脂联素对转化生长因子β1致肝星状细胞低表达内皮型一氧化氮合酶的阻断作用

Adiponectin inhibits the activation of hepatic stellate cells induced by TGF β1 via up-regulatlng the expression of eNOS

目的探讨脂联素在非酒精性脂肪性肝纤维化中抑制肝星状细胞活化的分子机制。方法体内实验：用高脂饮食（标准饲料＋2％胆固醇＋10％猪油＋5％玉米油）喂养大鼠建立非酒精性脂肪性肝纤维化大鼠模型，应用RT-PCR法和Westernblot法检测大鼠肝组织中脂联素和I型胶原mRNA及蛋白的表达。体外实验：用促进脂肪细胞分化的培养液诱导人肝星状细胞株LX-2细胞获得静止表型，分别用转化生长因子D1（TGF01）、脂联素、TGFD1＋脂联素处理静止型LX-2细胞，RT-PCR法和Westernblot法检测各组LX-2细胞中α-平滑肌肌动蛋白（α-SMA）、I型胶原、腺苷酸活化蛋白激酶（AMPK）、诱导型一氧化氮合酶（iNOS）、内皮型NOS（eNOS）mRNA及蛋白的变化。对多组间数据进行单因素方差分析，两两比较采用Student-Newman-Keuls法l相关分析采用直线相关分析法。结果随造模时间延长，大鼠肝组织脂肪变性、炎症及纤维化程度逐渐加重，I型胶原mRNA表达逐渐增加；与对照组相比，模型24周组大鼠血清脂联素水平降低（2．49±0．86与5．81±0．87，P值〈0．05），肝组织脂联素mRNA及蛋白表达均减少（0．26±0．04与0．72±0．0810．21±0．07与0．64±0．07，P值均〈0．05），且与I型胶原mRNA及蛋白表达呈负相关（相关系数分别为-0．47，-0．44，-0．88和～0．89，P值均〈0．01）。体外实验，与对照组相比，TGFp1能促进静止型LX-2细胞活化（α-sMA蛋白由0．13±0．03升高至0．58±0．07，P〈0．05），减少eNOSmRNA及蛋白表达（0．30±0．10与0．44±0．08，0．30±0．09与0．46±0．07，P值均〈0．05），增加iNOSmRNA和蛋白表达（0．53±0．07与0．37±0．04；0．55±0．07与0．39±0．05，P值均〈0．05），减弱AMPK活性（0．24±0．04与0．43士0．07，P〈0．05）；脂联素能阻断此过程，上调eNOSmRNA和蛋白表达（0．43±0．08与0．30±0．10，0．42±0．07与0．30±0．09，P值均〈0．05），下调iNOStuRNA和蛋白表达（0．44±0．05与0．53±0．0710．46±0．07与0．55±0．07，P值均〈0．05），增加AMPK涪性（0．43±0．07与0．24±0．04，P值〈0．05）。结论脂联素通过上调由TGFD1导致的LX-2细胞低表达eNOS的表达而阻止LX-2细胞活化，减少I型胶原生成，这可能与脂联素增加LX-2细胞中AMPK活性有关。

Objective To investigate the molecular mechanism of adiponectin inhibiting activation of hepatic stellate cells in non-alcoholic fatty liver fibrosis. Methods The rat models of non-alcoholic fatty liver fibrosis were successfully established by fat-rich diet administration. The expression of adiponectin mRNA and protein were respectively detected by reverse transcription-polymerase chain reaction （RT-PCR） and Westem blot. LX-2 cells were cultured in an adipogenic differentiation mixture to induce quiescent adipocyticphenotypes, and then they were treated with TGF β1, adiponectin and TGF β1 ＋ adiponectin, respectively. RT- PCR and Western blot were used to determine the expressions of mRNAs and proteins of a-smooth muscle actin （α-SMA）, Collagen I, adenosine monophosphate-activated protein kinase （AMPK）, inducible nitric oxide synthase （iNOS）, and endothelial NOS （eNOS）. The results were analyzed using one-way ANOVA, Student-Newman-Keuls test, and linear correlation analysis. A P value of less than 0.05 was considered as statistically significant. Results In vivo, with the progress of non-alcoholic fatty liver fibrosis, the model rats gradually showed hepatic steatosis, inflammation, necrosis and fibrosis. Compared with the control group, the level of serum adiponectin （2.49 ± 0.86 vs 5.81 ±0.87, P 〈 0.05） and hepatic expressions of adiponectin mRNA and protein （0.26 ± 0.04 vs 0.72 ± 0.08; 0.64 ±0.07 vs 0.21 ± 0.07, all P 〈 0.05） were all decreased in the 24th week group, and were negatively correlated with the level of Collagen I which increased gradually. In vitro, TGF 131 could activate quiescent LX-2 cells by decreasing mRNA and protein expression of eNOS （0.30 ± 0.10 vs 0.44 ± 0.08; 0.30 ± 0.09 vs 0.46 ±0.07, allP〈 0.05） and increasing the expression of iNOS （0.53 ± 0.07 vs 0.37 ± 0.04; 0.55 ± 0.07 vs 0.39 ± 0.05, allP 〈 0.05）, Recombinant adiponectin not only maintained the quiescent phenotype of LX-2 cells but also inhibited LX-2 cells activation due to TGF 131 by increasing the expression of eNOS （0.43 ± 0.08 vs 0.30 ± 0.10; 0.42 ± 0.07 vs 0.30 ± 0.09, allP 〈 0.05） and phosphorylation of AMPK （0.43 ± 0.07 vs 0.24 ± 0.04,P 〈 0.05） and decreasing the expression ofiNOS （0.44± 0.05 vs 0.53 ± 0.07; 0.46 ± 0.07 vs 0.55 ± 0.07, all P 〈 0.05）. Conclusions Data suggested that adiponectin could play a protective role on the pathogenesis of non-alcoholic fatty liver fibrosis by inhibiting the activation of hepatic stellate cells via up-regulating the expression of eNOS, which might associate with increased phosphorylation of AMPK.