辛伐他汀抑制肝星状细胞活化及其对腺苷酸活化蛋白激酶活性的影响

Simvastatin inhibits activation of hepatic stellate cells and promotes activation of adenosine monophosphate- activated protein kinase

目的研究辛伐他汀治疗非酒精性脂肪性肝纤维化的作用机制。方法高脂饲料喂养大鼠复制非酒精性脂肪性肝纤维化模型，辛伐他汀灌胃治疗后，病理学染色观察肝组织病理改变，RT-PCR和Western blot法测定肝组织腺苷酸活化蛋白激酶（AMPK）α的mRNA和蛋白质表达水平，同时测定血清胆固醇（TC）、甘油三酯（TG）、ALT、AST和血清肿瘤坏死因子（TNF）α水平。体外用促进脂肪细胞分化的培养液诱导入肝星状细胞株LX-2获得静止表型，分别用转化生长因子β1（TGFβ1）、辛伐他汀、TGFβ1＋辛伐他汀处理静止型LX-2细胞，RT-PCR和Western blot法检测AMPKα的mRNA及蛋白质表达变化。多组间数据比较用单因素方差分析或析因设计方差分析，两组间数据比较用q检验。结果高脂饮食24周成功复制大鼠非酒精性脂肪性肝纤维化模型。随造模时间的延长，肝组织炎症、脂肪变和纤维化程度加重，血清TC、TG、ALT、AST和TNF0t水平逐渐升高妒值均〈0．01），肝组织AMPK仅的mRNA相对表达量及活性逐渐降低（P〈0．01或P〈0．05）。与模型对照组相比，辛伐他汀治疗组TC、TG、ALT、AST和TNFα水平明显下降（P值均〈0．05），AMPKα mRNA（0．31±0．05比0．24±0．07）和AMPKα活性（0．45±0．07比0．27±0．07）明显升高（q值分别为3．22和6．44，P〈0．01或P〈0．05）；肝星状细胞的AMPKα活性明显增强（静止型为0．93±0．10比0．72±0．09，活化型为0．72±0．10比0．54±0．10，q值分别为7．00和6．00，P值均〈0．01），α-平滑肌动蛋白的mRNA和蛋白质表达明显减少（分别为0．30±0．02比0．36±0．02和0．30±0．03比0．38±0．02，q值分别为11．245和11．216，P值均〈0．01），Ⅰ型胶原的mRNA和和蛋白质表达也明显减少（分别为0．30±0．03比0．37±0．03和0．25±0．03比0．33±0．03，q值分别为8．791和11．163，P值均〈0．01）。结论辛伐他汀可以通过提高AMPK活性，抑制肝星状细胞活化，延缓非酒精性脂肪性肝纤维化的发生和发展。

Objective To investigate the underlying molecular mechanism of the cholesterol-blocking drug, simvastatin, in treating nonalcoholic fatty liver fibrosis. Method A rat model of nonalcoholic fatty liver fibrosis was established by feeding Wistar rats a fat-rich diet. After treatment with simvastatin （4 mg/kg/day）, liver histological specimens were stained with hematoxylin-eosin and Masson＇s trichrome for microscopic analysis. Expression of adenosine monophosphate-activated protein kinase-alpha （AMPKa） was evaluated by reverse transcription-polymerase chain reaction （RT-PCR; for mRNA） and Western blotting （protein）. The levels of serum total cholesterol （TC）, triglycerides （TG）, alanine aminotransferase （ALT）, aspartate aminotransferase （AST）, and tumor necrosis factor-alpha （TNFa） were measured by standard biochemical assays. The human hepatic steUate cell line, LX-2 （quiescent or activated）, was treated with transforming growth factor-beta 1 （TGF-β1） alone, simvastatin alone, or TGF-β1 ＋simvastatin. RT-PCR and Western blotting were used to determine changes in AMPKa mRNA and protein expression, respectively. Results In the rat model of nonalcoholic fatty liver fibrosis, the extent of pathological changes in hepatic tissues correlated with severity of disease progression. The levels of serum TC, TG, ALT, AST and TNFa were increased significantly in model rats （vs. healthy controls; all, P〈 0.01）. AMPKa mRNA expression and activity was significantly decreased in model rats （vs. healthy controls; P 〈 0.01 and P 〈 0.05, respectively）. Simvastatin, treatment significantly improved all of these parameters in model rats （vs. untreated model rats; all, P〈 0.05）. In vitro simvastatin treatment of human HSCs significantly increased AMPKa activity （quiescent LX-2：0.93 ± 0.10 vs. 0.72±0.09, activated LX-2：0.72±0.10 vs. 0.54±0.10, q= 7.00, 6.00; all, P〈0.01）, decreased a-smooth muscle actin expression （mRNA： 0.30±0.02 vs. 0.36±0.02, protein： 0.30±0.03 vs. 0.38±0.02, q= 11.245, 11.216; all, P〈0.01）, and decreased collagen I expression （mRNA： 0.30±0.03 vs. 0.37±0.03, protein： 0.25±0.03 vs. 0.33＋0.03, q= 8.791, 11.163; all, P〈0.01）. Conclusion Simvastatin may improve nonalcoholic fatty liver fibrosis by inducing AMPK phosphorylation.