冠心平片含药血清对（H2）（O2）诱导人血管内皮细胞凋亡及NF-κB蛋白表达的影响

作者	单位
严士海	江苏省中医院科技处
李七一	江苏省中医院科技处
王海丹	江苏省中医院科技处
万盟	江苏省中医院科技处

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中文摘要:

目的探讨冠心平片含药血清抗H2O2诱导血管内皮细胞(vascular endothelialcells,VEC)凋亡作用及对核因子-κB(nuclear factor kappa B,NF-κB)蛋白表达的影响。方法将大白兔随机分为正常组(生理盐水,10mL/kg)、维拉帕米组(0.02g/kg,10mL/kg)、冠心平小剂量组(2.8g/kg,10mL/kg)、冠心平中剂量组(5.6g/kg,10mL/kg)、冠心平大剂量组(11.2g/kg,10mL/kg),每组3只。灌胃给药,连续3天,最后1天灌胃2次,间隔2h,末次给药后1h,耳缘静脉采血,放置1h,2500r/min离心30min,吸取血清,56℃、30min灭活,制备含药血清。采用100μmol/LH2O2作用于对数生长期的VEC建立细胞凋亡损伤模型。造模后分为6组,每组5个样本:正常组(10%空白血清培养液)、模型组(10%空白血清培养液+100μmol/LH2O2)、维拉帕米组(10%维拉帕米血清培养液+100μmol/LH2O2)、冠心平低剂量组(10%低剂量冠心平血清培养液+100μmol/LH2O2)、冠心平中剂量组(10%中剂量冠心平血清培养液+100μmol/LH2O2)、冠心平高剂量组(10%高剂量冠心平血清培养液+100μmol/LH2O2)。采用流式细胞术检测VEC凋亡率,采用Westernblot检测NF-κB蛋白表达。结果模型组VEC凋亡率(9.00%±1.18%)、NF-κB蛋白表达(0.39%±0.06%)较正常组升高(P<0.01);与模型组比较,维拉帕米组(6.00%±0.18%)及冠心平高剂量组(5.30%±0.08%)、中剂量组(6.83%±0.51%)VEC凋亡率明显降低,各给药组NF-κB蛋白表达显著降低(维拉帕米组:0.28%±0.03%,冠心平低剂量组:0.33%±0.03%,冠心平中剂量组:0.30%±0.03%,冠心平高剂量组:0.28%±0.04%,P<0.01,P<0.05)。结论冠心平片可以拮抗H2O2诱导VEC的凋亡效应,其机制可能与调节NF-κB有关。

英文摘要:

Objective To study the effects of Guanxinping Tablet(GT) containing serum on H2O2-induced apoptosis and the nuclear factor kappa B(NF-κB) expression in vascular endothelial cells(VECs).Methods Rabbits were randomly divided into the normal control group(treated with normal saline,10 mL/kg),the verapamil group(0.02 g/kg,10 mL/kg),the small dose GT group(2.8 g/kg,10 mL/kg),the middle dose GT group(5.6 g/kg,10 mL/kg),and the large dose GT group(11.2 g/kg,10 mL/kg),3 in each group.The medication was given to rabbits by gastrogavage for 3 successive days.The gastrogavage was performed twice on the last day with an interval of 2 h.One h after the last medication the peripheral blood was sampled from the vein of the ear edge.The blood was put for 1 h and centrifuged at 2 500 r/min for 30 min.The serum was extracted and deactivated at 56 ℃ for 30 min to prepare the drug containing serum.The apoptosis injury model was established using 100 μmol/L H2O2 induced VECs in the log phase growth.After modeling they were divided into 6 groups,5 samples in each group,i.e.,the normal group(10% vehicle serum culture solution),the model group(10% vehicle serum culture solution+100 μmol/L H2O2),the verapamil group(10% verapamil serum culture solution +100 μmol/L H2O2),the low dose GT group(10% low dose GT culture solution +100 μmol/L H2O2),the middle dose GT group(10% middle dose GT culture solution+100 μmol/L H2O2),and the high dose GT group(10% high dose GT culture solution+100 μmol/L H2O2).THE VEC apoptotic rate was detected using flow cytometry.The protein expression of NF-κB was detected using Western blot.Results The VEC apoptosis rate(9.00%±1.18%) and the protein expression of NF-κB(0.39%±0.06%) increased more in the model group than in the normal control group(P<0.01).Compared with the model group,the VEC apoptosis rate of the verapamil group(6.00%±0.18%),the large dose GT group(5.30%±0.08%),and the middle dose GT group(6.83%±0.51%) were obviously lower.The expression of NF-κB of each treatment group significantly decreased(the verapamil group:0.28%±0.03%;the small dose GT group:0.33%±0.03%;the middle dose GT group:0.30%±0.03%;the large dose GT group:0.28%±0.04%,P<0.01,P<0.05).Conclusions GT could fight against H2O2-induced VEC cell apoptosis.Its mechanism might be correlated with regulating the expression of NF-κB protein.

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