参莲提取物通过调控Nrf2/Keap1信号通路减轻TNF-α诱导的ECV304损伤  

Shenlian extract attenuates TNF-α-induced ECV304 injury by regulating Nrf2/Keap1 signaling pathway

作　　者：王春淼 李玉洁[1] 李晶晶 臧远龙 崔羲和 宋敏 杨庆[1] 陈颖[1] 李琦[1] 蔡维艳[1] 翁小刚[1] 王娅杰[1] 朱晓新[1] WANG Chun-miao;LI Yu-jie;LI Jing-jing;ZANG Yuan-long;CUI Xi-he;SONG Min;YANG Qing;CHEN Ying;LI Qi;CAI Wei-yan;WENG Xiao-gang;WANG Ya-jie;ZHU Xiao-xin(Institute of Chinese Materia Medica,China Academy of Chinese Medical Sciences,Beijing 100700,China)

机构地区：[1]中国中医科学院中药研究所,北京100700

出　　处：《中国中药杂志》2021年第13期3402-3409,共8页China Journal of Chinese Materia Medica

基　　金：国家“重大新药创制”科技重大专项(2017ZX09301012002);中央级公益性科研院所基本科研业务费专项(ZXKT20026);国家自然科学基金项目(81673640)。

摘　　要：探讨参莲提取物对肿瘤坏死因子α(TNF-α)诱导的血管内皮细胞(ECV304)损伤的干预作用及可能的作用机制。以TNF-α诱导ECV304细胞损伤模型,噻唑蓝(MTT)法检测细胞生存活力;采用流式细胞仪检测细胞内活性氧(ROS)水平;生化法和酶联免疫吸附(ELISA)法检测细胞上清液中超氧化物歧化酶(SOD)、丙二醛(MDA)、一氧化氮(NO)、内皮素-1(ET-1)和白细胞介素-1β(IL-1β)含量;通过Western blot法检测Kelch样环氧氯丙烷相关蛋白-1(Keap1)、核因子E2相关因子2(Nrf2)、醌氧化还原酶(NQO1)、血红素加氧酶1(HO-1)、半胱天冬酶3(caspase-3)、半胱天冬酶9(caspase-9)、B淋巴细胞瘤-2基因(Bcl-2)和Bcl-2相关X蛋白(Bax)的表达水平。结果显示,与对照组相比,50μg·L^(-1)的TNF-α能够导致ECV304细胞活力显著下降(P<0.01),引起ECV304细胞ROS产生增加,SOD活性降低,MDA、NO、ET-1、IL-1β含量显著增加(P<0.01),Keap1、caspase-9、Bax蛋白表达显著上调,NQO1、Bcl-2蛋白表达显著降低(P<0.05);与模型组比较,参莲提取物可以提高ECV304细胞存活率,抑制细胞ROS产生,提高SOD活性,降低MDA、NO、ET-1、IL-1β含量(P<0.01或P<0.05),下调Keap1、caspase-3、caspase-9、Bax蛋白表达水平,提高Nrf2、NQO1、HO-1、Bcl-2蛋白表达(P<0.01或P<0.05)。提示参莲提取物可能通过调控Nrf2/Keap1信号通路降低TNF-α诱导的ECV304细胞氧化应激损伤,减少炎症反应和细胞凋亡。This study aimed to investigate the effect and the possible mechanism of Shenlian(SL)extract on tumor necrosis factor-α(TNF-α)-induced ECV304 injury.After the establishment of TNF-α-induced ECV304 cells injure model,MTT assay was used to detect cell viability and the level of reactive oxygen species(ROS)was measured by flow cytometry.The contents of superoxide dismutase(SOD),malondialdehyde(MDA),nitric oxide(NO),endothelin-1(ET-1)and interleukin-1β(IL-1β)in the supernatant were detected by biochemical method and enzyme linked immunosorbent assay(ELISA).The expression levels of apoptosis-related proteins B-lymphoma-2 gene(Bcl-2),Bcl-2 associated X protein(Bax),caspase-3,caspase-9 and nuclear factor E2 associated factor2(Nrf2)/Kelch like epichlorohydrin associated protein-1(Keap1)signaling pathway related proteins Nrf2,Keap1,quinone oxidoreductase(NQO1)and heme oxygenase 1(HO-1)were detected by Western blot.The results showed that 50μg·L^(-1) TNF-α significantly damaged ECV304 cells,induced the impairment of cell viability(P<0.01),the increase of ROS production,the decrease of SOD activity,and the increase of MDA,NO,ET-1 and IL-1β(P<0.01),meanwhile,it caused the up-regulation of Keap1,caspase-9 and Bax protein expression,and down-regulation of NQO1 and Bcl-2 protein expression(P<0.05)compared with the control group.Compared with the model group,SL extract reduced the damage of ECV304 cells induced by TNF-α,improved cell viability,reduced ROS production,increased SOD activity and decreased MDA,NO,ET-1,IL-1β content(P<0.01 or P<0.05).In addition,SL extract also down-regulated the protein expression levels of Keap1,caspase-3,caspase-9 and Bax,and increased the protein expressions of Nrf2,NQO1,HO-1 and Bcl-2(P<0.01 or P<0.05).The above results indicate that SL extract can provide protective effect on ECV304 cells injury induced by TNF-α,alleviate oxidative stress injury,inflammation and apoptosis,and its mechanism may be related to regulating Nrf2/Keap1 signaling pathway.

关 键 词：参莲提取物 血管内皮细胞 氧化应激 炎症反应 细胞凋亡 

分 类 号：R285[医药卫生—中药学]