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目的探讨冠心平片抗过氧化氢(H2O2)诱导人血管内皮细胞(VEC)的凋亡作用及可能机制。方法选择新西兰大白兔15只,随机分成正常组、维拉帕米组、冠心平小剂量组、冠心平中剂量组、冠心平大剂量组,每组3只,分别给予生理盐水、维拉帕米缓释片0.02g/kg、冠心平片2.8g/kg、冠心平片5.6g/kg、冠心平片11.2g/kg,用蒸馏水溶解配置灌胃给药,10ml/kg,每天1次,连续3天,末次给药后1h,耳缘静脉采血,制备含药血清。采用100μmol/L浓度H2O2作用于对数生长期的VEC建立细胞凋亡损伤模型,建模同时用不同剂量冠心平含药血清进行干预。处理后收集细胞,进行流式细胞术检测VEC的凋亡率;采用免疫细胞化学法检测B细胞淋巴瘤/白血病-2相关联的x基因(bax)与B细胞淋巴瘤/白血病-2基因(bcl-2)蛋白的表达;采用PCR检测bax与bcl-2 mRNA的相对量。结果与模型组比较,冠心平大剂量组、冠心平中剂量组、维拉帕米组细胞凋亡率明显降低(P<0.05或P<0.01);维拉帕米组、冠心平小剂量组、冠心平中剂量组、冠心平大剂量组bax蛋白面密度、bax/bcl-2平均面密度与模型组比较均显著下降(P<0.05),bcl-2蛋白面密度显著上升(P<0.05或P<0.01),冠心平中剂量组与大剂量组细胞bax/bcl-2比值降低(P<0.05或P<0.01)。结论冠心平含药血清可以拮抗H2O2诱导VEC的凋亡效应,其机制可能与调节凋亡基因bax、bcl-2的表达有关。
Objective To investigate the anti-hydrogen peroxide (H2O2)-induced apoptosis and its possible mechanism of Guanxin Tablet in human vascular endothelial cells (VEC). Methods Fifteen New Zealand white rabbits were randomly divided into normal group, verapamil group, Guanxinping low-dose group, Guanxinping middle-dose group, and Guanxinping high-dose group. Three rats in each group were given normal saline and verapagin respectively. Sustained-release tablets of 0.02g/kg, Guanxin tablets 2.8g/kg, Guanxin tablets 5.6g/kg, Guanxin tablets 11.2g/kg, dissolved in distilled water and administered by gavage, 10ml/kg, once daily. For 3 consecutive days, 1 hour after the last administration, blood was collected from the marginal vein to prepare drug-containing serum. 100μmol/L H2O2 was applied to the VECs in logarithmic growth phase to establish apoptotic injury model. At the same time, different doses of Guanxinping-containing serum were used for intervention. After treatment, cells were harvested, flow cytometry was used to detect the apoptosis rate of VEC, and immunocytochemistry was used to detect B cell lymphoma/leukemia-2 associated x gene (bax) and B cell lymphoma/leukemia-2 gene ( Bcl-2) protein expression; the relative amount of bax and bcl-2 mRNA was detected by PCR. Results Compared with the model group, the apoptotic rate of Guanxinping large-dose group, Guanxinping middle-dose group, verapamil group was significantly lower (P<0.05 or P<0.01); Verapamil group, Guanxinping small-dose group The surface area density of bax and the average areal density of bax/bcl-2 in the Guanxinping middle-dose group and high-dose Guanxinping group were significantly lower than those in the model group (P<0.05). The areal density of bcl-2 protein increased significantly (P<0.05). Or P<0.01), the ratio of bax/bcl-2 in the Guanxinping middle-dose group and the high-dose group decreased (P<0.05 or P<0.01). Conclusion Guanxinping-containing serum can antagonize the apoptosis effect of H2O2-induced VEC, and its mechanism may be related to the regulation of apoptosis gene bax, bcl-2 expression.