基于TRPV1通道分析诃子制草乌减轻H9c2心肌细胞毒性的作用机制  

Mechanism of Aconiti Kusnezoffii Radix Processed with Chebulae Fructus Against H9c2 Cardiomyocyte Toxicity Based on TRPV1 Channel

作　　者：韩舒 包丽媛 刘凯洋 韩喜桃 唐雅楠 刘子琴 王宏月 范盎然[1] 李飞[1] 杜红[1] HAN Shu;BAO Li-yuan;LIU Kai-yang;HAN Xi-tao;TANG Ya-nan;LIU Zi-qin;WANG Hong-yue;FAN Ang-ran;LI Fei;DU Hong(Beijing University of Chinese Medicine,Beijing 100029,China)

机构地区：[1]北京中医药大学,北京100029

出　　处：《中国实验方剂学杂志》2022年第1期173-181,共9页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金项目(81774004);国家重点研发计划项目(2018YFC1706303)。

摘　　要：目的:探究瞬时受体电位香草酸亚型1(TRPV1)通道在诃子制草乌减轻心肌细胞毒性中的作用机制。方法:以体外培养的H9c2心肌细胞为模型,采用噻唑蓝(MTT)法检测细胞活力;实时荧光定量聚合酶链式反应(Real-time PCR)检测心肌细胞中TRPV1 mRNA的表达水平;酶联免疫吸附测定试验(ELISA)检测细胞中乳酸脱氢酶(LDH)漏出率,细胞核,活性氧(ROS),线粒体膜电位及钙离子(Ca^(2+))含量的变化。结果:与空白组比较,当质量浓度≥0.5 g·L^(-1)时,生草乌、诃子制草乌均可显著降低细胞活力(P<0.01),细胞中LDH漏出率,ROS及Ca^(2+)释放量增加,线粒体膜电位降低,细胞核固缩甚至是破碎。当质量浓度≥0.5 g·L^(-1)时,与同质量浓度生草乌组相比,诃子制草乌组细胞活力显著上升(P<0.01),细胞中LDH漏出率,ROS及Ca^(2+)释放量减少,线粒体膜电位升高,细胞核形态有所改善。与作用于未经处理的H9c2心肌细胞比较,同质量浓度的生草乌作用于TRPV1抑制剂BCTC预处理后的H9c2心肌细胞后,其细胞活力明显上升,细胞中LDH漏出率,ROS及Ca^(2+)释放量减少,线粒体膜电位升高,细胞核固缩程度得到改善;而同质量浓度的诃子制草乌作用于BCTC预处理后的H9c2心肌细胞,其细胞活力下降,细胞中LDH漏出率,ROS及Ca^(2+)释放量增加,线粒体膜电位降低。Real-time PCR结果表明,草乌和辅料诃子汤均能使心肌细胞中TRPV1 mRNA表达量增多,且呈现浓度依赖性。结论:生草乌可通过激活TRPV1通道诱导心肌细胞凋亡,引发心脏毒性;而诃子制草乌可通过TRPV1通道介导起到减毒作用,其减毒作用的发挥可能与诃子中的酸性成分和草乌中的生物碱协同作用于TRPV1通道有关。Objective:To explore the role of transient receptor potential vanilloid 1(TRPV1)channel in reducing cardiomyocyte toxicity of Aconiti Kusnezoffii Radix processed with Chebulae Fructus. Method:H9c2 cardiomyocytes cultured in vitro were used as a model to assess cell viability by methyl thiazolyl tetrazolium(MTT)assay,the expression of TRPV1 mRNA was detected by real-time fluorescence quantitative polymerase chain reaction(Real-time PCR),and the leakage rate of lactate dehydrogenase(LDH),the changes of nucleus,reactive oxygen species(ROS),mitochondrial membrane potential and Ca^(2+) contents were detected by enzyme linked immunosorbent assay(ELISA). Result: Compared with the blank group, when the concentration was ≥0.5 g·L^(-1),the cell viability was significantly decreased(P<0.01),the leakage rate of LDH,the release of ROS and Ca^(2+) were increased,the mitochondrial membrane potential was decreased,and the nucleus was pyknosis or even broken in raw Aconiti Kusnezoffii Radix and Aconiti Kusnezoffii Radix processed with Chebulae Fructus groups. When the concentration was ≥0.5 g·L^(-1), compared with the same mass concentration of raw Aconiti Kusnezoffii Radix group,the cell viability increased significantly(P<0.01),the leakage rate of LDH,the release of ROS and Ca^(2+) decreased,the mitochondrial membrane potential increased,and the nuclear morphology improved in Aconiti Kusnezoffii Radix processed with Chebulae Fructus group.Application of the same mass concentration of raw Aconiti Kusnezoffii Radix to H9c2 cardiomyocytes pretreated with the TRPV1 inhibitor BCTC significantly increased cell viability,decreased leakage rate of LDH,ROS and Ca^(2+) release, increased mitochondrial membrane potential and improved nuclear pyknosis compared with untreated H9c2 cardiomyocytes. Application of the same mass concentration of Aconiti Kusnezoffii Radix processed with Chebulae Fructus to H9c2 cardiomyocytes pretreated with BCTC decreased cell viability,increased LDH leakage rate,ROS and Ca^(2+) release,reduced mitochondr

关 键 词：生草乌 诃子制草乌 瞬时受体电位香草酸亚型1(TRPV1) H9C2心肌细胞 炮制减毒 聚合酶链式反应(PCR) 乳酸脱氢酶(LDH) 

分 类 号：R22[医药卫生—中医基础理论] R943.1[医药卫生—中医学]