广西中华眼镜蛇的蛇毒蛋白natrin对过氧化氢损伤乳大鼠原代心肌细胞钙超载及相关基因表达的影响  

Effect of natrin from Naja naja atra on calcium overload and expression of related genes in neonatal rat primary cardio myocytes exposed to hydrogen peroxide

作　　者：梁永红[1] 苏延旭[2] 马兴才 张洪也[1] 蒋兴明[1] 陆世银 苏志恒[1] 郑华[1] 宋慧[1] 

机构地区：[1]广西医科大学药学院,广西南宁530021 [2]中国人民解放军第303医院药剂科,广西南宁530021 [3]南宁第七人民医院,广西南宁530022

出　　处：《中国药理学与毒理学杂志》2016年第2期95-100,共6页Chinese Journal of Pharmacology and Toxicology

基　　金：广西壮族自治区科技厅科学基金(0991127);广西壮族自治区科技厅科学基金资助项目(0542043)~~

摘　　要：目的观察广西中华眼镜蛇（Naja naja atra）的蛇毒蛋白（natrin）对SD大鼠原代心肌细胞钙超载的影响,并探讨natrin对心肌的保护作用及对心肌钙、钾离子通道影响的可能作用机制。方法分别用natrin 5,25和125 mg·L^-1预处理原代SD乳大鼠心肌细胞24 h后,再以H2O20.3 mmol·L^-1诱导损伤,以Fluo-3为Ca^2＋荧光探针,应用激光共聚焦显微镜动态检测细胞内游离钙离子浓度变化;另外用natrin 5,25和125 mg·L^-1及维拉帕米（Ver）5 nmol·L^-1预处理24 h后,再以H2O20.3 mmol·L^-1诱导损伤15 min,FQPCR法检测心肌细胞钙离子通道Cav1.2,Calm,Ry R2和钾离子通道Kir6.2的m RNA表达情况。结果激光共聚焦显微镜检测结果发现,与正常对照组比较,H2O2作用3-15 min心肌细胞内钙荧光平均峰值增强49.37%（P〈0.01）;而给予natrin 5,25,125 mg·L^-1预处理24 h组该平均峰值分别增强27.52%,12.71%和5.15%,显著低于H2O2组（P〈0.01）。PCR结果表明,H2O2损伤组其心肌细胞Cav1.2,Calm,Ry R2及Kir6.2的m RNA表达分别为正常对照组的1.78倍,1.26倍,4.34倍和0.79倍（P〈0.01）;而给予natrin及维拉帕米干预组,细胞Cav1.2,Calm和Ry R2的m RNA表达较H2O2损伤组均显著降低（P〈0.01）,而Kir6.2的m RNA表达较H2O2损伤组显著升高（P〈0.05）。结论广西中华眼镜蛇natrin可明显减轻H2O2所致的心肌细胞钙超载,对心肌细胞过氧化损伤有良好的保护作用,其作用机制可能与降低H2O2损伤的心肌细胞的Cav1.2,Calm和Ry R2的m RNA表达及增加Kir6.2的m RNA表达有关。OBJECTIVE To observe the effect of natrin from Naja naja atra （Chinese cobra） on intracellular free calcium overload,and to discuss the protective effect and the possible mechanism of natrin on myocardium calcium （Ca^2＋） and potassium （K^＋） ion channels in the primary cardiomyocytes of SD neonatal rats.METHODS The primary cardiomyocytes of SD neonatal rats were used,which were respectively pretreated with natrin 5,25 and 125 mg·L^-1 for 24 h before injury was induced by H2O2 0.3 mmol·L^-1.The dynamic variation of intracellular calcium was monitored by laser confocal microscopy using Fluo-3 as Ca^2＋fluorescence probe.Additionally,the cardio myocytes of neonatal rats were pretreated for 24 h using different concentrations of natrin 5,25,125 mg·L^-1 and verapamil 5 nmol·L^-1,followed by exposure to H2O2 0.3 mmol·L^-1 for 15 min.Then,the mRNA expressions of calcium channels subunits Cav1.2,Calm,RyR2 and potassium channel Kir6.2 were analyzed by FQPCR method.RESULTS Laser confocal microscopy revealed that H2O2 obviously caused calcium overload in cardiomyocytes,giving rise to 49.37% fluorescence increase in intracellular calcium compared with the control group （P〈0.01）.However,natrin 5,25 and 125 mg·L^-1 resulted in 27.52%,12.71% and 5.15% fluorescence increase in intracellular calcium,respectively,compared with the control group （P〈0.01）.Moreover,the PCR results showed that the mRNA expressions of Cav1.2,Calm and RyR2 in the myocardial cells treated with H2O2 were increased 2.78,2.26,and 5.34 times as compared with the control group,while Kir6.2 displayed a 1.79-fold expression level （P〈0.01）.By contrast,the combination of natrin and verapamil significantly decreased the mRNA expression of Cav1.2,Calm and RyR2,compared to the H2O2-treated group （P〈0.01）.Meanwhile,the expression of Kir6.2 was considerably higher than that of the H2O2-treated group （P〈0.05）.CONCLUSION Natrin can reduce the intracellular calcium overload of cardiomyocytes induced by H2O2 and shows a

关 键 词：natrin 钙超载 肌细胞 心脏 离子通道 

分 类 号：R285.5[医药卫生—中药学]