[1]温艳君,张雪蕊,韦严,等.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[J].眼科新进展,2022,42(4):262-266.[doi:10.13389/j.cnki.rao.2022.0053]
　WEN Yanjun,ZHANG Xuerui,WEI Yan,et al.Effects of Ndufa4 mitochondrial complex associated like 2 on 661W cells exposed to high glucose[J].Recent Advances in Ophthalmology,2022,42(4):262-266.[doi:10.13389/j.cnki.rao.2022.0053]
点击复制

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/HTML
分享到：

《眼科新进展》[ISSN:1003-5141/CN:41-1105/R]

卷:
42卷
期数:
2022年4期
页码:
262-266
栏目:
实验研究
出版日期:
2022-04-05

文章信息/Info

Title:
Effects of Ndufa4 mitochondrial complex associated like 2 on 661W cells exposed to high glucose
作者:
温艳君; 张雪蕊; 韦严; 赵培泉
200092　上海市杨浦区，上海交通大学医学院附属新华医院眼科(温艳君，张雪蕊，赵培泉)；200031　上海市徐汇区，复旦大学附属眼耳鼻喉科医院眼科(韦严)
Author(s):
WEN Yanjun1; ZHANG Xuerui1; WEI Yan2; ZHAO Peiquan1
1.Department of Ophthalmology,Xinhua Hospital Affiliated to Medicine School of Shanghai Jiaotong University,Shanghai 200092,China
2.Department of Ophthalmology,EYE&ENT Hospital of Fudan University,Shanghai 200031,China
关键词:
糖尿病视网膜病变; 线粒体; Ndufa4线粒体复合体相关蛋白2; 活性氧
Keywords:
diabetic retinopathy;  mitochondria;  Ndufa4 mitochondrial complex associated like 2;  reactive oxygen species
分类号:
R774.1
DOI:
10.13389/j.cnki.rao.2022.0053
文献标志码:
A
摘要:
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。
Abstract:
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.

参考文献/References:

［1］　FRANK R N.Diabetic retinopathy［J］.N Engl J Med,2004,350(1):48-58.
［2］　ANTONETTI D A,KLEIN R,GARDNER T W.Diabetic retinopathy［J］.N Engl J Med,2012,366(13):1227-1239.
［3］　MADSEN-BOUTERSE S A,MOHAMMAD G,KANWAR M,KOWLURU R A.Role of mitochondrial DNA damage in the development of diabetic retinopathy,and the metabolic memory phenomenon associated with its progression［J］.Antioxid Redox Signal,2010,13(6):797-805.
［4］　TAURINO F,STANCA E,SICULELLA L,TRENTADUE R,PAPA S,ZANOTTI F,et al.Mitochondrial proteome analysis reveals depression of the ndufs3 subunit and activity of complex I in diabetic rat brain［J］.J Proteomics,2012,75(8):2331-2341.
［5］　PESTA D,JELENIK T,ZAHARIA O P,BOBROV P,GRGENS S,BDIS K,et al.Ndufb6 polymorphism is associated with physical activity-mediated metabolic changes in type 2 diabetes［J］.Front Endocrinol (Lausanne),2021,12:693683.
［6］　DURAISAMY A J,MOHAMMAD G,KOWLURU R A.Mitochondrial fusion and maintenance of mitochondrial homeostasis in diabetic retinopathy［J］.Biochim Biophys Acta Mol Basis Dis,2019,1865(6):1617-1626.
［7］　TEWARI S,SANTOS J M,KOWLURU R A.Damaged mitochondrial DNA replication system and the development of diabetic retinopathy［J］.Antioxid Redox Signal,2012,17(3):492-504.
［8］　KANWAR M,CHAN P S,KERN T S,KOWLURU R A.Oxidative damage in the retinal mitochondria of diabetic mice:possible protection by superoxide dismutase［J］.Invest Ophthalmol Vis Sci,2007,48(8):3805-3811.
［9］　VINOGRADOV A D,GRIVENNIKOVA V G.Oxidation of NADH and ROS production by respiratory complex I［J］.Biochim Biophys Acta,2016,1857(7):863-871.
［10］　XU W N,YANG R Z,ZHENG H L,JIANG L S,JIANG S D.Ndufa4l2 regulated by hif-1α promotes metastasis and epithelial-mesenchymal transition of osteosarcoma cells through inhibiting ROS production［J］.Front Cell Dev Biol,2020,8:515051.
［11］　YUAN Y,GAO H,ZHUANG Y,WEI L,YU J,ZHANG Z,et al.Ndufa4l2 promotes trastuzumab resistance in her2-positive breast cancer［J］.Ther Adv Med Oncol,2021,13:177-182.
［12］　CHEN Z,WEI X,WANG X,ZHENG X,CHANG B,SHEN L,et al.Ndufa4l2 promotes glioblastoma progression,is associated with poor survival,and can be effectively targeted by apatinib［J］.Cell Death Dis,2021,12(4):377.
［13］　LIU Y,NIE X,ZHU J,WANG T,LI Y,WANG Q,et al.Ndufa4l2 in smooth muscle promotes vascular remodeling in hypoxic pulmonary arterial hypertension［J］.J Cell Mol Med,2021,25(2):1221-1237.
［14］　ZHENG J,ZHANG M,WENG H.Induction of the mitochondrial ndufa4l2 protein by hif-1a regulates heart regeneration by promoting the survival of cardiac stem cell［J］.Biochem Biophys Res Commun,2018,503(4):2226-2233.

相似文献/References:

[1]杜玮 刘子扬 周艳艳 雒雷鸣.糖尿病视网膜病变与血清胆红素水平的关系[J].眼科新进展,2012,32(5):000.
[2]范松涛 卢建民.阿司匹林与糖尿病患者玻璃体出血以及玻璃体切割术疗效的相关性研究[J].眼科新进展,2012,32(11):000.
[3]李艳 李筱荣 袁佳琴 潘斌.糖尿病大鼠视网膜中VEGF、PEDF的表达与血-视网膜屏障损伤[J].眼科新进展,2013,33(1):000.
[4]李朝晖 崔治华 胡晓英 孟丽珠 张敬维.糖尿病视网膜病变激光面积与疗效的分析[J].眼科新进展,2013,33(2):000.
[5]冯冬梅 朱鸿 施彩虹.CXC趋化因子及其受体在糖尿病视网膜病变中的作用[J].眼科新进展,2013,33(6):000.
[6]牛淑玲.糖尿病视网膜病变患者HbAlc、FPG与血小板参数的变化及危险因素分析[J].眼科新进展,2013,33(7):000.
[7]毕春潮 王睿 王建洲 雷春灵 董晓娟 王小莉 薛晓辉.Ad-PEDF对糖尿病视网膜病变大鼠视网膜新生血管的抑制作用[J].眼科新进展,2013,33(8):000.
[8]杨萍 孙书明 李晓鹏.辛伐他汀对糖尿病视网膜病变和炎症因子的影响[J].眼科新进展,2013,33(8):000.
[9]罗文婷 孙大卫.血管黏附蛋白-1在眼科疾病中的研究进展[J].眼科新进展,2013,33(8):000.
[10]李小璐 马雅玲.糖尿病视网膜病变大鼠视网膜VEGF和PEDF的动态表达[J].眼科新进展,2013,33(9):000.
[11]王紫郦,魏婷婷,谢田华,等.线粒体功能紊乱在糖尿病视网膜病变中作用的研究进展[J].眼科新进展,2022,42(12):999.[doi:10.13389/j.cnki.rao.2022.0204]
　WANG Zili,WEI Tingting,XIE Tianhua,et al.Research progress on the role of mitochondrial dysfunction in diabetic retinopathy[J].Recent Advances in Ophthalmology,2022,42(4):999.[doi:10.13389/j.cnki.rao.2022.0204]
[12]张媛媛,李筱荣,邵彦.糖尿病视网膜病变“代谢记忆”与线粒体功能相关性研究进展[J].眼科新进展,2023,43(12):1005.[doi:10.13389/j.cnki.rao.2023.0200]
　ZHANG Yuanyuan,LI Xiaorong,SHAO Yan.Research progress on the correlation between metabolic memory and mitochondrial function in diabetic retinopathy[J].Recent Advances in Ophthalmology,2023,43(4):1005.[doi:10.13389/j.cnki.rao.2023.0200]

备注/Memo

备注/Memo:
国家自然科学基金项目（编号：81770964,81770933）
更新日期/Last Update: 2022-04-05