敲减心磷脂酰基转移酶1对肝细胞脂肪变和氧化应激的影响及其机制  

Effect and mechanism of Acyl-CoA:lysocardiolipin acyltransferase 1 knockdown on hepatocyte steatosis and oxidative stress

作　　者：胡晓娜 董方元 姜鑫[2] 纪雪莹 陈洁 于晓峰 保志军 Hu Xiaona;Dong Fangyuan;Jiang Xin;Ji Xueying;Chen Jie;Yu Xiaofeng;Bao Zhijun(Department of Gastroenterology,Huadong Hospital Affiliated to Fudan University,Shanghai 200040,China;Department of Geriatrics,Huadong Hospital Affiliated to Fudan University,Shanghai 200040,China)

机构地区：[1]复旦大学附属华东医院消化内科,上海200040 [2]复旦大学附属华东医院老年科,上海200040

出　　处：《中华消化杂志》2020年第8期546-554,共9页Chinese Journal of Digestion

基　　金：国家重点研发计划(2018YFC2002000);国家自然科学基金(81701374);上海市卫生系统优秀青年人才培养计划(2018YQ58)。

摘　　要：目的探讨心磷脂酰基转移酶1(ALCAT1)对脂肪肝细胞模型中肝细胞脂肪变和氧化应激的影响及其作用机制。方法以游离脂肪酸(FFA;亚油酸和棕榈酸混合物)诱导建立脂肪肝细胞模型,采用实时荧光定量PCR和蛋白质印迹法检测ALCAT1在脂肪肝细胞模型中的表达情况。构建空载小干扰RNA(siRNA)质粒和ALCAT1 siRNA质粒。以空载siRNA质粒转染人正常肝细胞(L-02细胞)24 h,然后与FFA共培养24 h,设立脂肪肝细胞模型组;以ALCAT1 siRNA质粒转染L-02细胞24 h,然后与FFA共培养24 h,设立ALCAT1干预组;以常规培养基培养的L-02细胞作为空白对照组。在透射电子显微镜下观察各组的脂滴沉积、线粒体形态,采用蛋白质印迹法测定自噬体标志物微管相关蛋白1轻链3-Ⅱ(LC3-Ⅱ)、酵母ATG6同源物(Beclin1)和哺乳动物雷帕霉素靶蛋白(mTOR)信号通路关键蛋白mTOR和磷酸化丝氨酸/苏氨酸激酶(AKT)的表达水平,采用ELISA法测定氧化应激产物丙二醛、4-羟基壬烯醛(4-HNE)、活性氧和炎症因子IL-6、TNF-α的表达。采用独立样本t检验进行统计学分析。结果脂肪肝细胞模型中ALCAT1的mRNA和蛋白质的表达水平均高于阴性对照组(9.26±0.83比1.02±0.12、0.35±0.02比0.17±0.01),差异均有统计学意义(t=9.82、6.83,P均<0.05)。透射电子显微镜结果显示,脂肪肝细胞模型组和ALCAT1干预组的脂滴沉积量均高于空白对照组(17.67±3.52和7.67±0.33比4.33±0.33),ALCAT1干预组脂滴沉积量低于脂肪肝细胞模型组(7.67±0.33比17.67±3.52),差异均有统计学意义(t=3.76、7.07、2.82,P均<0.05);脂肪肝细胞模型组线粒体肿胀程度高于空白对照组,而ALCAT1干预组线粒体肿胀程度低于脂肪肝细胞模型组。蛋白质印迹法结果显示,脂肪肝细胞模型组的LC3-Ⅱ表达水平高于空白对照组(0.43±0.01比0.28±0.02),差异有统计学意义(t=7.32,P<0.05);脂肪肝细胞模型组的Beclin1表达水平与空白对照组比较(0.93±0.05Objective To investigate the effect and mechanism of Acyl-CoA:lysocardiolipin acyltransferase 1(ALCAT1)on hepatocyte steatosis and oxidative stress in fatty liver cell model.Methods A fatty liver cell model was established and induced by free fatty acids(FFA).The expression of ALCAT1 in fatty liver cell model was detected by quantitative real-time polymerase chain reaction(qRT-PCR)and Western blotting.The empty siRNA plasmid and ALCAT1 siRNA plasmid were constructed.For the fatty liver cell model group,human normal hepatocytes(L-02 cells)were transfected with empty siRNA plasmid for 24 hours,and then cultured with FFA for 24 hours.For the ALCAT1 interfering group,L-02 cells were transfected with ALCAT1 siRNA plasmid for 24 hours,and then cultured with FFA for 24 hours.And L-02 cells cultured in common medium were used as as blank control group.Lipid droplet deposition and mitochondrial morphology were observed under transmission electron microscopy.The expression levels of autophagy-associated proteins(microtubule-associated protein 1 light chain 3(LC3)-Ⅱand Beclin1)and key proteins of autophagy signal pathway(mammalian target of rapamycin(mTOR)and serine/threonine kinase(AKT))were measured by Western blotting.The expression levels of oxidative stress products(malondialdehyde,4-hydroxynonenal(4-HNE)and reactive oxygen species(ROS))and inflammatory factors(interleukin-6(IL-6)and tumor necrosis factor(TNF)-α)were detected by enzyme-linked immunosorbent assay(ELISA)kits.Independent sample t test was used for statistical analysis.Results The mRNA and protein expression levels of ALCAT1 of the fatty liver cell model group were both higher than that of negative control group(9.26±0.83 vs.1.02±0.12,0.35±0.02 vs.0.17±0.01),and the differences were statistically significant(t=9.82 and 6.83,both P<0.05).The results of electron microscopy indicated that the deposition of lipid droplets of the fatty liver cell model group and ALCAT1 interfering group were both higher than that of blank control group(17.67±3.52 and 7.

关 键 词：氧化性应激 心磷脂酰基转移酶1 肝细胞脂肪变 自噬 

分 类 号：R575.5[医药卫生—消化系统]