高糖环境下Ndufa4线粒体复合体相关蛋白2(Ndufa4l2)对小鼠视网膜感光细胞661W的影响

Effects of Ndufa4 mitochondrial complex associated like 2 on 661W cells exposed to high glucose

目的探讨高糖环境下Ndufa4线粒体复合体相关蛋白2(Ndufa4l2)对视网膜感光细胞661W功能的影响及其相关机制。方法体内实验选取C57BL/6J小鼠12只,采用随机数字表法分为糖尿病组和正常组,每组各6只,糖尿病组小鼠采用腹腔注射链脲佐菌素造模,正常组小鼠腹腔注射等体积柠檬酸缓冲液。采用免疫组织化学法检测Ndufa4l2蛋白在正常组和糖尿病组小鼠视网膜中的表达情况。体外实验将661W细胞随机分组为对照组(细胞采用正常培养基培养24 h)、高糖组(细胞采用含葡萄糖浓度为50 mmol·L^(-1)高糖培养基培养24 h)、si-NC组(细胞转染si-NC无序序列后,采用正常培养基培养24 h)、si-NC+HG组(细胞转染si-NC无序序列后,采用含50 mmol·L^(-1)葡萄糖的培养基培养24 h)和si-Ndufa4l2+HG组(细胞转染si-Ndufa4l2特异敲低序列后,采用含50 mmol·L^(-1)葡萄糖的培养基培养24 h);采用qRT-PCR和Western blot检测对照组和高糖组细胞中Ndufa4l2的表达情况。CCK-8法检测si-Ndufa4l2对661W细胞活性的影响,采用活性氧(ROS)试剂盒检测si-Ndufa4l2对661W细胞中ROS水平的影响,采用乳酸检测试剂盒检测si-Ndufa4l2对661W细胞中乳酸水平的影响。结果体内实验结果表明,与正常组小鼠相比,糖尿病组小鼠视网膜中Ndufa4l2蛋白表达降低。体外实验结果显示,对照组和高糖组661W细胞中Ndufa4l2 mRNA相对表达量分别为1.001±0.069和0.356±0.071,Ndufa4l2蛋白相对表达量分别为1.000±0.078和0.795±0.070,与对照组相比,高糖组661W细胞中的Ndufa4l2 mRNA和蛋白达表水平均下降(均为P<0.05)。CCK-8法检测发现,si-Ndufa4l2+HG组661W的细胞活力低于si-NC+HG组(P<0.001)。ROS检测结果表明,si-Ndufa4l2+HG组661W细胞的荧光亮度高于si-NC+HG组。细胞上清乳酸检测结果显示,si-NC组、si-NC+HG组和si-Ndufa4l2+HG组细胞上清中乳酸含量分别为(10.470±0.140)mmol·L^(-1)、(7.480±0.270)mmol·L^(-1)和(6.080±0.240)mmol·L^(-1),si-Ndufa4l2+HG组与si-NC+HG组相比,细胞上清中乳酸含量进一步降低(P<0.001)。结论线粒体Ndufa4l2蛋白在高糖条件下低表达,Ndufa4l2水平下降会通过氧化磷酸化途径损伤细胞的代谢功能从而进一步抑制661W的细胞活力。

Objective To investigate the effects of Ndufa4 mitochondrial complex associated like 2(Ndufa4l2)on the function of the retinal photoreceptor cell line 661W in a high glucose(HG)environment and its related mechanisms.Methods In vivo experiment twelve C57BL/6J mice were randomly divided into the diabetes mellitus(DM)group and normal group,with six in each group.The diabetic mice were intraperitoneally injected with streptozotocin(STZ),and the normal mice were intraperitoneally injected with equivalent citric acid buffer.The protein expression of Ndufa4l2 in retinal tissues was detected by immunohistochemistry.In vitro experiment 661W cells were randomly grouped as follows control group(cells were cultured in normal medium for 24 h),HG group(cells were cultured in 50 mmol·L^(-1) high glucose medium for 24 h),si-NC group(cells were transfected with si-NC first and then cultured in normal medium for 24 h),si-NC+HG group(cells were transfected with si-NC first and then cultured in 50 mmol·L^(-1) high glucose medium for 24 h),and si-Ndufa4l2+HG group(cells were transfected with si-Ndufa4l2 knockdown sequence first and then cultured in 50 mmol·L^(-1) high glucose medium for 24 h).The expression of Ndufa4l2 in the control group and HG group was detected by the quantitative real-time polymerase chain reaction(qRT-PCR)and Western blot.CCK-8 was used to detect the effect of si-Ndufa4l2 on the activity of 661W cells,the reactive oxygen species(ROS)assay kit was used to detect the effect of si-Ndufa4l2 on the level of ROS in 661W cells,and the lactic acid(LA)assay kit was used to detect the effect of si-Ndufa4l2 on the level of LA in 661W cells.Results In vivo experiment results showed that Ndufa4l2 protein expression was reduced in the retinas of diabetic mice compared with normal mice.In vitro experiment results showed that the mRNA levels of Ndufa4l2 in the control group and HG group were 1.001±0.069 and 0.356±0.071,respectively,while the protein levels of Ndufa4l2 were 1.000±0.078 and 0.795±0.070,respectively,and compared with the control group,the mRNA and protein levels of Ndufa4l2 in 661W cells of mice in the HG group were decreased(both P<0.05).CCK-8 assay results showed that cell viability in the si-Ndufa4l2+HG group was lower than that in the si-NC+HG group(P<0.001).ROS assay results showed that the fluorescence intensity in the si-Ndufa4l2+HG group was higher than that in the si-NC+HG group.LA assay results showed that LA content in the supernatant of 661W cells in the si-NC,si-NC+HG,and si-Ndufa4l2+HG groups were(10.470±0.140)mmol·L^(-1),(7.480±0.270)mmol·L^(-1),and(6.080±0.240)mmol·L^(-1),respectively,and compared with the si-NC+HG group,LA content in the si-Ndufa4l2+HG group was decreased(P<0.001).Conclusion Exposed to HG,the protein level of mitochondrial Ndufa4l2 is decreased.Down-regulation of Ndufa4l2 inhibits the viability of 661W cells by affecting their metabolic function through oxidative phosphorylation pathway.