黄芩苷调节STIM1介导的钙超载减轻脂多糖诱导的心肌细胞凋亡  

Baicalin regulates STIM1-mediated calcium overload and reduces apoptosis of cardiomyocytes induced by lipopolysaccharide

作　　者：李萌芳[1] 胡系意 陈隆望[1] 连洁[1] 赵光举[1] 洪广亮[1] 卢中秋[1] Li Mengfang;Hu Xiyi;Chen Longwang;Lian Jie;Zhao Guangju;Hong Guangliang;Lu Zhongqiu(Emergency Department,the First Affiliated Hospital of Wenzhou Medical University,Wenzhou 325000,China)

机构地区：[1]温州医科大学附属第一医院急诊医学中心,温州325000

出　　处：《中华医学杂志》2019年第40期3176-3182,共7页National Medical Journal of China

基　　金：国家自然科学基金(81772112,81571937,81871583);浙江省自然科学基金(LQ15H150003);浙江省医药卫生科研项目(2015KYA152);浙江省中医药管理局项目(2015ZA120);温州市科技计划项目(Y20170177,Y20170185,Y20150182)。

摘　　要：目的探讨黄芩苷对脂多糖(LPS)诱导的H9C2心肌细胞凋亡的保护作用及可能机制。方法培养H9C2心肌细胞,分为正常组、黄芩苷组(10μmol/L的黄芩苷预处理12 h)、LPS组(1μg/ml,刺激作用6 h)、LPS+黄芩苷组(10μmol/L的黄芩苷预处理12 h,再加入终浓度为1μg/ml刺激6 h),收集细胞样本。CCK-8(The Cell Counting Kit-8)检测细胞活力,TUNEL(Terminal-deoxynucleoitidyl Transferase Mediated Nick End Labeling)法检测细胞凋亡,共聚焦显微镜观察细胞钙库操控性钙离子内流(SOCE)水平的变化,免疫印迹法检测细胞cleaved-caspase3、Bcl-2及Bax蛋白表达水平的变化,荧光定量聚合酶链反应(PCR)、免疫印迹法检测细胞上蛋白基质交感分子1(STIM1)分别在mRNA和蛋白表达水平的变化。结果与对照组相比,LPS诱导后H9C2心肌细胞存活率降低[(93.8±1.7)%与(65.6±1.2)%,P<0.05],凋亡百分率升高[(8.7±1.2)%,LPS组为(47.4±5.2)%,P<0.05],钙离子内流增加(P<0.05),cleaved-caspase3、Bax蛋白表达水平升高(P<0.05),Bcl-2蛋白表达降低(P<0.05),STIM1 mRNA及蛋白水平升高(P<0.05)。与LPS组相比,黄芩苷干预组H9C2细胞心肌细胞存活率升高(P<0.05),凋亡百分率降低(P<0.05),钙离子内流减少(P<0.05),cleaved-caspase3、Bax蛋白水平降低(P<0.05),Bcl-2蛋白表达升高(P<0.05),STIM1 mRNA及蛋白水平降低(P<0.05)。结论黄芩苷具有减轻LPS诱导心肌细胞凋亡的能力,其通过改善其调控STIM1-SOCE钙超载减轻LPS引起的心肌细胞凋亡。Objective To investigate the protective effect of Baicalin on apoptosis induced by lipopolysaccharide in H9C2 cardiomyocytes and its possible mechanism.Methods In order to establish apoptosis model of H9C2 cardiomyocytes,H9C2 cardiomyocytes were cultured and divided into four groups:the control group;the baicalin group was treated with baicalin at the final concentration of 10μmol/L for 12 hours;the LPS group was stimulated with LPS at the final concentration of 1μg/ml for 6 hours;The LPS+baicalin group was stimulated with LPS at the final concentration of 1μg/ml for 6 hours within treated with baicalin at the final concentration of 10μmol/L for 12 hours.Collecting cell samples,CCK-8(The Cell Counting Kit-8)was used to detect cell activity,and Terminal-deoxynucleoitidyl Transferase Mediated Nick End Labeling(TUNEL)was used to detect the expression levels of apoptosis.Laser Scanning Confocal Microscopy was used to detect the expression levels of store-operated calcium entry in H9C2 cardiomyocytes.Western blot was used to detect the protein expression levels of STIM1,cleaved-caspase3,Bax and Bcl-2.Fluorogenic quantitative PCR was used to detect the mRNA expression level of STIM1.Results Compared with the control group,LPS-induced H9C2 cardiomyocyte survival rate decreased(P<0.05),the expression level of apoptosis increased(P<0.05),the internal flow of calcium increased(P<0.05),the expression levels of cleaved-caspase3,Bax protein levels increased(P<0.05),Bcl-2 protein level decreased(P<0.05),the expression of STIM1 mRNA and protein level increased(P<0.05).Compared with LPS group,the survival rate of H9C2 cardiomyocytes in baicalin intervention group increased(P<0.05),the expression level of apoptosis decreased(P<0.05),the internal flow of calcium decreased(P<0.05),the expression levels of cleaved-caspase3,Bax protein decreased(P<0.05),and the level of Bcl-2 protein increased(P<0.05),the expression of STIM1 mRNA and protein level decreased(P<0.05).Conclusion Baicalin may alleviate LPS-induced cardiomyocyte apopt

关 键 词：黄芩苷 脂多糖 心肌细胞 凋亡 钙库操控性钙离子内流 STIM1 

分 类 号：R28[医药卫生—中药学]