环维黄杨星D调控Ca^(2+)/CaMKⅡ信号改善醛固酮诱导心肌细胞损伤的实验研究  

Cyclovirobuxine D ameliorates aldosterone-induced primary neonatalrat cardiac myocyte injury via Ca^(2+)/CaMKⅡsignaling pathway

作　　者：张光琼 王声全 付凌云[1,2] 徐旖旎[1,2] 罗红[1,2] 陶玲[1,2] 沈祥春[1,2] 李玲[1,2] ZHANG Guang-qiong;WANG Sheng-quan;FU Ling-yun;XU Yi-ni;LUO Hong;TAO Ling;SHEN Xiang-chun;LI Ling(the State Key Laboratory of Functions and Applications of Medicinal Plants,School of Pharmaceutical Sciences,Guizhou Medical University,Guiyang 550025,China;the High Efficacy Application of Natural Medicinal Resources Engineering Center of Guizhou Province,Guizhou Medical University,Guiyang 550025,China)

机构地区：[1]贵州医科大学省部共建药用植物功效与利用国家重点实验室,贵州贵阳550025 [2]贵州医科大学贵州省特色天然药物高效利用工程中心药学院,贵州贵阳550025

出　　处：《中国药理学通报》2021年第4期564-570,共7页Chinese Pharmacological Bulletin

基　　金：国家自然科学基金资助项目(No 82060729);国家自然科学基金喀斯特中心项目(No U1812403-4-4);贵州省高层次创新型人才百层次人才(No黔科合人才[2015]4029号);贵州省药学国际科技合作基地(No黔科合平台人才[2017]5802);贵阳市科技计划项目(No筑科合同[2017]30-17号);贵州医科大学2018年度学术新苗培养及创新探索专项项目(No黔科合平台人才[2018]5779-66)。

摘　　要：目的探讨环维黄杨星D(cyclovirobuxine D,CVB-D)对醛固酮(aldosterone,ALD)诱导原代大鼠心肌细胞(PNRCMs)损伤的保护作用及其机制。方法通过胰酶消化法提取PNRCMs,以ALD(10μmol·L^(-1))制备PNRCMs损伤模型,给予不同剂量的CVB-D处理后,采用噻唑蓝(MTT)法检测细胞活力,Giemsa染色观察细胞形态变化,Flou-4AM荧光探针检测胞内Ca^(2+)浓度,Western blot检测钙调蛋白激酶Ⅱ(CaMKⅡ、p-CaMKⅡ)和凋亡相关蛋白Bcl-2、Bax、Cleaved caspase-9的表达水平。进一步采用钙离子螯合剂(BAPTA-AM),钙离子载体(A23187)及CaMKⅡ抑制剂(KN93)进行干预处理,分析Bcl-2,Bax,Cleaved caspase-9蛋白的表达变化。结果与ALD组比较,CVB-D可显著恢复细胞活力,改善细胞的病理形态变化,上调Bcl-2/Bax的比值,下调Cleaved caspase-9和p-CaMKⅡ的表达并抑制胞内钙离子累积。进一步研究表明,BAPTA-AM和KN93能促进CVB-D上调Bcl-2/Bax的比值,下调Cleaved caspase-9的表达;而A23187可抑制CVB-D上调Bcl-2/Bax的比值,下调Cleaved caspase-9表达。结论CVB-D可改善ALD诱导的原代心肌细胞损伤,其作用机制与Ca^(2+)/CaMKⅡ信号通路相关。Aim To investigate the protective effect of cyclovirobuxine D(CVB-D)on aldosterone(ALD)-induced primary neonatal rat cardiac myocytes(PNRCMs)injury and the possible mechanism.Methods PNRCMs were extracted by trypsin,and the PNRCMs injury model was established by ALD(10μmol·L^(-1)).After treatment with different doses of CVB-D,cell viability was detected by MTT assay,cell morphological changes were observed by Giemsa staining,intracellular Ca^(2+)concentration was detected by Flou-4AM fluorescence probe,and the expression of calmodulin kinaseⅡ(CaMKⅡ,p-CaMKⅡ)and apoptosis-related proteins Bcl-2,Bax,Cleaved caspase-9 were measured by Western blot.Furthermore,calcium chelating agent(BAPTA-AM),calcium ionophore(A23187)and CaMKⅡinhibitor(KN93)were used to further analyze the expression changes of Bcl-2,Bax and Cleaved caspase-9 protein.Results Compared with ALD group,CVB-D could significantly restore cell viability,improve cell pathomorphology,up-regulate the ratio of Bcl-2/Bax,down-regulate the expression of Cleaved caspase-9 and p-CaMKⅡ,and inhibit intracellular calcium accumulation.In ALD environment,BAPTA-AM and KN93 could promote CVB-D to up-regulate the ratio of Bcl-2/Bax and down-regulate the expression of Cleaved caspase-9,while A23187 could inhibit CVB-D from up-regulating the ratio of Bcl-2/Bax and down-regulating the expression of Cleaved caspase-9.Conclusions CVB-D can ameliorate the injury of PNRCMs induced by ALD,and its mechanism is related to Ca^(2+)/CaMKⅡsignaling pathway.

关 键 词：环维黄杨星D 醛固酮 心肌细胞损伤 凋亡 钙离子 钙调蛋白激酶Ⅱ 

分 类 号：R-33[医药卫生] R284.1