慢病毒介导长链非编码RNA H19过表达对非酒精性脂肪性肝病大鼠的影响

Effect of lentivirus-mediated overexpression of long-chain non-coding RNA H19 on rats with non-alcoholic fatty liver disease

目的探究慢病毒介导长链非编码RNA H19(lncRNA H19)过表达对非酒精性脂肪性肝病(NAFLD)大鼠的影响。方法采用饲喂高脂饲料法建立大鼠NAFLD模型,将造模成功的30只大鼠随机分为模型组、空载体组、过表达H19组,每组10只,另取10只大鼠为对照组。实验12周后将大鼠全部处死,根据体质量、肝脏湿重计算肝脏指数;酶联免疫吸附法(ELISA)检测大鼠肝功能、血脂、氧化应激指标;生物化学法检测氧化应激指标;苏木精-伊红(HE)染色后观察肝组织的病理改变;实时荧光定量聚合酶链反应(RT-PCR)检测肝组织中还原型辅酶Ⅱ(NADPH)氧化酶4(NOX4)mRNA、转化生长因子-β1(TGF-β1)mRNA、结缔组织生长因子(CTGF)mRNA的表达水平;蛋白质免疫印迹法(Western Blotting)检测各组肝组织中NOX4蛋白的水平。结果模型组及空载体组的肝功能减弱、肝细胞发生病变、肝组织中超氧化物歧化酶(SOD)及谷胱甘肽(GSH)的水平低于对照组,而肝组织中丙二醛(MDA)的水平、NOX4 mRNA、TGF-β1 mRNA、CTGF mRNA及蛋白的表达水平均高于对照组,差异有统计学意义(P<0.05)。过表达H19组大鼠肝功能增强,肝细胞病变状态改善,肝组织中SOD及GSH水平均高于模型组及空载体组,而MDA水平、NOX4 mRNA、TGF-β1 mRNA、CTGF mRNA表达水平及NOX4蛋白表达水平均低于模型组及空载体组,差异有统计学意义(P<0.05)。结论过表达lncRNA H19可抑制NOX4的表达以抵抗氧化应激的扩大,进而对高脂饮食诱导的NAFLD大鼠的肝损伤起到保护作用。

Objective An attempt is made in this paper to explore the effect of lentivirus-mediated overexpression of long-chain non-coding RNA H19(lncRNA H19)on non-alcoholic fatty liver disease(NAFLD)rats.Methods A high-fat diet was used to establish a rat NAFLD model.Thirty rats successfully modeled were randomly divided into a model group,an empty vector group,and an over-expressing H19 group,10 rats in each group,and another 10 rats were used as the controls group.After 12 weeks of experiment,all rats were sacrificed,and liver index was calculated according to body weight and wet weight of liver.Enzyme-linked immunosorbent assay(ELISA)was used to detect liver function,blood lipid.Biochemical method was used to detect oxidative stress index.Hematoxylin-eosin(HE)staining was used to observe the pathological changes of liver tissue.The reduced coenzymeⅡ(NADPH)oxidase 4(NOX4)mRNA,transforming growth factor-β1(TGF-β1)mRNA,and connective tissue growth factor(CTGF)mRNA expression in the liver tissue were detected by real-time fluorescent quantitative polymerase chain reaction(RT-PCR),while the level of NOX4 protein in the liver tissue of each group was detected using Western Blotting.Results The liver function of the model group and the empty vector group were weakened,the liver cell lesions,the levels of superoxide dismutase(SOD),and the glutathione(GSH)in the liver tissue were lower than those in the control group,while the levels of malondialdehyde(MDA),NOX4 mRNA,TGF-β1 mRNA,CTGF mRNA,and protein in the liver tissue were higher than those in the control group,with a statistically significant difference of(P<0.05).The liver function of the over-expressing H19 group was enhanced,and the state of hepatocytes was improved.The levels of SOD and GSH in the liver tissue were higher than those in the model group and the empty vector group,while the levels of MDA,NOX4 mRNA,TGF-β1 mRNA,CTGF mRNA,and NOX4 protein were lower than those in the model group and the empty vector group,with a statistically significant differences of(P<0.05).Conclusion Overexpression of lncRNA H19 can inhibit the expression of NOX4 to resist the expansion of oxidative stress,and protect the liver damage of NAFLD rats induced by high-fat diet.