摘要
目的：通过真武汤抗大鼠心肌细胞凋亡机制的体外实验研究,阐述真武汤抗心肌细胞凋亡的具体机制。方法：（1）采用MTT法分别观察于3 h、6 h、12 h三个时间点异丙肾上腺素对心肌细胞活性的影响,测定细胞存活率,选择建立体外心肌细胞损伤模型所需异丙肾上腺素的最佳时间及剂量;（2）筛选真武汤含药血清的最佳浓度;（3）实验分为模型对照组[异丙肾上腺素（10-6 mol/L）]、空白对照组、真武汤组（异丙肾上腺素＋10%含药血清）、卡托普利组[异丙肾上腺素＋卡托普利（10^-6mol/L）];（4）AO/EB双重荧光染色和流式细胞仪检测心肌细胞凋亡;（5）Western blotting方法测定培养心肌细胞凋亡相关蛋白Caspase-3的表达。结果：（1）异丙肾上腺素对心肌细胞的生长有抑制作用;异丙肾上腺素10μmol/L作用6h后,细胞存活率降低明显。（2）真武汤含药血清浓度的选择：各组心肌细胞与相应干预措施作用48h后,各干预组细胞活性均低于空白组,差异有统计学意义（P〈0.05）;卡托普利组及不同浓度含药血清组细胞活性均高于异丙肾上腺素组（P〈0.05）;20%含药血清组、10%含药血清组与卡托普利组相比细胞存活率相似,差异无统计学意义,但考虑到中药的细胞毒作用及成本,选择10%含药血清组进行后续实验。（3）AO/EB双重荧光染色检测细胞凋亡：与空白组比较,真武汤组及西药组可见明显抑制心肌细胞凋亡。（4）流式细胞仪检测心肌细胞凋亡：模型对照组细胞凋亡率明显升高,与空白对照组比较有显著差异（P〈0.01）;卡托普利组及真武汤组与模型对照组相比细胞凋亡明显降低,有统计学差异（P〈0.05）。（5）Western Blotting检测结果：真武汤组和卡托普利组与模型对照组相比较,Caspase-3蛋白表达降低（P〈0.05）。结论：真武汤能降低心肌细胞损伤,抑制心肌细胞凋亡,其机制可能与�
Objective：Through the in vitro experimental study on the mechanism of Zhenwu Decoction against cardiomyocyte apoptosis in rats,the specific mechanism of Zhenwu Decoction against cardiomyocyte apoptosis was described. Methods：1. The effects of isoproterenol on the activity of cardiomyocytes at 3 h,6 h and 12 h were observed by MTT method. The survival rate of cells was determined. The best time for establishing isoproterenol for in vitro cardiomyocyte injury model was selected. 2 We selected the best concentration of Zhenwu Decoction containing serum. 3. The experiment was divided into model control group [isoproterenol（10^-6 mol/L）],blank control group,Zhenwu Decoction group（ isoproterenol ＋ 10% drug-containing serum） and captopril [isoproterenol ＋ captopril（10^-6 mol/L）]. 4. AO/EB dual fluorescent staining and flow cytometry detected myocardial cell apoptosis. 5. Western blotting method determined the expression of apoptosis-related protein Caspase-3 in cultured cardiomyocytes. Results：1. Isoproterenol inhibited the growth of cardiomyocytes. After 6 hours of isoproterenol 10 μmol/L,the cell survival rate decreased significantly. 2. The concentration of Zhenwu Decoction containing serum concentration：after 48 hours of myocardial cells in each group and corresponding intervention measures,the activity of cells in each intervention group was lower than that in the blank group,and the difference was statistically significant（ P〈0.05）. Captopril group＇s cell viability of different concentrations of drug-containing serum was higher than that of isoproterenol（ P〈0.05）. The cell viability was similar between20% drug-containing serum and 10% drug-containing serum and captopril. There was no statistical significance. But considering the cytotoxicity and cost of traditional Chinese medicine,we selected the 10% drug-containing serum group for follow-up experiments. 3. Apoptosis was detected by double fluorescent staining of AO/EB. Compared with the blank group,Zhenwu Decoction group an
作者
王宇宏
李文杰
李峥
WANG Yuhong;LI Wenjie;LI Zheng(Liaoning University of TCM,Shenyang 110032,Liaoning,China;The Affiliated Hospital of Liaoning University of TCM,Shenyang 110032,Liaoning,China)
出处
《辽宁中医杂志》
CAS
2018年第7期1550-1553,I0005,共5页
Liaoning Journal of Traditional Chinese Medicine
基金
国家自然科学基金面上项目(81173184)
