p38 MAPK通路在TNF-α诱导人脐静脉内皮细胞分泌ET-1与eNOS中的作用及通心络干预影响

The role of p38MAPK signal pathway in the course of TNF-α inducing HUVEC to secrete ET-1 and eNOS and the effect of Tongxinluo

目的观察p38 MAPK信号通路在肿瘤坏死因子-α(TNF-α)诱导人脐静脉内皮细胞(HUVEC)表达内皮素-1(ET-1)与内皮型一氧化氮合酶(eNOS)中的作用及通心络干预影响。方法分别采用放免法及ELISA法测定不同浓度TNF-α(0、2.5、5、10、15、20μg·L-1)在不同时间点(0、1、2、4、8、12、24h)干预后HUVEC培养上清液中ET-1和eNOS含量;分别采用Western blot和Realtime RT-PCR方法检测TNF-α干预24h后HUVEC中ET-1、eNOS蛋白及mRNA表达;采用Western blot方法检测TNF-α干预10min、30min、60min后HUVEC磷酸化p38 MAPK蛋白表达。结果不同浓度TNF-α随时间延长均明显增加HUVEC培养上清液中ET-1含量,降低eNOS含量;TNF-α升高细胞中ET-1蛋白及mRNA水平、降低eNOS蛋白及mRNA水平、在各时间点均可升高细胞p-p38 MAPK蛋白表达。通心络可降低TNF-α诱导的HUVEC培养上清ET-1含量、降低细胞中ET-1蛋白及mRNA的异常升高;增加HUVEC培养上清中eNOS的表达、增加细胞中eNOS蛋白及mRNA的表达;明显抑制TNF-α诱导的细胞p-p38 MAPK表达。结论p38 MAPK信号通路参与了TNF-α诱导HUECV细胞分泌ET-1和eNOS,通心络对内皮细胞保护作用机制与抑制该通路有关。

Aim To observe the role of p38MAPK signal pathway which was in the course of TNF-α inducing HUVEC to secrete ET-1 and eNOS and the effect of Tongxinluo.Methods The contents of ET-1 and eNOS derived from HUVEC culture supernatant after exerting into different concentration TNF-α（0,2.5,5,10,15,20 μg·L-1） in different time points（0,1,2,4,8,12,24 h） were detected by the method of radioimmunity and ELISA,respectively.The protein and mRNA expression of ET-1/eNOS in HUVEC lysate were detected by the method of Western blot and Real time quantitive RT-PCR after 24 hours added with TNF-α.The expression of HUVEC phosphorylation p38MAPK protein was detected by the method of Western blot after 10 min,30 min,60 min incorporated into TNF-α,respectively.Results The different concentrations of TNF-α could increase the ET-1 contents in HUVEC cultural supernatant with the time prolonging,and decrease the eNOS contents inversely.Meanwhile,TNF-α could increase the level of ET-1 protein and mRNA in HUVEC lysate significantly,the same thing,decrease the level of eNOS protein and mRNA.In addtion,it could increase p-p38 MAPK protein expression at different time points.Tongxinluo could decrease ET-1 in HUVEC cultural supernatant and the expression of ET-1 protein and mRNA in cell lysate induced by TNF-α,on the contrary,it could increase eNOS in HUVEC cultural supernatant and the expression of eNOS protein and mRNA in cell lysate,at the same time,Tongxinluo could restrain p-p38 MAPK expression in HUVEC.Conclusions The p38MAPK signal pathway plays an important role in the course of TNF-α inducing HUVEC to secrete ET-1 and eNOS,the protective mechanism of Tongxinluo is related to the p38MAPK signal pathway for endothelial cells.