CaMKⅡ调控脂多糖诱导的心肌细胞内钙、ROS的作用机制研究  

The mechanism of CaMKⅡregulating cytoplasmic calcium and ROS induced by LPS in cardiomyocytes

作　　者：巩奇明 李德慧 龚元勋 姜艳 Gong Qiming;Li Dehui;Gong Yuanxun;Jiang Yan(The Affiliated Hospital of Youjiang Medical University for Nationalities,Baise 53000,Guangxi,China;Science Experimental Center,Youjiang Medical University for Nationalities,Baise 53000,Guangxi,China)

机构地区：[1]右江民族医学院附属医院,广西百色533000 [2]右江民族医学院科学实验中心,广西百色533000

出　　处：《右江民族医学院学报》2021年第3期297-301,共5页Journal of Youjiang Medical University for Nationalities

基　　金：广西自然科学基金项目(2020GXNSFBA297052);广西高校中青年教师基础能力提升项目(2018KY0435)。

摘　　要：目的探究钙调蛋白依赖性蛋白激酶Ⅱ(CaMKⅡ)调控脂多糖(LPS)诱导的心肌细胞胞浆Ca^(2+)、活性氧(ROS)的分子作用机制。方法大鼠心肌细胞(H9C2)经10.0μg/ml的LPS处理48 h,构建心肌炎体外细胞模型。实验分为对照组、LPS组(10.0μg/ml的LPS作用48 h)、KN93+LPS组[5.0μmol/L的KN93(CaMKⅡ抑制剂)预处理1 h,再加入10.0μg/ml的LPS刺激48 h]。采用荧光探针法,观察细胞内Ca^(2+)、ROS含量水平;RT-qPCR法检测CaMKⅡ、受磷蛋白(PLB)、心肌细胞肌浆网Ca^(2+)-ATP酶(SERCA)、无翅型小鼠乳腺肿瘤病毒整合位点家族成员4(Wnt4)、β-连环蛋白(β-catenin)、原癌基因(c-Myc)、G1/S-特异性周期蛋白D1(Cyclin D1)、诱导型一氧化氮(iNOS)、白介素10(IL-10)基因mRNA表达水平;Western Blot检测细胞内CaMKⅡ蛋白含量。结果与对照组比较,LPS组胞浆Ca^(2+)以及细胞内ROS水平增加;CaMKⅡ的mRNA及蛋白表达增加(P<0.01);PLB和SERCA mRNA水平降低(P<0.01);Wnt4、β-catenin、c-Myc、Cyclin D1、IL-10以及iNOS的mRNA表达增加(P<0.01)。与LPS组相比,KN93+LPS组胞浆Ca^(2+)降低,ROS含量降低;CaMKⅡ的mRNA及蛋白表达均减少(P<0.01);PLB和SERCA mRNA水平增加(P<0.01);Wnt4、Cyclin D1、IL-10以及iNOS的mRNA表达降低(P<0.05),而β-catenin、c-Myc的mRNA水平差异无统计学意义(P>0.05)。结论CaMKⅡ可能通过调控细胞内钙调控蛋白PLB、SERCA,Wnt通路蛋白Wnt4、Cyclin D1,以及细胞因子iNOS、IL-10的表达,从而抑制LPS诱导的心肌胞浆Ca^(2+)及ROS水平增加。Objective To investigate the molecular mechanism of calmodulin-dependent protein kinaseⅡ(CaMKⅡ)regulating cytoplasmic Ca^(2+)and reactive oxygen species(ROS)induced by lipopolysaccharide(LPS)in cardiomyocytes.Methods Rat cardiomyocytes(H9C2)were treated with 10.0μg/ml LPS for 48 h to establish myocarditis cell models in vitro.There were three groups in this experiment:the control group,the LPS group(10.0μg/ml LPS stimulation for 48 h),the KN93+LPS group[pretreated with 5.0μmol/L KN93(the inhibitor of CaMKⅡ)for 1 h,and then stimulated with 10.0μg/ml LPS for 48 h].The content of Ca^(2+)and ROS was observed by fluorescence probe.RT-qPCR was employed to detect the expressions of CaMKⅡ,phospholamban(PLB),sarco endoplasmic reticulum Ca^(2+)-ATPase(SERCA),member 4 of the wingless mouse mammary tumor virus integration site family(Wnt4),β-catenin,c-Myc,G1/S-specific cyclin D1(Cyclin D1),inducible nitric oxide synthase(iNOS)and interleukin 10(IL-10)mRNA.The content of CaMKⅡprotein in cells was detected by Western Blot.Results Compared with the control group,the LPS group had increased levels of cytoplasmic Ca^(2+)and intracellular ROS as well as raised mRNA and protein expressions of CaMKⅡ(P<0.01);but the PLB and SERCA mRNA levels decreased(P<0.01).In the LPS group,the mRNA expressions of Wnt4,β-catenin,c-Myc,Cyclin D1,IL-10 and iNOS also increased(P<0.01).Compared with the LPS group,the KN93+LPS group had decreased cytoplasmic Ca^(2+)and ROS content and declined mRNA and protein expressions of CaMKⅡ(P<0.01).Furthermore,this group had increased mRNA expressions of PLB and SERCA(P<0.01)and decreased mRNA expressions of Wnt4,Cyclin D1,IL-10 and iNOS(P<0.01).However,there was no statistically significant difference in the mRNA expressions ofβ-catenin and c-Myc between two groups(P>0.05).Conclusion CaMKⅡmay inhibit the increase of Ca^(2+)and ROS levels induced by LPS in myocardial cytoplasm.The inhibition may be by regulating the expressions of PLB and SERCA,protein Wnt4 of Wnt pathway and Cyclin D1,as w

关 键 词：钙调蛋白依赖性蛋白激酶Ⅱ 脂多糖 心肌细胞 胞浆钙 活性氧 

分 类 号：R392[医药卫生—免疫学]