氧化型脂蛋白(a)通过抑制细胞色素b表达促进血管内皮细胞焦亡  

Oxidized lipoprotein(a)induces endothelial cell pyroptosis by inhibiting the expression of cytochrome b

作　　者：曹子彤 陈彦君 谈世铭 饶羽祝 王晶晶 蔡泽民[2] 王佐[1] CAO Zitong;CHEN Yanjun;TAN Shiming;RAO Yuzhu;WANG Jingjing;CAI Zeming;WANG Zuo(Institute of Cardiovascular Disease,University of South China&Key Laboratory of Arterial Hard Chemistry of Hunan Province&Hunan Province Arteriosclerotic Disease International Scientific and Technological Innovation Cooperation Base;The First Affiliated Hospital of University of South China,Hengyang,Hunan 421001,China)

机构地区：[1]南华大学心血管疾病研究所,动脉硬化学湖南省重点实验室,湖南省动脉硬化性疾病国际科技创新合作基地 [2]南华大学附属第一医院,湖南省衡阳市421001

出　　处：《中国动脉硬化杂志》2024年第7期558-566,共9页Chinese Journal of Arteriosclerosis

基　　金：国家自然科学基金项目(81970389);湖南省科技创新计划项目(2021SK51801)。

摘　　要：[目的]探讨氧化型脂蛋白(a)[oxLp(a)]诱发血管内皮细胞焦亡及其机制。[方法]人脐静脉血管内皮细胞(HUVEC)经100 mg/L oxLp(a)孵育24 h后,通过Western blot和RT-qPCR检测焦亡相关蛋白、促炎细胞因子、线粒体相关蛋白核呼吸因子1(NRF1)、核呼吸因子2(NRF2)、过氧化物酶体增殖物激活受体γ共激活因子1α(PGC-1α)及线粒体基因细胞色素b(CYTB)蛋白表达水平,ELISA检测炎症因子水平,扫描电镜检测细胞膜破裂,透射电镜检测线粒体形态,Hoechst33342/PI染色检测细胞凋亡,MitoSOX探针检测线粒体活性氧(mtROS),Flou-4AM探针检测钙离子,JC-1探针检测线粒体膜电位(MMP),Calcein AM染色检测线粒体膜通透性转换孔(mPTP)开放。向HUVEC转染CYTB过表达慢病毒,分析其对oxLp(a)诱发焦亡与线粒体功能的影响。[结果]经oxLp(a)处理后,焦亡相关分子NLRP3、pro-Caspase-1、Caspase-1、消皮素D(GSDMD)、GSDMD-N蛋白水平显著升高(P<0.05);CYTB、促炎细胞因子IL-1β和IL-18蛋白和mRNA水平均显著升高(P<0.05);细胞膜上出现细小裂孔,PI染色阳性细胞百分比显著增加(P<0.05)。oxLp(a)抑制线粒体相关蛋白NRF1、NRF2、PGC-1α的表达及线粒体基因CYTB的表达,促使mtROS生成增加、钙离子超载、ATP水平下降、MMP下降、mPTP值升高、线粒体形态异常。pHelper 2.0慢病毒载体转染过表达CYTB后,oxLp(a)诱发HUVEC焦亡与线粒体形态功能异常被过表达CYTB部分逆转。[结论]oxLp(a)通过下调CYTB促进线粒体形态功能异常而诱发HUVEC焦亡。Aim To explore the mechanism of oxidized lipoprotein(a)(oxLp(a))inducing pyroptosis of vascular endothelial cells.Methods After incubating human umbilical vein endothelial cells(HUVEC)with 100 mg/L ox-Lp(a)for 24 hours,Western blot and RT-qPCR was used to detect pyroptosis related proteins,pro-inflammatory cytokines,mitochondrial related proteins NRF1,NRF2,PGC-1αand mitochondrial gene cytochrome b(CYTB),ELISA was used to detect the levels of inflammatory factors,scanning electron microscopy was used to detect cell membrane rupture,transmission electron microscopy was used to detect mitochondrial morphology,Hoechst33342/PI staining was used to detect cell apoptosis,MitoSOX probe was used to detect mitochondrial reactive oxygen species(mtROS),Flu-4AM probe was used to detect calcium ions,JC-1 probe was used to detect mitochondrial membrane potential(MMP),and Calcein AM staining was used to detect mitochondrial permeability transition pore(mPTP).Transfecting HUVEC with CYTB overexpressing lentivirus and analyzing its effects on oxLp(a)induced pyroptosis and mitochondrial function.Results After treatment with oxLp(a),the expression of NLRP3,pro-Caspase-1,Caspase-1,GSDMD and GSDMD-N proteins related to pyroptosis were significantly increased(P<0.05);the protein and mRNA levels of CYTB and pro-inflammatory cytokine IL-1β,IL-18 were significantly increased(P<0.05).Small pores appeared on the cell membrane,the percentage of PI stained positive cells significantly increased(P<0.05).OxLp(a)significantly inhibited the expression of mitochondrial related proteins NRF1,NRF2 and PGC-1α,and the expression of mitochondrial gene CYTB,promoted an increase in mtROS generation,Ca2+overload,a decrease in ATP levels,a decrease in MMP,an increase in mPTP values,and abnormal mitochondrial morphology.After transfection with pHelper 2.0 lentivirus vector overexpressing CYTB,it was found that oxLp(a)induced HUVEC pyroptosis and mitochondrial morphological and functional abnormalities were partially reversed by overexpression of CYTB.Con

关 键 词：细胞焦亡 氧化型脂蛋白(a) 线粒体功能 血管内皮细胞 细胞色素B 

分 类 号：R363[医药卫生—病理学] R5[医药卫生—基础医学]