冠心平片含药血清对(H_2)(O_2)诱导人血管内皮细胞凋亡及NF-~κB蛋白表达的影响

Effects of Guanxinping Tablet Containing Serum on(H_2)(O_2)-induced Apoptosis and NF-~κB Expressions in Vascular Endothelial Cells

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。

Objective To study the effects of Guanxinping Tablet （GT） containing serum on H202-in- duced apoptosis and the nuclear factor kappa B （NF-KB） 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 extrac- ted and deactivated at 56 C for 30 min to prepare the drug containing serum. The apoptosis injury model was es- tablished using 100 pmol/L H202 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 ,mol/L H202）, the verapamil group （10% verapamil serum culture solution ＋100 pmol/L H202）, the low dose GT group （10% low dose GT culture solution ＋100 μmol/L H202） , the middle dose GT group （10% middle dose GT culture solution ＋ 100 μmol/L H202）, and the high dose GT group （10% high dose GT culture solution ＋ 100 tJmol/L H202）. THE VEC apoptotic rate was detected using flow cytometry. The protein expression of NF-KB was detected using Western blot. Results The VEC apoptosis rate （9.00% ＋1.18% ） and the protein expression of NF-KB （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 ver- apamil 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-KB of each treatment group significantly de- creased （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）. Conclusiens GT could fight against H202-induced VEC cell apoptosis. Its mechanism might be correlated with regulating the ex- pression of NF-KB protein.