作者：史喜德，陈江炜，张旭涛，李亚娟，李飞，刘峰舟

单位：

1.710032陕西省西安市，空军军医大学第一附属医院心血管内科 2.710032陕西省西安市，空军军医大学第三附属医院医疗康复科 3.710032陕西省西安市，空军军医大学航空航天医学系航空航天临床医学中心 通信作者：李飞，E-mail：lifei01@fmmu.edu.cn 刘峰舟，E-mail：liufengzhou1986@163.com

Units：

1.Department of Cardiology, First Affiliated Hospital, Air Force Medical University, Xi'an 710032, China 2.Department of Medical Rehabilitation, Third Affiliated Hospital, Air Force Medical University, Xi'an 710032, China 3.Center of Clinical Aerospace Medicine, Aerospace Medicine School, Air Force Medical University, Xi'an 710032, China Corresponding author: LI Fei, E-mail: lifei01@fmmu.edu.cn; LIU Fengzhou, E-mail: liufengzhou1986@163.com

关键词：

缺血再灌注损伤; MICU1; 缺氧/复氧; 内皮细胞; 炎症反应; 凋亡;

Keywords：

Ischemia-reperfusion injury；MICU1; Hypoxia/reoxygenation; Endothelial cells; Inflammatory response;Apoptosis

CLC：

DOI：

10.12114/j.issn.1008-5971.2022.00.328

Funds：

国家自然科学基金资助项目（82100310，81870284，81900242）；陕西省重点研发计划一般项目（2021SF-146，2021SF-253）

摘要：

目的 探讨调控线粒体钙离子摄入蛋白1(MICU1)表达对血管内皮细胞缺氧/复氧（H/R）损伤的影响及机制。方法 本研究时间为2021年12月至2022年7月。将人脐静脉内皮细胞（HUVECs）分成对照组和模型组，对照组细胞正常培养，不加任何干预；模型组细胞构建H/R损伤模型，检测各组MICU1 mRNA、蛋白。将HUVECs分成对照组、siCtrl+模型组及siMICU1+模型组，对照组细胞正常培养，不加任何干预；siCtrl+模型组细胞转染对照小干扰RNA后构建H/R损伤模型；siMICU1+模型组细胞转染MICU1小干扰RNA后构建H/R损伤模型。检测各组MICU1蛋白、活性氧（ROS）、NF-κB p65蛋白、炎性因子[肿瘤坏死因子α(TNF-α)和白介素6(IL-6)]。将HUVECs分成对照组、Ad-Ctrl+模型组及Ad-MICU1+模型组，对照组细胞正常培养，不加任何干预；Ad-Ctrl+模型组细胞感染对照腺病毒后构建H/R损伤模型；Ad-MICU1+模型组细胞感染MICU1腺病毒后构建H/R损伤模型，检测各组MICU1蛋白、ROS、NF-κB p65蛋白、炎性因子（TNF-α和IL-6）及细胞凋亡率。将HUVECs分成siCtrl+模型组、siMICU1+模型组及siMICU1+N,N’-二甲基硫脲（DMTU）+模型组，siCtrl+模型组细胞转染对照小干扰RNA后构建H/R损伤模型；siMICU1+模型组细胞转染MICU1小干扰RNA后构建H/R损伤模型；siMICU1+DMTU+模型组细胞转染MICU1小干扰RNA后加入ROS清除剂DMTU，并构建H/R损伤模型。检测各组NF-κB p65蛋白、炎性因子（TNF-α和IL-6）及细胞凋亡率。结果 模型组MICU1 mRNA、蛋白低于对照组（P<0.05）。siCtrl+模型组MICU1蛋白低于对照组，ROS、NF-κB p65蛋白、TNF-α、IL-6高于对照组（P<0.05）;siMICU1+模型组MICU1蛋白低于siCtrl+模型组，ROS、NF-κB p65蛋白、TNF-α、IL-6高于siCtrl+模型组（P<0.05）。Ad-Ctrl+模型组MICU1蛋白低于对照组，ROS、NF-κB p65蛋白、TNF-α、IL-6、细胞凋亡率高于对照组（P<0.05）;Ad-MICU1+模型组MICU1蛋白高于Ad-Ctrl+模型组，ROS、NF-κB p65蛋白、TNF-α、IL-6、细胞凋亡率低于Ad-Ctrl+模型组（P<0.05）。siMICU1+模型组NF-κB p65蛋白、TNF-α、IL-6、细胞凋亡率高于siCtrl+模型组，siMICU1+DMTU+模型组NF-κB p65蛋白、TNF-α、IL-6、细胞凋亡率低于siMICU1+模型组（P<0.05）。结论 下调MICU1表达可诱导内皮细胞H/R损伤模型发生炎症反应及细胞凋亡，而上调MICU1表达的作用相反，分析其机制可能与调控MICU1表达可影响依赖ROS的NF-κB通路激活有关。

Abstract：

【Abstract】　Objective To investigate the effect and possible mechanism of regulation of mitochondrial calcium uptake 1 (MICU1) expression on hypoxia/reoxygenation (H/R) injury in vascular endothelial cell. Methods The study was conducted from December 2021 to July 2022. Human umbilical vein endothelial cells (HUVECs) were divided into control group and model group. Cells in the control group were cultured normally without any intervention; cells in the model group were used to construct the H/R injury model, and the MICU1 mRNA and MICU1 protein in each group were detected. HUVECs were divided into control group, siCtrl+model group and siMICU1+model group. Cells in the control group were cultured normally without any intervention; cells in the siCtrl+model group were transfected with control small interfering RNA after construction of the H/R injury model; cells in the siMICU1+model group were transfected with MICU1 small interfering RNA after construction of the H/R injury model. The MICU1 protein, reactive oxygen species (ROS) , NF-κB p65 protein, inflammatory factors [tumor necrosis factor α (TNF-α) , interleukin-6 (IL-6) ] in each group were detected. HUVECs were divided into control group, Ad-Ctrl+model group and Ad-MICU1+model group. Cells in the control group were cultured normally without any intervention; cells in Ad-Ctrl+model group were infected with control adenovirus after construction of the H/R injury model; cells in Ad-MICU1+model group were infected with MICU1 adenovirus after construction of the H/R injury model. The MICU1 protein, ROS, NF-κB p65 protein, inflammatory factors (TNF-α, IL-6) and apoptosis rate in each group were detected. HUVECs were divided into siCtrl+model group, siMICU1+model group and siMICU1+N, N'-dimethylthiourea (DMTU) +model group. Cells in the siCtrl+model group were transfected with control small interfering RNA after construction of the H/R injury model; cells in siMICU1+model group were transfected with MICU1-small interfering RNA after construction of the H/R injury model; cells in siMICU1+DMTU+model group were transfected with MICU1-small interfering RNA and then added with ROS scavenger DMTU after construction of the H/R injury model. The NF- κB p65 protein, inflammatory factors (TNF-α, IL-6) and apoptosis rate in each group were detected. Results MICU1 mRNA and protein in model group were lower than those in control group ( P < 0.05) . The MICU1 protein in siCtrl+model group was lower than that in control group, ROS, NF-κB p65 protein, TNF-α, IL-6 were higher than those in control group (P < 0.05) . The MICU1 protein in siMICU1+model group was lower than that in siCtrl+model group, ROS, NF- κB p65 protein, TNF-α, IL-6 were higher than those in siCtrl+model group (P < 0.05) . The MICU1 protein in Ad-Ctrl+model group was lower than that in control group, ROS, NF-κB p65 protein, TNF-α, IL-6 and apoptosis rate were higher than those in control group (P < 0.05) . The MICU1 protein in Ad-MICU1+model group was higher than that in Ad-Ctrl+model group, and ROS, NF-κB p65 protein, TNF-α, IL-6 and apoptosis rate were lower than those in Ad-Ctrl+model group (P < 0.05) . The NF-κB p65 protein, TNF-α, IL-6 and apoptosis rate in siMICU1+model group were higher than those in siCtrl+model group, and NF- κB p65 protein, TNF-α, IL-6 and apoptosis rate in siMICU1+DMTU+model group were lower than those in siMICU1+model group (P < 0.05) . Conclusion In H/R endothelial cell injury model, downregulation of MICU1 expression can induce inflammation and apoptosis, while upregulation of MICU1 expression has the opposite effect. The mechanism may be related to that regulation of MICU1 expression can affect the activation of ROS dependent NF- κB pathway.

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