基于内皮损伤学说的通阳化浊方干预冠心病细胞实验研究

Experimental Study on Tongyang Huazhuo Decoction(通阳化浊方) in Intervention of Coronary Heart Disease Based on Theory of Endothelial Injury

目的基于内皮损伤学说,探讨通阳化浊方干预冠心病的作用机制。方法培养人主动脉血管内皮细胞,以灌服蒸馏水的大鼠血清为对照,在人主动脉血管内皮细胞培养基中加入H2O2,并在实验体系中加入灌服通阳化浊方、四妙勇安汤灌胃处理的大鼠血清。收集各组细胞上清液,用酶联免疫吸附双抗体夹心法测各组细胞培养液上清中SOD、MDA和NO的含量;流式细胞仪Annexin V-PI双染检测人主动脉血管内皮细胞的凋亡发生率,噻唑蓝(MTT)法检测人主动脉血管内皮细胞的增殖活性,鬼笔环肽染色细胞骨架。结果 H2O2显著降低人主动脉血管内皮细胞培养上清液中NO、SOD的含量,升高MDA含量,降低人主动脉血管内皮细胞增殖活性,促进内皮细胞凋亡,使内皮细胞皱缩、细胞骨架排列紊乱。给药组能显著降低MDA含量,升高NO、SOD含量,与四妙勇安汤相比,通阳化浊方组血清增加内皮细胞活性效果更好,降低内皮细胞凋亡率,使内皮细胞骨架更加清晰;而灌服蒸馏水的大鼠血清则无此作用。结论通阳化浊方组的大鼠血清对H2O2造成的人主动脉内皮细胞损伤具有更好保护作用。

Objective To investigate the therapeutic mechanism of Tongyang Huazhuo Decoction(通阳化浊方) for coronary heart disease intervention based on the theory of endothelial injury.Methods The human aortic vascular endothelial cells were cultured.The serum of rats which were treated with distilled water was as the control.H2O2 was added to the culture medium of human aortic vascular endothelial cells,and the serum of rats treated with Tongyang Huazhuo Decoction and Simiao Yong’an Decoction(四妙勇安汤) were added to the experimental system.We collected the cell supernatant of each group,and used enzyme-linked immunosorbent assay(ELISA) double antibody sandwich method to detect the contents of SOD,MDA and NO in the cell supernatant of each group.Annexin V-PI flow cytometry double staining was used to detect the apoptosis rate of vascular endothelial cells in human aorta and the proliferative activity of endothelial cells in human aorta was detected by MTT method.The cytoskeleton was stained with the ghost pen ring peptide.Results H2O2 significantly reduced the contents of NO and SOD in the supernatant of human aortic endothelial cell culture,increased the contents of MDA,reduced the proliferation activity of human aortic endothelial cells and promoted endothelial cell apoptosis.It also can cause endothelial cells to contract and cytoskeleton arrangement disorder.The content of MDA was significantly decreased and the contents of NO and SOD were increased in the drug administration groups.Compared with that of the SimiaoYong ’An Decoction group,the serum of Tongyang Huazhuo Formula group can increase the activity of endothelial cells,reduce the rate of apoptosis of endothelial cells,and make the endothelial cytoskeleton clearer.The serum of rats treated with distilled water had no such effect.Conclusion The serum of Tongyang Huazhuo Formula group had more protective effect on the injury of human aortic endothelial cells caused by H2O2.