银杏内酯B通过抑制内质网应激拮抗血管内皮损伤的作用机制  

Mechanism of ginkgolide B antagonizing vascular endothelial injury by inhibiting endoplasmic reticulum stress

作　　者：马长淞 黄帅 瓦庆德 陈伟之 汪洋[1] 令狐熙涛 唐欲博[3] MA Changsong;HUANG Shuai;WA Qingde;CHEN Weizhi;WANG Yang;LINGHU Xitao;TANG Yubo(不详;Department of Orthopedics,the Second Affiliated Hospital of Guangzhou Medical University,Guangzhou 510230,China)

机构地区：[1]广州医科大学附属第二医院骨科,广州510230 [2]遵义医科大学第二附属医院骨外科,贵州遵义563000 [3]中山大学附属第一医院药学部,广州510080

出　　处：《实用医学杂志》2023年第24期3175-3181,共7页The Journal of Practical Medicine

基　　金：广东省自然科学基金面上项目(编号:2021A1515011257);广东省自然科学基金青年提升项目(编号:2023A1515030091);广东省基础与应用基础研究基金-区域联合基金重点项目(编号:2020B1515120094,2021B1515120053)。

摘　　要：目的探究银杏内酯B(ginkgolide B,GB)是否通过拮抗内质网应激(endoplasmic reticulum stress,ERS)发挥其拮抗血管内皮损伤作用及其相关分子机制。方法建立衣霉素(tunicamycin,TM)诱导的人骨髓来源内皮祖细胞(bone marrow derived-endothelial progenitor cells,EPCs)ERS损伤模型,用MTS检测细胞增殖能力;Calcein-AM/EthD-I双染法检测细胞活性状态;Transwell实验检测细胞迁移能力;DCFH-DA染色检测活性氧(reactive oxygen species,ROS)的水平;ELISA法测定NADPH活性;JC-1和DiOC6染色定性和定量检测线粒体膜电位;qRT-PCR检测mRNA的水平;Western Blot检测蛋白表达水平。结果GB浓度依赖性减轻衣霉素对hEPCs造成的ERS内皮损伤(细胞活性、细胞迁移率和血管形成率的降低)(P<0.01);降低ROS及NADPH水平(P<0.01);呈浓度依赖性抑制ERS介导的线粒体膜电位下降(P<0.01);抑制ERS相关蛋白(GRP78、ATF4、CHOP等)表达,并调控细胞凋亡相关蛋白(Bcl-xl、Bax、cleaved caspase-4、cytochrome c)的表达水平,拮抗内质网应激介导的细胞损伤。结论银杏内酯B能通过拮抗内质网应激,发挥血管内皮保护作用,其机制可能与降低细胞内活性氧水平,抑制ERS相关蛋白(CHOP,GRP78、ATF4)表达及调节细胞凋亡蛋白(Bcl-xl、Bax、cleaved caspase-4、cytochrome c)的表达水平有关。Objective To investigate the potential of ginkgolide B(GB)in mitigating vascular endothelial injury by antagonizing endoplasmic reticulum stress(ERS)and elucidate its underlying molecular mechanism.Methods An injury model of human bone marrow-derived endothelial progenitor cells(EPCs)induced by tunica-mycin(TM)was established.Cell proliferation was assessed using MTS assay,while cell viability was determined through Calcein-AM/EthD-I double staining.Transwell assay was employed to evaluate cell migration ability.DCFH-DA staining was utilized to measure intracellular ROS levels,and NADPH activity was quantified via ELISA.JC-1 and DiOC6 staining were performed for qualitative and quantitative assessment of mitochondrial membrane potential respectively.Qrt-pcr analysis was conducted to determine mRNA expression levels,whereas western blot analysis enabled detection of protein expression levels in the cells.Results GB dose-dependently attenuated tunicamycin-induced ERS-mediated endothelial injury in hEPCs,as evidenced by decreased cell viability,impaired cell migration,and angiogenesis inhibition(P<0.01).Furthermore,GB treatment significantly reduced ROS production and NADPH levels within the cells(P<0.01),while also inhibiting ERS-mediated decline in mitochondrial membrane potential concentration-dependently(P<0.01).Additionally,GB inhibited the expression of ERS-related proteins such as GRP78,ATF4,CHOP etc.,regulated apoptosis-related protein Bcl-xl,Bax cleaved caspase-4 cytochrome c;thereby effectively counteracting endoplasmic reticulum stress-induced cellular damage.Conclusions GB exerts a protective effect on vascular endothelium by antagonizing endoplasmic reticulum stress;this mechanism may be attributed to its ability to reduce intracellular reactive oxygen species levels.It also suppresses the expression of ERS-related proteins(CHOP78 and ATF4),and modulates apoptosis-associated proteins(Bcl-xl,Bax,cleaved caspase-4,and cytochrome c).

关 键 词：银杏内酯B 内质网应激 血管内皮损伤 线粒体功能障碍 骨修复 

分 类 号：R966[医药卫生—药理学]