RhoA/ROCK-NF-κB信号传导通路介导内脂素诱导的心肌细胞炎性反应机制研究  

The action mechanism of RhoA/ROCK-NF-κB signaling pathway mediating visfatin induced cardiomyocyte inflammatory reaction in vitro

作　　者：常亮[1] 徐璐 刘素云[1] 郭炳彦[1] 金鑫[1] 贾妍[1] 李拥军 CHANG Liang;XU Lu;LIU Suyun(Department of Cardiology,The Second Hospital of Hebei Medical University,Hebei,Shijiazhuang 050000,China)

机构地区：[1]河北医科大学第二医院心内科,石家庄市050000

出　　处：《河北医药》2020年第24期3685-3690,共6页Hebei Medical Journal

基　　金：国家自然科学基金面上基金项目(编号:81570345);国家自然科学基金青年科学基金项目(编号:81400217);河北省自然科学基金项目(编号:H2014206389);河北省科技支撑计划项目(编号:132777186)。

摘　　要：目的研究内脂素引起心肌细胞炎性反应相关机制。方法提取2~3 d SD乳鼠原代心肌细胞,培养后按照实验设计分组为(1)正常对照组,10、50、100 ng/ml浓度的重组内脂素,干预24 h;(2)正常对照组,100 ng/ml重组内脂素分别干预12、24、48、72 h;(3)正常对照组、重组内脂素100 ng/ml、SN50(20μmol/L)、SN50(20μmol/L)+重组内脂素100 ng/ml,干预48 h;(4)正常对照组、重组内脂素100 ng/ml、Y27632(10μmol/L)、Y27632(10μmol/L)+重组内脂素100 ng/ml、辛伐他汀(1μmol/L)、辛伐他汀(1μmol/L)+重组内脂素100 ng/ml,干预48 h。(5)正常对照组、重组内脂素100 ng/ml、sirtinol(200μmol/L)、sirtinol(200μmol/L)+重组内脂素100 ng/ml,干预24 h。,并予相应的干预。应用Real time-PCR检测心肌细胞TNF-αmRNA表达,应用MTT法检测心肌细胞活力,Western blot法检测心肌细胞Sirt1蛋白表达。结果心肌细胞TNF-αmRNA表达随内脂素浓度的升高而升高,100 ng/ml时最高;OD值在浓度为10 ng/ml时最高,50 ng/ml、100 ng/ml时降低,且100 ng/ml时明显低于正常对照组(P<0.05)。100 ng/ml内脂素干预心肌细胞48 h时TNF-αmRNA达到峰值,OD值在12 h时与正常对照组比较,差异无统计学意义(P>0.05),24 h后开始降低,与正常对照组比较,差异有统计学意义(P<0.05)。应用sirtinol预干预心肌细胞后用100 ng/ml内脂素干预心肌细胞24 h,sirtinol+重组内脂素干预组心肌细胞OD值明显低于正常对照组,高于100 ng/ml内脂素干预组(P<0.05)。心肌细胞Sirt1蛋白表达水平随着内脂素干预浓度的升高而升高,100 ng/ml内脂素干预48 h后达到峰值,72 h时继续升高,但是较48 h组差异无统计学意义(P>0.05)。加用SN50、sirtinol和Y27632预干预组的心肌细胞TNF-αmRNA水平明显低于单纯内脂素干预组,高于正常对照组(P<0.05)。结论内脂素可以直接诱导心肌细胞炎性反应,可能与RhoA/ROCK2-NF-κB信号有关。内脂素诱导的适量SIRT1表达增加对心肌细胞具有正性Objective To investigate the action mechanism of RhoA/ROCK-NF-κB signaling pathway mediating visfatin induced cardiomyocyte inflammatory reaction in vitro.Methods The primary myocardial cells of SD suckling rats(2~3 of age)were extracted,which,after cultured,were divided into groups accrding to experiment design.Real time-PCR was used to detect the expression levels of TNF-αmRNA in cardiomyocytes,MTT was used to detect the viability of cardiomyocytes,and Western blot was used to detect the expression levels of Sirt1 protein in cardiomyocytes.Results The expression levels of TNF-αmRNA in cardiomyocytes were increased with the increase of lipoprotein concentration,which was the highest at 100ng/ml,and the OD value was highest at the concentration of 10ng/ml,which was decreased at 50ng/ml and 100ng/ml,which in the concentration of 100ng/ml was significantly lower than that in control group(P<0.05).TNF-αmRNA reached a peak at 100ng/ml in cardiomyocytes after 48-hour intervention,and the OD value was not significantly different from that in control group after 12-hour intervention(P>0.05),but which was decreased after 24-hour intervention,which was significantly different from that in control group(P<0.05).After the preintervention of cardiomyocytes with SIRT1,then the cardiomyocytes were interfered by 100 ng/ml lipoprotein for 24h,the OD value in simvastatin+recombinant lipidone intervention group was significantly lower than that in control group and higher than that in 100ng/ml lipidone intervention group(P<0.05).Moreover the expression levels of Sirt1 protein were increased with the increase of the concentration of lipolipin,and the peak of 100ng/ml lipoprotein was at 48h after intervention,which continued to increase at 72h,but there were no significant differences,as compared with those in 48h group(P>0.05).In addition the levels of TNF-αmRNA in preintervention group with SN50,sirtinol and Y27632 were significantly lower than those in lipidone-treated group,which were significantly higher than those in cont

关 键 词：RhoA/ROCK-NF-κB信号传导通路 心肌 炎症反应 内脂素 肿瘤坏死因子Α 

分 类 号：R542.2[医药卫生—心血管疾病]