基于线粒体ATP合成功能探讨人参皂苷Rb1改善缺氧/复氧诱导的内皮细胞渗漏的作用与机制  

Exploration on the effect and mechanism of ginsenoside Rb1 in ameliorating hypoxia/reoxygenation-induced endothelial cell leakage based on mitochondrial ATP synthesis function

作　　者：王美跃 田贵华[1] 贺珂 苏友祥 龙俊燚 WANG Meiyue;TIAN Guihua;HE Ke;SU Youxiang;LONG Junyi(Dongzhimen Hospital,Beijing University of Chinese Medicine,Beijing 100700,China)

机构地区：[1]北京中医药大学东直门医院,北京100700

出　　处：《中华中医药杂志》2024年第12期6479-6484,共6页China Journal of Traditional Chinese Medicine and Pharmacy

基　　金：国家自然科学基金项目(No.82305024);中央高校基本科研业务费专项资金(No.2021-BUCMXJKY002)。

摘　　要：目的:探讨人参皂苷Rb1对缺氧/复氧(H/R)损伤的人脐静脉内皮细胞(HUVEC)连接屏障是否有保护作用及相关机制。方法:以HUVEC细胞建立连续缺氧2 h/复氧2 h的H/R损伤模型。设置对照组、Rb1本底组、模型组、Rb1干预组(1×10^(-3)、1×10^(-4)、1×10^(-5)mol/L)和Rb1+寡霉素干预组。异硫氰酸荧光素-葡聚糖示踪法检测内皮细胞的通透性,荧光探针以及ATP检测试剂盒评估细胞内ATP水平,免疫荧光和Western Blot检测ZO-1、Occludin和Claudin-5的含量,荧光探针法观察细胞线粒体形态,ELISA测定线粒体呼吸链复合体酶Ⅰ和Ⅴ的活性。结果:与模型组比较,1×10^(-4)mol/L Rb1干预组显著抑制复氧后细胞渗透性增加(P<0.05),显著提高细胞ZO-1、Occludin和Claudin-5蛋白表达(P<0.05),1×10^(-4)mol/L Rb1干预组细胞内ATP水平显著增加(P<0.05),线粒体呼吸链复合物Ⅰ和Ⅴ的活性显著增强(P<0.05),细胞线粒体分裂状态明显减少。与1×10^(-4)mol/L Rb1干预组比较,Rb1+寡霉素干预组内皮细胞的渗透性显著升高(P<0.05),ZO-1、Occludin、Claudin-5蛋白表达均显著降低(P<0.05)。结论:人参皂苷Rb1通过改善H/R后内皮细胞线粒体的结构和功能,增加ATP合成,从而提高内皮细胞连接蛋白表达,对内皮屏障发挥保护作用。Objective:To investigate whether ginsenoside Rb1 protects against hypoxia/reoxygenation(H/R)injury-induced HUVEC barrier dysfunction and its related mechanisms.Methods:HUVEC cells were used to establish a H/R injury model by continuous hypoxia 2 h/reoxygenation 2 h.A control group,a Rb1 background group,a model group,an Rb1 intervention group(1×10^(-3),1×10^(-4),1×10^(-5) mol/L),and an Rb1+oligomycin group were set up.The permeability of the endothelial cell monolayer was assessed using FITC-Dextran tracer method,and the ATP expression level in cells was evaluated using fluorescent probe labeling and ATP assay kit.The expression levels of ZO-1,Occludin,and Claudin-5 were detected by immunofluorescence and Western Blot.The morphology of mitochondria in cells was observed using mito-Tracker Red CMXRos fluorescent probe method,and the activities of mitochondrial respiratory chain complexes I and V were determined by ELISA.Results:Compared with the model group,1×10^(-4) mol/L Rb1 intervention group significantly ameliorated the increased permeability of HUVEC cells after reoxygenation(P<0.05)and increased cellular ZO-1,Occludin and Claudin-5 protein expression(P<0.05),1×10^(-4) mol/L Rb1 intervention group significantly increased intracellular ATP levels(P<0.05),improved the activity of mitochondrial respiratory chain complexes Ⅰ and Ⅴ(P<0.05),and markedly reduced cellular mitochondrial division status.The permeability of the endothelium was elevated in the Rb1+oligomycin group compared with the 1×10^(-4) mol/L Rb1 intervention group(P<0.05),and the expression of ZO-1,Occludin,and Claudin-5 proteins were all reduced(P<0.05).Conclusion:Ginsenoside Rb1 exerts a protective effect on the endothelial barrier by improving the structure and function of mitochondria in endothelial cells after H/R,increasing ATP synthesis,and enhancing the expression of endothelial cell junction proteins.

关 键 词：缺氧/复氧 人参皂苷RB1 线粒体 ATP 内皮细胞 连接蛋白 机制 能量代谢 

分 类 号：R28[医药卫生—中药学]