化瘀祛痰方药及其拆方对H_2O_2诱导EA.hy926细胞凋亡的影响及机制研究

Effect and Mechanism of Huayu Qutan Recipe and Its Separated Recipes on EA.hy926 Cells Apoptosis Induced by H_2O_2

目的探讨化瘀祛痰方药及其拆方含药血清对过氧化氢(H2O2)诱导人脐静脉内皮细胞EA.hy926凋亡的影响及其机制。方法 40只SD大鼠随机分为5组,即全方组、补气组、化瘀组、祛痰组、空白血清对照组。各组大鼠分别以相应中药煎剂或生理盐水连续灌胃9 d,末次灌胃给药2 h后,腹主动脉采血,离心后分离血清。体外培养EA.hy926细胞,随机分为7组,即①正常对照组、②H2O2刺激组、③全方组、④补气组、⑤化瘀组、⑥祛痰组、⑦空白血清对照组。其中②组加入终浓度为300μmol.L-1H2O2,③～⑦组用各组含药血清(浓度为10%)预处理24 h后加入终浓度为300μmol.L-1H2O2,各组细胞培养24 h后进行各项指标测定。采用流式细胞术检测EA.hy926细胞凋亡;实时定量反转录—聚合酶链反应(Real-time PCR)定量分析bcl-2、bax、caspase-3 mRNA表达;比色法检测caspase-3活性;蛋白质免疫印记技术(Western-blot)检测bcl-2、bax蛋白表达。结果与正常对照组相比,H2O2刺激后EA.hy926细胞凋亡率、bax、caspase-3表达显著增加,而bcl-2表达显著减少。全方组细胞凋亡率、bax、caspase-3水平较H2O2刺激组相比显著降低,而bcl-2水平显著升高,且全方组干预作用强于各拆方组。拆方各组中,化瘀组、祛痰组细胞凋亡率显著降低,化瘀组bax、cas-pase-3表达显著减少,而bcl-2表达显著增加。结论化瘀祛痰方药全方及化瘀拆方可显著抑制内皮细胞凋亡,影响凋亡相关基因bcl-2、bax、caspase-3的表达,这可能是其抗AS的作用机制之一。

Objective To study the influence of Huayu Qutan recipe and its separated recipes on cultured EA.hy926 cell apoptosis induced by H2O2.Methods Fifty SD rats were equally divided into five groups in random： Huayu Qutan recipe group,Buqi recipe group,Huayu recipe group,Qutan recipe group and normal serum control group.The rats in all of the medication groups were intragastrically perfused daily with the decoction of the corresponding Chinese herbs,and the normal control group was given normal saline.Blood was collected from the abdominal aorta of rats 2 h after the last perfusion on the 9th day,and the serum was separated for testing.The cultured EA.hy926 cells were divided into 7 groups in random： the normal control group,H2O2 stimulating group,Huayu Qutan recipe group,Buqi recipe group,Huayu recipe group,Qutan recipe group and normal serum control groups.H2O2 stimulating group was cultured with final concentration of 300 μmol·L-1 H2O2,and the other groups were pretreated with 10 % drug-containing serum for 24 h and then were given 300 μmol·L-1 H2O2.After culture for 24 h,EA.hy926 cells apoptosis was detected by Annexin V-FITC/PI double-dyed flow cytometry,the mRNA expression of bcl-2,bax and caspase-3 were tested by real-time PCR,the activity of caspase-3 was detected by spectrophotometry technique,and protein expression of bcl-2 and bax were tested by western-blot method.Results Compared to the normal control group,the apoptotic rate and expression levels of bax and caspase-3 were significantly increased while bcl-2 expression level was significantly decreased after stimulation of H2O2.The apoptotic rate and expression levels of bax and caspase-3 were obviously decreased while bcl-2 expression level was obviously increased in Huayu Qutan recipe group as compared to H2O2 stimulating group.In the separated recipe groups,the apoptotic rate was decreased in Huayu recipe group and Qutan recipe group,and expression levels of bax and caspase-3 were decreased while bcl-2 expression level was increased in Huayu recipe group Conclusion Huayu Qutan recipe and its separated recipes show certain inhibitory effect on the apoptosis of the endothelial cells,and through regulating the expression levels of apoptosis-related genes such as bcl-2,bax and caspase-3 may be one of its anti-atherosclerosis molecular mechanism.