消退素E1调控高尔基体应激抑制低氧内皮细胞损伤的作用机制

Mechanism of RvE1 inhibiting hypoxic endothelial cell injury by regulating Golgi stress

目的探究高尔基体应激在低氧诱导血管内皮细胞损伤中的作用,并探讨消退素E1(RvE1)对高尔基体应激的调控作用。方法人脐静脉内皮细胞(HUVEC)分为20.9%O_(2)、2%O_(2)、2%O_(2)+si-NC、2%O_(2)+si-GRASP65、2%O_(2)+RvE1、2%O_(2)+RvE1+OE-GRASP65-NC、2%O_(2)+RvE1+OE-GRASP65组。采用透射电镜观察HUVEC高尔基体形态变化,CCK-8检测细胞活力,流式细胞术检测活性氧(ROS)水平,ELISA检测细胞培养上清内皮素1(ET-1)、前列环素-2(PGI-2)、RvE1的变化,实时荧光定量PCR(qRT-PCR)及Western blotting检测一氧化氮合成酶(eNOS)、高尔基体外周膜蛋白P65抗体(GRASP65)mRNA和蛋白表达。结果与20.9%O_(2)组比较,低氧可致血管内皮细胞高尔基体破裂甚至重构,显著抑制HUVEC活力(P<0.01),显著上调ROS生成(P<0.01),显著抑制eNOS、PGI-2、RvE1表达(P<0.01),显著促进ET-1、GRASP65、p-GRASP65表达(P<0.01);与2%O_(2)组比较,si-GRASP65、RvE1可以改善低氧HUVEC高尔基体应激反应,显著增强HUVEC活力(P<0.01),显著下调ROS生成(P<0.01),显著增加eNOS、PGI-2表达(P<0.01),显著抑制ET-1、GRASP65、p-GRASP65表达(P<0.01);OE-GRASP65对RvE1的药效有明显的抵消作用。结论RvE1具有拮抗内皮细胞低氧损伤作用,其机制可能与其抑制低氧所致高尔基体应激有关。

Objective To explore the role of Golgi stress in the injury of hypoxic vascular endothelial cells,and to explore the regulatory effect of fading hormone E1(RvE1)on Golgi stress.Methods Human umbilical vein endothelial cells were divided into 20.9%O_(2),2%O_(2),2%O_(2)+si-NC,2%O_(2)+si-GRASP65,2%O_(2)+RvE1,2%O_(2)+RvE1+OE-GRASP65-NC and 2%O_(2)+RvE1+OEGRASP65 groups.The morphological changes of HUVEC Golgi apparatus were observed by transmission electron microscopy,cell viability was detected by CCK-8,reactive oxygen species(ROS)levels were detected by flow cytometry,and the changes of endothelin-1(ET-1),prostacyclin-2(PGI-2)and RvE1 were detected by ELISA.Real-time quantitative fluorescent PCR(qRT-PCR)and Western blotting were used to detect the mRNA and protein expression of nitric oxide synthetase(eNOS)and Golgi in vitro peripheral membrane protein P65 antibody(GRASP65).Results Compared with 20.9%O_(2)group,hypoxia could cause rupture or even remodeling of the Golgi apparatus of vascular endothelial cells,significantly inhibit HUVEC activity(P<0.01),significantly up-regulate ROS production(P<0.01),and significantly inhibit the expressions of eNOS,PGI-2 and RvE1(P<0.01).Significantly promoted the expression of ET-1,GRASP65,p-GRASP65(P<0.01);Compared with 2%O_(2)group,si-GRASP65 and RvE1 could improve the stress response of hypoxic HUVEC Golgi apparatus,significantly enhance HUVEC activity(P<0.01),significantly down-regulate ROS production(P<0.01),and significantly increase the expressions of eNOS and PGI-2(P<0.01).The expression of ET-1,GRASP65 and p-GRASP65 was significantly inhibited(P<0.01).OE-GRASP65 has a significant cancelling effect on the efficacy of RvE1.Conclusions RvE1 can antagonize hypoxic injury of endothelial cells possibly by inhibiting hypoxia-induced Golgi stress.