CHIP参与高糖介导下的血管内皮细胞损伤  

CHIP participates in high glucose-induced vascular endothelial cell injury

作　　者：乔先栋 刘婷[3] 洪丽英 付勇南 QIAO Xian-dong;LIU ting;HONG Li-ying;FU Yong-nan(Nanchang University,The First Affiliated Hospital of Nan-chang University,Nanchang 330006,China;Hypertension Institute of Jiangxi,The First Affiliated Hospital of Nan-chang University,Nanchang 330006,China;Department of Cardiology,The First Affiliated Hospital of Nan-chang University,Nanchang 330006,China)

机构地区：[1]南昌大学,江西南昌330006 [2]江西省高血压研究所,江西南昌330006 [3]南昌大学第一附属医院心内科,江西南昌330006

出　　处：《中国病理生理杂志》2020年第8期1368-1374,共7页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.81260049,No.81860086)。

摘　　要：目的:探讨热休克蛋白70羧基末端相互作用蛋白(CHIP)在高糖(HG)介导的血管内皮细胞损伤中的作用。方法:培养原代人脐静脉内皮细胞(HUVECs),利用RNA干扰技术下调CHIP表达,再以5.5 mmol/L正常浓度葡萄糖(NG)或25.5 mmol/L高浓度葡萄糖(HG)处理24 h,实验前用甘露醇排除渗透压对实验的干扰,接着将细胞分成NG+siRNA NC组、NG+siRNA CHIP组、HG+siRNA NC组和HG+siRNA CHIP组。MTT法和TUNEL染色法分别检测细胞活力和凋亡率;ELISA试剂盒检测内皮素1(ET-1)的表达水平;二氢乙啶荧光染料法检测细胞内活性氧簇(ROS)水平;一氧化氮(NO)、超氧化物岐化酶(SOD)及诱导型一氧化氮合酶(iNOS)试剂盒分别检测细胞内NO水平及SOD、iNOS活性;RT-qPCR检测白细胞介素8(IL-8)和单核细胞趋化蛋白1(MCP-1)的mRNA表达量;Western blot检测CHIP、NADPH氧化酶(NOX)2、NOX4、p38、p65、p-p38、p-p65、Bax和Bcl-2蛋白表达水平。结果:与NG+siRNA NC组相比,HG+siRNA NC组HUVECs死亡率、凋亡率及IL-8和MCP-1 mRNA表达水平均升高(P<0.05),细胞内ROS水平升高(P<0.05),SOD活性下降(P<0.05),ET-1、NO水平及iNOS活性升高(P<0.05),pp38、p-p65和Bax蛋白水平升高(P<0.05)。与HG+siRNA NC组相比,HG+siRNA CHIP组HUVECs炎症反应和氧化应激反应明显增强,凋亡率显著升高(P<0.05),p-p38和p-p65蛋白水平升高(P<0.05)。结论:下调CHIP表达可加重HG介导的血管内皮细胞损伤。AIM:To investigate the effects of carboxy terminus of heat shock protein 70-interacting protein(CHIP)on high glucose(HG)-induced vascular endothelial cell injury.METHODS:Human umbilical vein endothelial cells(HUVECs)were cultured in vitro and treated with 5. 5 mmol/L glucose(normal glucose,NG)or 25. 5 mmol/L glucose(HG)for 24 h. Down-regulation of CHIP expression by RNA interference was conducted. Before the experiment,mannitol was used to eliminate the interference of osmotic pressure. Subsequently,the cells was divided into 4 groups:NG+siRNA NC group,NG+siRNA CHIP group,HG+siRNA NC group,and HG+siRNA CHIP group. Additionally,MTT assay and TUNEL staining were used to detect the viability and apoptosis. The level of endothelin-1(ET-1)was measured by ELISA,and the level of reactive oxygen species(ROS)was detected by fluorescence probe dihydroethidium. The level of nitric oxide(NO),and the activity of superoxide dismutase(SOD)and inducible nitric oxide synthase(iNOS)in the cells were detected by their respective kits. The mRNA expression of interleukin-8(IL-8)and monocyte chemotactic protein 1(MCP-1)was detected by RT-qPCR. The protein levels of CHIP,NADPH oxidase(NOX)2,NOX4,p38,p65,pp38,p-p65,Bax and Bcl-2 were determined by Western blot.RESULTS:Compared with NG+siRNA NC group,the cell viability was decreased,the apoptosis rate,the mRNA expression of IL-8 and MCP-1,and the level of ROS were increased(P<0. 05),the activity of SOD was decreased(P<0. 05),while the levels of ET-1,NO and iNOS and the protein levels of p-p38,p-p65 and Bax were increased in HG+siRNA NC group(P<0. 05). Compared with HG+siRNA NC group,the inflammatory response,the oxidative stress,the apoptosis rate,and the protein levels of p-p38,p-p65 and Bax were significantly increased in HG+siRNA CHIP group(P<0. 05).CONCLUSION:Down-regulation of CHIP expression aggravates HG-induced vascular endothelial cell injury.

关 键 词：糖尿病 热休克蛋白70羧基末端相互作用蛋白 人脐静脉内皮细胞 细胞凋亡 氧化应激 

分 类 号：R329.25[医药卫生—人体解剖和组织胚胎学] R363.2[医药卫生—基础医学]