微小RNA-378调控小鼠急性肝衰竭作用机制的研究

Research on the mechanism underlying the regulation of miR-378 on acute liver failure in mice

目的探究微小RNA-378(miR-378)调控小鼠急性肝衰竭的作用机制。方法将36只雄性巴比塞小鼠以随机数字表法均分为实验组和对照组。实验组腹腔注射D氨基半乳糖(D-GalN)800 mg/kg联合脂多糖10μg/kg诱导急性肝衰竭,对照组为腹腔内注射等量的生理盐水。两组在诱导后0、1、3、5、7、9 h处死小鼠取静脉血和肝组织,每个时间点有3只实验鼠。检测两组在各时间点血清中丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST)水平,酶联免疫吸附测定(ELISA)检测血清中肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)水平,逆转录聚合酶链反应(RT-PCR)测定肝组织中的胱天蛋白酶-3(Caspase-3)、TNF-α及IL-6 mRNA表达量,探针法确定肝组织中miR-378表达量,蛋白质印迹法(Westernblotting)检测Caspase-3蛋白表达量。结果实验组ALT/AST水平逐渐升高且在诱导后7 h达到峰值,对照组ALT/AST未见明显变化。相较对照组,实验组在诱导后1 h和7 h TNF-α/IL-6血清浓度及mRNA水平明显升高;实验组在诱导后5 h[(0.0038±0.0003)比(0.0054±0.0003),P<0.05]和7 h[(0.0014±0.0005)比(0.0054±0.0003),P<0.05]miR-378表达量明显降低,Caspase-3 mRNA[5 h(0.0052±0.0003),7 h(0.0070±0.0006)比(0.0033±0.0001),均P<0.05]和蛋白[5 h(0.867±0.012),7 h(1.062±0.039)比(0.577±0.035),均P<0.05]表达量升高。结论在小鼠D-GalN联合脂多糖诱导的急性肝衰竭模型中,miR-378表达量的下调促进了Caspase-3的活化,增强了肝细胞凋亡活动,进而调控急性肝衰竭的发展。miR-378可作为治疗急性肝衰竭新靶点。

Objective To investigate the effects of microRNA(miR)-378 on mouse acute liver failure.Methods Thirty-six male BALB/c mice were randomly divided into experimental group and control group according to the random number table method.Mice in the experimental were intraperitoneally injected with 800 mg/kg D-galactosamine(D-GalN)and 10μg/kg lipopolysaccharide(LPS),while mice in the control group were injected with equal amount of saline.The mice of the two groups were sacrificed at different time points(0,1,3,5,7,9 h)for blood and liver tissue,with 3 mice at each time point.The levels of serum aminotransferase(ALT&AST)at different time points were measured.The levels of tumor necrosis factor(TNF)-αand interleukin(IL)-6 were measured by ELISA.The expression of Caspase-3,TNF-αand IL-6 mRNA in liver tissue were examined by reverse transcription-polymerase chain reaction(RT-PCR).The expression of miR-378 was measured by TaqMan Universal PCR Master Mix.We determined the expression of Caspase-3 by Western blotting.Results The enzyme levels(ALT/AST)in the experimental group elevated gradually,peaking at 7 h,while ALT/AST levels in the control group were not significantly changed.Compared with those in the control group,the mRNA levels and serum concentrations of TNF-α/IL-6 were higher at 1 h and 7 h in experimental group.In the experimental group,miR-378 was down-regulated at 5 h[(0.0038±0.0003)vs.(0.0054±0.0003)](P<0.05)and 7 h[(0.0014±0.0005)vs.(0.0054±0.0003)](P<0.05),while the mRNA[5 h(0.0052±0.0003),7 h(0.0070±0.0006)vs.(0.0033±0.0001),both P<0.05]and protein[5 h(0.867±0.012),7 h(1.062±0.039)vs.(0.577±0.035),both P<0.05]of Caspase-3 were up-regulated.Conclusions In the mouse ALF model induced by D-GalN/LPS,the down-regulation of miR-378 promotes the activation of Caspase-3,enhances hepatocytes apoptosis in liver.MiR-378 may serve as a potential therapeutic target for the treatment of ALF.