六味地黄丸含药血清对KCl诱导PC12细胞内Ca^(2+)离子浓度及钙调蛋白依赖性蛋白激酶Ⅱ的影响

Effect of Serum Containing Liuwei Dihuang Wan on Calcium Concentration and Level of CaMKⅡ mRNA in PC12 Cells Induced by KCl

目的:研究六味地黄丸含药血清对KCl诱导PC12细胞内Ca2+浓度及钙调蛋白依赖性蛋白激酶Ⅱ(CaMKⅡ)mRNA水平的影响。方法:取对数生长期的PC12细胞株(大鼠肾上腺嗜铬肿瘤细胞株),经10%六味地黄丸含药血清预保护48 h后,加入150 mmol.L-1KCl刺激后,应用细胞活性检测(MTT法)观察细胞活性和应用激光共聚焦等技术方法观察六味地黄丸含药血清对PC12细胞Ca2+浓度变化的影响,RT-PCR法检测各组PC12细胞CaMKⅡmRNA表达水平。结果:10%六味地黄丸血清[细胞吸光度(A)为(1.15±0.09)]能显著提升细胞存活率,与模型组[细胞吸光度(A)为(0.47±0.11)]相比有显著性差异(P<0.01);PC12细胞经10%六味地黄丸血清预保护48 h后静息钙强度与空白对照组、模型组相比有显著性差异(P<0.05),150 mmol.L-1KCl刺激后,经10%六味地黄丸血清预保护48 h的细胞内Ca2+平均荧光强度明显低于模型组(P<0.05),同时细胞内CaMKⅡmRNA表达显著升高(P<0.05)。结论:六味地黄丸含药血清对KCl诱导PC12细胞内钙超载有抑制作用,且可提高细胞内CaMKⅡmRNA表达水平。

Objective： To investigate the effect of serum containing Liuwei Dihuang Wan on calcium concentration and the level of calmodulin-dependent protein kinaseⅡ（CaMKⅡ ） mRNA in PC12 cells induced by KCl. Method： The models of PC12 cells was established （rat adrenal pheochromocytoma cell line） by being stimulated by 150 mmol ,L-1 KC1. Then Dulbecco＇s modified Eagle＇s medium （DMEM） containing 10% Liuwei Dihuang Wan serum was added to exponentially-growing PC12 ceils for pre-protection. After 48 h, the proliferation of PC12 cells was detected by MTT and the calcium concentration in PC12 cells was observed by laser scanning confocal microscope. The level of CaMKⅡ mRNA was analyzed by RT-PCR. Result： It can significantly improve the activity of cells in traditional Chinese medicine treatment group （ 1.15 ± 0.09）, compared with the model group （0.47 ± 0. 11 ）, （P 〈 0.01 ）. It can significantly reduced the mean fluorescence intensity of calcium in PC12 ceils induced by KC1 （150 mmol. L-1） in traditional Chinese medicine treatment group compared with the model group （P 〈 0.05）. The basic calcium intensity in PC12 cells was down-regulated in traditional Chinese medicine pre-protection group compared with the control group and the model group. In addition, the level of CaMKⅡ mRNA in traditional Chinese medicine treatment group was up-regulated compared with that in model group （P 〈 0.05）. Conclusion： The serum containing with 10% Liuwei Dihuang Wan can effectively inhibit the intracellular calcium overload induced by KC1 and improve the expression of CaMK Ⅱ mRNA in PC12 cells.