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

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±0.05比0.98±0.05),差异无统计学意义(P>0.05);ALCAT1组的LC3-Ⅱ、Beclin1表达水平均高于脂肪肝细胞模型组和空白对照组(0.95±0.04比0.42±0.01和0.28±0.02、2.07±0.06比0.93±0.05和0.98±0.05),差异均有统计学意义(t=13.30、15.63、14.05、13.02,P均<0.05)。脂肪肝细胞模型组和ALCAT1干预组的mTOR表达水平均低于空白对照组(1.44±0.02和0.74±0.01比1.93±0.10),ALCAT1干预组mTOR的表达水平低于脂肪肝细胞模型组(0.74±0.01比1.44±0.02),差异均有统计学意义(t=4.83、12.04、32.14,P均<0.05);脂肪肝细胞模型组和ALCAT1干预组的磷酸化AKT的表达水平均低于空白对照组(0.14±0.01和0.07±0.01比0.28±0.01),ALCAT1干预组磷酸化AKT的表达水平低于脂肪肝细胞模型组(0.07±0.01比0.14±0.01),差异均有统计学意义(t=8.59、14.10、5.96,P均<0.05)。ELISA检测结果显示,脂肪肝细胞模型组和ALCAT1干预组的活性氧、丙二醛、4-HNE、IL-6和TNF-α的表达水平均高于空白对照组[(11.44±0.30)和(5.84±0.36)g/L比(1.72±0.38)g/L、(19.94±2.47)和(11.95±1.55)μmol/L比(1.47±0.18)μmol/L、(5.00±0.43)和(2.99±0.37)ng/L比(1.46±0.23)ng/L、(203.40±5.16)和(92.07±11.98)ng/L比(23.32±3.33)ng/L、(123.70±8.38)和(67.42±4.88)ng/L比(47.18±4.57)ng/L],差异均有统计学意义(t=19.86、7.86、7.45、6.74、7.22、3.49、29.34、5.53、8.02、3.03,P均<0.05)。ALCAT1干预组的活性氧、4-HNE、IL-6和TNF-α的表达水均低于脂肪肝细胞模型组[(5.84±0.36)g/L比(11.44±0.30)g/L、(2.99±0.37)ng/L比(5.00±0.43)ng/L、(92.07±11.98)ng/L比(203.40±5.16)ng/L、(67.42±4.88)ng/L比(123.70±8.38)ng/L],差异均有统计学意义(t=11.99、3.51、8.54、5.81,P均<0.05);ALCAT1干预组丙二醛的表达水平与脂肪肝细胞模型组比较[(11.95±1.55)μmol/L比(19.94±2.47)μmol/L],差异无统计学意义(P>0.05)。结论ALCAT1在脂肪肝细胞模型肝细胞中表达上调,敲减ALCAT1可抑制mTOR通路相关蛋白的表达,激活自噬,减轻肝细胞脂肪变、氧化应激和炎症反应。

Objective 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 7.67±0.33 vs.4.33±0.33),the quantity of lipid droplets deposition of ALCAT1 interfering group was lower than that of fatty liver cell model group(7.67±0.33 vs.17.67±3.52),and the differences were statistically significant(t=3.76,7.07 and 2.82,all P<0.05).The degree of mitochondria swelling of fatty liver cell model group was higher than that of blank control group and the degree of mitochondria swelling of ALCAT1 interfering group was lower than that of fatty liver cell model group.The results of Western blotting showed that the expression level of LC3-Ⅱof the fatty liver cell model group was higher than that of the blank control group(0.43±0.01 vs.0.28±0.02),and the difference was statistically significant(t=7.32,P<0.05).However there was no significant difference in the expression level of Beclin1 between fatty live cell model group and blank control group(0.93±0.05 vs.0.98±0.05,P>0.05).The expression levels of LC3-Ⅱand Beclin1 of the ALCAT1 interfering group were both higher than those of the fatty liver cell model group and blank control group(0.95±0.04 vs.0.42±0.01 and 0.28±0.02,2.07±0.06 vs.0.93±0.05 and 0.98±0.05),and the differences were statistically significant(t=13.30,15.63,14.05 and 13.02,all P<0.05).The expression levels of mTOR of the fatty liver cell model group and ALCAT1 interfering group were both lower than that of the blank control group(1.44±0.02 and 0.74±0.01 vs.1.93±0.10),the expression level of mTOR of the ALCAT1 interfering group was lower than that of the fatty liver cell model group(0.74±0.01 vs.1.44±0.02),and the differences were statistically significant(t=4.83,12.04 and 32.14,all P<0.05).The expression levels of phosphorylated AKT of the fatty liver cell model group and ALCAT1 interfering group were both lower than that of the blank control group(0.14±0.01 and 0.07±0.01 vs.0.28±0.01),while the expression level of phosphorylated AKT of the ALCAT1 interfering group was lower than that of the fatty liver cell model group(0.07±0.01 vs.0.14±0.01),and the differences were statistically significant(t=8.59,14.10 and 5.96,all P<0.05).The results of ELISA indicated that the expression levels of ROS,malondialdehyde,4-HNE,IL-6 and TNF-αof the fatty liver cell model group and the ALCAT1 interfering group were all higher than those of the blank control group((11.44±0.30)and(5.84±0.36)g/L vs.(1.72±0.38)g/L;(19.94±2.47)and(11.95±1.55)μmol/L vs.(1.47±0.18)μmol/L;(5.00±0.43)and(2.99±0.37)ng/L vs.(1.46±0.23)ng/L;(203.40±5.16)and(92.07±11.98)ng/L vs.(23.32±3.33)ng/L;(123.70±8.38)and(67.42±4.88)ng/L vs.(47.18±4.57)ng/L),and the differences were all statistically significant(t=19.86,7.86,7.45,6.74,7.22,3.49,29.34,5.53,8.02 and 3.03,all P<0.05).While the expression levels of ROS,4-HNE,IL-6 and TNF-αof the ALCAT1 interfering group were all lower than those of the fatty liver cell model group((5.84±0.36)g/L vs.(11.44±0.30)g/L,(2.99±0.37)ng/L vs.(5.00±0.43)ng/L,(92.07±11.98)ng/L vs.(203.40±5.16)ng/L and(67.42±4.88)ng/L vs.(123.70±8.38)ng/L),and all the differences were statistically significant(t=11.99,3.51,8.54 and 5.81,all P<0.05).There was no statistically significant difference in the expression of malondialdehyde between ALCAT1 interfering group and fatty liver cell model group((11.95±1.55)μmol/L vs.(19.94±2.47)μmol/L,P>0.05).Conclusions The expression of ALCAT1 is up-regulated in fatty liver cell model.Knockdown of ALCAT1 can inhibit the expression of mTOR pathway proteins,activate autophagy,alleviate hepatocyte steatosis,oxidative stress and inflammatory response.