广西眼镜蛇毒蛋白Natrin对H_2O_2所致大鼠心肌细胞钙超载的影响

Effects of Natrin from Guangxi cobra venom on H_2O_2-induced intracellular calcium overload in cultured cardiac myocytes of neonatal rats

目的观察广西眼镜蛇毒蛋白Natrin对H2O2诱导的原代培养大鼠乳鼠心肌细胞内钙超载的拮抗作用。方法采取SD大鼠乳鼠心肌细胞原代培养,将心肌细胞分正常对照组:不加任何处理;钙超载模型组:检测时加入终浓度为0.3 mmol.L-1的H2O2;药物处理组:预先分别给以不同剂量的Natrin孵育24 h,检测时加入终浓度为0.3 mmol.L-1的H2O2。以Fluo-3/AM荧光指示剂负载,应用激光共聚焦显微镜技术,动态检测细胞内[Ca2+]i变化。结果动态监测15 min发现,与正常对照组比较,模型组细胞内平均荧光峰值增加49.37%,高于正常对照组(P<0.01)。而高、中、低药物浓度组平均峰荧光值比正常对照组分别增加27.52%、12.71%、5.15%。与模型组比较,药物组细胞内平均峰荧光强度值均降低(P<0.01)。结论广西眼镜蛇毒蛋白Natrin对心肌细胞钙离子通道有较好的阻断作用,能减轻H2O2诱导的心肌细胞内[Ca2+]i超载,且药物组随着药物浓度的逐渐升高,细胞内钙荧光强度逐渐下降,并呈现剂量依赖性。

Aim To examine the effects of hydrogen peroxide （H202） on intracellular free calcium concen- tration （ [ Ca2 ＋ ] i） in cultured cardiac myocytes and its antagonism by Natrin from Guangxi cobra venom. Methods A cell culture model of SD neonatal rat car- diac myocytes was used, and the experiment was divid- ed into five groups： normal control group; hydrogen peroxide damage group; drug treatment groups： treated with three different doses of Natrin for 24h before hy- drogen peroxide damage. Intracellular [ Ca2＋ ]i was measured by laser scanning confocal microscope when the cells were loaded with Ca2~ sensitive fluorescent in- dicator Fluo-3/AM. Results 15 mimutes＇ dynamic detecting showed that the peak fluorescence intensity values of the model group was 49.37% higher com-pared with the normal control group. And drug treat- ment groups of high, middle and low doses were in- creased respectively by 27.52%, 12.71%, 5.15% , they were significantly lower compared with the model group （ P 〈 0.01 ）. Conclusion Natrin has a better antagonizing effect on myocardial cells calcium ion channel. It can significantly reduce the myocardial in- tracellular Ca2＋ overload induced by I-t202. And the fluorescence intensity of the myocytes decreases gradu- ally in a dose-dependent manner with the increasing concentration of Natrin among the drug treatment groups