目的 探究高爾基體應(yīng)激在低氧誘導(dǎo)血管內(nèi)皮細(xì)胞損傷中的作用，并探討消退素E1（RvE1）對(duì)高爾基體應(yīng)激的調(diào)控作用。方法 人臍靜脈內(nèi)皮細(xì)胞（HUVEC）分為20.9% O₂、2% O₂、2% O₂＋si-NC、2% O₂＋si-GRASP65、2% O₂＋RvE1、2% O₂＋RvE1＋OE-GRASP65-NC、2% O₂＋RvE1＋OE-GRASP65組。采用透射電鏡觀察HUVEC高爾基體形態(tài)變化，CCK-8檢測(cè)細(xì)胞活力，流式細(xì)胞術(shù)檢測(cè)活性氧（ROS）水平，ELISA檢測(cè)細(xì)胞培養(yǎng)上清內(nèi)皮素1 （ET-1）、前列環(huán)素-2（PGI-2）、RvE1的變化，實(shí)時(shí)熒光定量PCR（qRT-PCR）及Western blotting檢測(cè)一氧化氮合成酶（eNOS）、高爾基體外周膜蛋白P65抗體（GRASP65）mRNA和蛋白表達(dá)。結(jié)果 與20.9% O₂組比較，低氧可致血管內(nèi)皮細(xì)胞高爾基體破裂甚至重構(gòu)，顯著抑制HUVEC活力（P＜0.01），顯著上調(diào)ROS生成（P＜0.01），顯著抑制eNOS、PGI-2、RvE1表達(dá)（P＜0.01），顯著促進(jìn)ET-1、GRASP65、p-GRASP65表達(dá)（P＜0.01）；與2% O₂組比較，si-GRASP65、RvE1可以改善低氧HUVEC高爾基體應(yīng)激反應(yīng)，顯著增強(qiáng)HUVEC活力（P＜0.01），顯著下調(diào)ROS生成（P＜0.01），顯著增加eNOS、PGI-2表達(dá)（P＜0.01），顯著抑制ET-1、GRASP65、p-GRASP65表達(dá)（P＜0.01）；OE-GRASP65對(duì)RvE1的藥效有明顯的抵消作用。結(jié)論 RvE1具有拮抗內(nèi)皮細(xì)胞低氧損傷作用，其機(jī)制可能與其抑制低氧所致高爾基體應(yīng)激有關(guān)。

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% O₂, 2% O₂+si-NC, 2% O₂+si-GRASP65, 2% O₂+RvE1, 2% O₂+RvE1+OE-GRASP65-NC and 2% O₂+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% O₂ 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% O₂ 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.

R965

國(guó)家自然科學(xué)基金資助項(xiàng)目（81470247）;西藏自治區(qū)科技廳區(qū)域科技協(xié)同創(chuàng)新專(zhuān)項(xiàng)（QYXTZX-RKZ2022-07）