NAME by increasing eNOS expressionCompared with the control group, quiescent LX-2 cells treated with L-NAME acquired the activated phenotypes, expressed moreα-SMA (0.41±0.04 vs. 0.12±0.02; 0.40±0.07 vs. 0.13±0.03, P<0.05), more Collagen ? (0.38±0.06 vs. 0.09±0.02; 0.38±0.05 vs. 0.08±0.03, P<0.05), less eNOS (0.19±0.05 vs. 0.44±0.08; 0.17±0.04 vs. 0.46±0.07, P<0.05), and less iNOS (0.22±0.05 vs. 0.37±0.04; 0.25±0.04 vs. 0.39±0.05, P<0.05) both in mRNA and protein levels. And the results, except iNOS, were similar to that of TGF-β1 group, which used as a positive control. Adiponectin antagonized this effect on eNOS mRNA (0.31±0.05 vs. 0.17±0.04, P<0.05), but neither on eNOS protein, although the protein level increased, nor on the expressions of iNOS both in mRNA and protein levels.3 Molecular mechanism of simvastatin ameliorating the progression of non-alcoholic fatty liver fibrosis3.1 Changes of serum levels of ALT, AST, TC, TG, and adiponectin in each group of rats:The serum levels of ALT, AST, TC, and TG in model rats were all increased with the consumption of high-fat diet, but the level of adiponectin was decreased. Compared with rats in the 24th week group, the levels of ALT, AST, TC, and TG were all declined in rats of simvastatin-treatment group (P<0.05 or P<0.01). The level of adiponectin was increased either, but with no significant differences.3.2 Pathological changes of hepatic tissue: Hepatic steatosis, inflammation, and fibrosis were aggravated with consumption of high-fat diet. At 24th week, all model rats exhibited sinusoidal fibrosis.. These conditions were improved in rats of simvastatin-treatment group.3.3 The expressions of adiponectin, eNOS, and CollagenⅠboth in mRNA and protein levels in the hepatic tissues of rats in each group: Compared with that in normal control group, the mRNA and protein expressions of adiponectin and eNOS were gradually decreased in livers of model rats, while CollagenⅠwas gradually increased (P<0.05 or P<0.01). Compared with rats in 24th week group, rats in simvastatin-treatment group had more adiponectin (0.34±0.04 vs. 0.27±0.05; 0.36±0.05 vs. 0.24±0.06, P<0.05) and eNOS (0.30±0.02 vs. 0.24±0.01; 0.45±0.04 vs. 0.22±0.02, P<0.05) mRNA and protein expressions, and less Collagen ? expression (0.14±0.01 vs. 0.25±0.01; 0.54±0.02 vs. 0.89±0.03, P<0.05). eNOS mRNA expression was negatively correlated with Collagen ? mRNA expression (r=-0.91, P<0.01), and their protein comparison showed the same result (r=-0.92, P<0.01).3.4 Simvastatin maintained the quiescent phenotype of LX-2 cells by increasing eNOS expressionCompared with that of the control group, eNOS mRNA and protein expressions (0.52±0.03 vs. 0.41±0.02; 0.52±0.02 vs. 0.45±0.03, P<0.05) were increased in LX-2 cells treated with simvastatin. And theirα-SMA and Collagen ? expressions were similar, with no significant differences.3.5 Simvastatin inhibited the activation of quiescent LX-2 cells caused by L-NAME via increasing eNOS expressionQuiescent LX-2 cells were pre-treated with L-NAME, and then were used for investigating the molecular mechanism of simvastatin inhibiting the activation of HSC. The results showed that, compared with the control group, the mRNA and protein expressions ofα-SMA (0.28±0.02 vs. 0.34±0.02; 0.26±0.02 vs. 0.36±0.02, P<0.05) and Collagen ? (0.30±0.02 vs. 0.40±0.03; 0.26±0.02 vs. 0.31±0.02, P<0.05) were reduced and that of eNOS (0.25±0.02 vs. 0.12±0.02; 0.23±0.03 vs. 0.12±0.02, P<0.05) were increased after treated with simvastatin. 3.6 Simvastatin increased the adiponectin expressions in LX-2 cells Quiescent LX-2 cells were treated with simvastatin of different doses and different times. The results showed that the expression of adiponectin both in mRNA and protein levels were gradually increased in LX-2 cells with simvastatin's concentration or duration increasing.Conclusions:1 Rats'models of non-alcoholic fatty liver fibrosis can be developed by high-fat diet administration, and the hepatic pathological changes of model rats reflect the progress of NAFLD in human.2 Adiponectin increases the phosphorylation of AMPK. They play important roles in the pathogenesis of non-alcoholic fatty liver fibrosis.3 The transformation of hepatic stellate cells'phenotypes can be accomodated by iNOS and eNOS. Adiponectin prevents the progress of liver fibrosis by inhibiting the activation of hepatic stellate cells via increasing the expression of eNOS, and decreasing the expression of iNOS.4 Simvastatin inhibits the activation of hepatic stellate cells by increasing the expression of adiponectin and eNOS. These results reveal the molecular mechanism of simvastatin in preventing and treating of non-alcoholic fatty liver fibrosis, and provide theoretical evidence for its clinical application.