过表达E1A激活基因阻遏子对OxLDL诱导的血管内皮细胞损伤的影响
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摘要
目的:研究过表达E1A激活基因阻遏子(CREG)对氧化低密度脂蛋白(Ox LDL)诱导的血管内皮细胞损伤的影响。方法:血管内皮细胞分为4组,空白对照组不处理; Ox LDL组用100 mg/L Ox LDL处理24 h;另2组分别感染对照逆转录病毒(阴性对照组)和p LNCX-CREG重组逆转录病毒(p LNCX-CREG组),之后均用100 mg/L OxLDL处理24 h。用Real-time PCR和Western blot法检测4组细胞CREG mRNA和蛋白的表达,用硫代巴比妥酸法检测培养液中丙二醛(MDA)含量,黄嘌呤氧化酶法检测培养液中超氧化物歧化酶(SOD)活性,DCFH-DA法检测培养液中活性氧(ROS)水平,流式细胞术检测细胞凋亡,Western blot法检测细胞中活化的Caspase-3(Cleaved Caspase-3)蛋白的表达水平。结果:与空白对照组比较,Ox LDL组细胞中CREG mRNA和蛋白表达水平均降低; p LNCXCREG组细胞中CREG mRNA和蛋白表达水平均较Ox LDL组升高(P <0. 05)。与空白对照组比较,Ox LDL组细胞SOD活性降低,MDA和ROS水平升高,细胞凋亡率和Cleaved Caspase-3蛋白表达水平均升高;与Ox LDL组比较,p LNCX-CREG组细胞氧化损伤指标的变化被逆转,细胞凋亡率及Cleaved Caspase-3蛋白表达水平均降低(P <0. 05)。结论:过表达CREG可以抑制Ox LDL诱导的血管内皮细胞氧化损伤并抑制细胞凋亡。
Aim: To study the effects of cellular repressor of E1 A-stimulated gene( CREG) overexpression on vascular endothelial cell injury induced by Ox LDL. Methods: Vascular endothelial cells were divided into four groups,the blank control group was not treated,the Ox LDL group was treated with 100 mg/L Ox LDL for 24 hours,the other two groups were infected with control retrovirus( negative control group) and p LNCX-CREG retrovirus( p LNCX-CREG group),and then treated with 100 mg/L Ox LDL for 24 hours. The expression of CREG was detected by Real-time PCR and Western blot.The content of malondialdehyde( MDA) in culture fluid was detected by thiobarbituric acid,the activity of superoxide dismutase( SOD) was detected by xanthine oxidase,and the level of reactive oxygen species( ROS) was detected by DCFHDA. The apoptosis was detected by flow cytometry,and the activated Caspase-3( Cleaved Caspase-3) was detected by Western blot. Results: Compared with the blank control group,the expression levels of CREG mRNA and protein in the Ox LDL group were decreased,while those in the p LNCX-CREG group were increased compared with the Ox LDL group( P < 0. 05).Compared with the blank control group,SOD activity decreased,MDA and ROS levels increased,apoptotic rate and Cleaved Caspase-3 protein expression increased in the Ox LDL group. Compared with the Ox LDL group,the changes of oxidative damage indexes in the p LNCX-CREG group were reversed,apoptotic rate and expression of Cleaved Caspase-3 protein were decreased( P < 0. 05). Conclusion: Overexpression of CREG can inhibit oxidative damage and reduce apoptosis of vascular endothelial cells induced by Ox LDL.
Aim: To study the effects of cellular repressor of E1 A-stimulated gene( CREG) overexpression on vascular endothelial cell injury induced by Ox LDL. Methods: Vascular endothelial cells were divided into four groups,the blank control group was not treated,the Ox LDL group was treated with 100 mg/L Ox LDL for 24 hours,the other two groups were infected with control retrovirus( negative control group) and p LNCX-CREG retrovirus( p LNCX-CREG group),and then treated with 100 mg/L Ox LDL for 24 hours. The expression of CREG was detected by Real-time PCR and Western blot.The content of malondialdehyde( MDA) in culture fluid was detected by thiobarbituric acid,the activity of superoxide dismutase( SOD) was detected by xanthine oxidase,and the level of reactive oxygen species( ROS) was detected by DCFHDA. The apoptosis was detected by flow cytometry,and the activated Caspase-3( Cleaved Caspase-3) was detected by Western blot. Results: Compared with the blank control group,the expression levels of CREG mRNA and protein in the Ox LDL group were decreased,while those in the p LNCX-CREG group were increased compared with the Ox LDL group( P < 0. 05).Compared with the blank control group,SOD activity decreased,MDA and ROS levels increased,apoptotic rate and Cleaved Caspase-3 protein expression increased in the Ox LDL group. Compared with the Ox LDL group,the changes of oxidative damage indexes in the p LNCX-CREG group were reversed,apoptotic rate and expression of Cleaved Caspase-3 protein were decreased( P < 0. 05). Conclusion: Overexpression of CREG can inhibit oxidative damage and reduce apoptosis of vascular endothelial cells induced by Ox LDL.
引文
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[2]CHANG MC,LEE JJ,CHEN YJ,et al.Lysophosphatidylcholine induces cytotoxicity/apoptosis and IL-8 production of human endothelial cells:Related mechanisms[J].Oncotarget,2017,8(63):106177
[3]华婷,张妍,王昕,等.E1A激活基因阻遏子抑制细胞凋亡机制的研究进展[J].中国实用眼科杂志,2016,34(3):206
[4]WANG N,HAN YL,TAO J,et al.Overexpression of CREGattenuates atherosclerotic endothelium apoptosis via VEGF/PI3K/AKT pathway[J].Atherosclerosis,2011,218(2):543
[5]刘艳霞,闫承慧,田孝祥,等.CREG基因启动子区甲基化调控在动脉粥样硬化发生中的机制研究[J].中国循环杂志,2016,31(z1):18
[6]王娜,韩雅玲,陶杰,等.p38MAPK介导E1A激活基因阻遏子蛋白调控人血管内皮细胞凋亡[J].中国病理生理杂志,2011,27(7):1329
[7]TSAI KL,HUNG CH,CHAN SH,et al.Baicalein protects against ox LDL-caused oxidative stress and inflammation by modulation of AMPK-alpha[J].Oncotarget,2016,7(45):72458
[8]TENG W,QIU C,HE Z,et al.Linc00152 suppresses apoptosis and promotes migration by sponging miR-4767 in vascular endothelial cells[J].Oncotarget,2017,8(49):85014
[9]TORISU K,SINGH KK,TORISU T,et al.Intact endothelial autophagy is required to maintain vascular lipid homeostasis[J].Aging Cell,2016,15(1):187
[10]HINDER LM,O'BRIEN PD,HAYES JM,et al.Dietary reversal of neuropathy in a murine model of prediabetes and metabolic syndrome[J].Dis Model Mech,2017,10(6):717
[11]LOPES-VIRELLA MF,HUNT KJ,BAKER NL,et al.High levels of AGE-LDL,and of Ig G antibodies reacting with MDA-lysine epitopes expressed by ox LDL and MDA-LDLin circulating immune complexes predict macroalbuminuria in patients with type 2 diabetes[J].J Diabetes Complications,2016,30(4):693
[12]MONDAL NK,ROYCHOUDHURY S,MUKHERJEE S,et al.Increased risk of cardiovascular disease in premenopausal female ragpickers of Eastern India:involvement of inflammation,oxidative stress,and platelet hyperactivity[J].Mol Cell Biochem,2016,419(1/2):193
[13]ZHOU F,LIU D,NING HF,et al.The roles of p62/SQSTM1 on regulation of matrix metalloproteinase-9 gene expression in response to ox LDL in atherosclerosis[J].Biochem Biophys Res Commun,2016,472(3):451
[14]DU L,MA L,QI F,et al.Characterization of a unique pathway for 4-cresol catabolism initiated by phosphorylation in corynebacterium glutamicum[J].J Biol Chem,2016,291(12):6583
[15]LIU J,QI Y,LI S,et al.CREG1 interacts with Sec8 to promote cardiomyogenic differentiation and cell-cell adhesion[J].Stem Cells,2016,34(11):2648
[16]KURTZ A,SELTMANN S,BAIROCH A,et al.A standard nomenclature for referencing and authentication of pluripotent stem cells[J].Stem Cell Reports,2018,10(1):1
[17]杨桂棠,韩雅玲,闫承慧.E1A激活基因阻遏子在动脉粥样硬化血管中的表达及其与炎症因子的关系[J].心脏杂志,2016,28(1):7
[18]张晓明,杨继红.Apo E与动脉粥样硬化的关系及Apo E基因敲除小鼠在动脉粥样硬化研究中的应用[J].昆明医学院学报,2012,33(S1):169
[19]QI J,ZHENG JB,AI WT,et al.Felodipine inhibits ox-LDL-induced reactive oxygen species production and inflammation in human umbilical vein endothelial cells[J].Mol Med Rep,2017,16(4):4871
[20]KALEEM S,SIDDIQUI S,SIDDIQUI HH,et al.Eupalitin induces apoptosis in prostate carcinoma cells through ROSgeneration and increase of caspase-3 activity[J].Cell Biol Int,2016,40(2):196
[21]SHIM JH,GIM H,LEE S,et al.Inductions of Caspase-,MAPK-and ROS-dependent apoptosis and chemotherapeutic effects caused by an ethanol extract of scutellaria barbata D.Don in human gastric adenocarcinoma cells[J].JPharmacopuncture,2016,19(2):129
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