葛根素通过改善线粒体呼吸功能减轻血管内皮细胞氧化损伤  

Puerarin reduces oxidative damage to vascular endothelial cells by improving mitochondrial respiratory function

作　　者：孙姝婵 龚迪菲 袁天翊 王守宝 方莲花 杜冠华 SUN Shu-chan;GONG Di-fei;YUAN Tian-yi;WANG Shou-bao;FANG Lian-hua;DU Guan-hua(Beijing Key Laboratory of Drug Targets Identification and Drug Screening,State Key Laboratory of Bioactive Substance and Function of Natural Medicines,Chinese Academy of Medical Sciences and Peking Union Medical College,Beijing 100050,China)

机构地区：[1]中国医学科学院、北京协和医学院药物研究所,药物靶点研究与新药筛选北京市重点实验室,天然药物活性物质与功能国家重点实验室,北京100050

出　　处：《药学学报》2022年第5期1352-1360,共9页Acta Pharmaceutica Sinica

基　　金：国家自然科学基金资助项目(81773935,82073853);中国医学科学院医学与健康科技创新工程项目(2021-I2M-1-005)。

摘　　要：本研究旨在探究黄酮类化合物葛根素对过氧化氢(H_(2)O_(2))诱导损伤的人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVEC)的影响及其机制。采用体外培养HUVEC细胞,设立空白组、模型组(H_(2)O_(2)400μmol·L^(-1))、不同浓度葛根素组(3、10、30、100μmol·L^(-1))。采用葛根素预孵育2 h,H_(2)O_(2)损伤HUVEC细胞24 h。CCK-8法检测细胞活力,Transwell小室观察细胞迁移能力,以JC-1为荧光探针检测线粒体膜电位。O2k线粒体功能检测系统测定线粒体呼吸功能。RT-PCR实验技术分析细胞中肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素-1β(interleukin-1β,IL^(-1)β)和白细胞介素-18(interleukin-18,IL^(-1)8)的mRNA表达水平;Western blot检测细胞焦亡相关蛋白氮端gasdermin D(N-GSDMD)、裂解天冬氨酸特异蛋白酶-1(cleaved-cysteinyl aspartate specific proteinase-1,cleaved-caspase-1)、核苷酸结合寡聚化结构域样受体蛋白3(NOD-like receptor protein 3,NLRP3)和嘌呤能离子通道型受体7(purinergic ligand-gated ion channel 7 receptor,P2X7R)的表达水平。结果显示,400μmol·L^(-1)H_(2)O_(2)处理24 h对HUVEC细胞有明显的损伤作用,与模型组比较,葛根素能够浓度依赖性地提高细胞活力,其中30和100μmol·L^(-1)效果最为显著;葛根素能够显著降低线粒体膜电位,改善线粒体呼吸功能,抑制H_(2)O_(2)诱导的细胞迁移,降低炎性因子的表达,下调焦亡相关蛋白的表达。上述研究结果表明,葛根素能够抑制HUVEC细胞的迁移,减轻H_(2)O_(2)诱导的HUVEC氧化损伤,其机制可能与改善线粒体呼吸功能、抑制细胞焦亡有关。This study investigated the effect of puerarin on human umbilical vein endothelial cells(HUVEC)injured with hydrogen peroxide(H_(2)O_(2)).HUVEC were divided into three groups:a control group,a model group(H_(2)O_(2)400μmol·L^(-1))and a puerarin-treated group(3,10,30 and 100μmol·L^(-1)).HUVEC were cultured with varied concentration of puerarin for 2 h and treated with H_(2)O_(2)for another 24 h.Cell proliferation was detected by a CCK-8 assay.The mitochondrial membrane potential was measured by a JC-1 fluorescent probe.A transwell chamber assay was adopted to observe cell migration ability.Mitochondrial respiratory function was measured in a two-chamber titration injection respirometer(Oxygraph-2k).The expression of interleukin-1β(IL^(-1)β),interleukin-18(IL^(-1)8)and tumor necrosis factor-α(TNF-α)was detected by quantitative real-time PCR.The expression of pyroptosis-mediated proteins,including cleaved-cysteinyl aspartate-specific proteinase-1(caspase-1),N-gasdermin D(N-GSDMD),NODlike receptor protein 3(NLRP3)and purinergic ligand-gated ion channel 7 receptor(P2X7R)was detected by Western blot.The results show that 400μmol·L^(-1)H_(2)O_(2)treatment for 24 h causes obvious damage to HUVEC.Compared with the model group,puerarin protected against cellular injury in a dose-dependent manner,with the greatest effect at a dose of 30 and 100μmol·L^(-1).Puerarin significantly decreased the mitochondrial membrane potential and improved mitochondrial function.Puerarin inhibited cell migration induced by H_(2)O_(2),suppressed the expression of IL^(-1)β,IL^(-1)8 and TNF-α,and down-regulated the pyroptosis-mediated protein.These changes are statistically significant(P<0.05).These findings demonstrate that puerarin has a protective effect against H_(2)O_(2)-induced oxidative damage of HUVEC by inhibiting the migration of HUVEC cells.The mechanism may be related to improved mitochondrial respiratory function and inhibition of pyroptosis.

关 键 词：葛根素 人脐静脉内皮细胞 作用机制 线粒体 细胞焦亡 

分 类 号：R966[医药卫生—药理学]