目的:研究补阳还五汤含药血清对同型半胱氨酸(Hey)致内皮细胞株(Eahy926)损伤的保护作用及其机制。方法:采用体外培养的内皮细胞株(Eahy926),测定不同的补阳还五汤含药血清对Hey致内皮细胞活力的影响,检测细胞中超氧化物歧化酶(SOD)、乳酸脱氢酶(LDH)活力和丙二醛(MDA)含量;ELISA法检测可溶性细胞间黏附分子(sICAM-1)和肿瘤坏死因子-a(TNF-a)的浓度;荧光显微镜检测核转录因子-KB(NF-tcB)的激活及Westernblotting法测I-KBtx蛋白表达。结果:10%,20%的补阳还五汤含药血清均能明显提高Hey损伤的内皮细胞的存活率、SOD[分别为(15.87±0.46),(16.31±0.96)U·mL-1]活力,与损伤组(11.44±1.00)U·mL-1相比,P〈0.01;降低LDH的释放和MDA[分别为(1.62±0.06),(1.47±0.18)nmol·L-1)的含量,与损伤组(1.89±0.25)nmol·L-1)相比,P〈0.05;以及细胞培养液中sICAM-1[分别为(0.93±0.11),(1.00±0.14)pg·L-1]和TNF-α[分别为(63.71±2.64),(66.22±4.43)ng·L-1]浓度,与损伤组(2.32±0.23)μg·L-1,(125.67±2.69)ng·L-1)相比,P〈0.05;同时抑制内皮细胞中NF-KB的激活,上调I-KBa蛋白表达量。结论:补阳还五汤含药血清对同型半胱氨酸致内皮细胞损伤具保护作用,其保护作用机制可能与抑制NF-KB的活化有关。
Objective: To observe the effects and mechanism of the serum containing Buyang Huanwu decoction on endothelial dysfunction induced by homoeysetine (Hey) in human umbilical vein endothelial cell. Method: Human umbilical vein endothelial cells (Eahy926) were incubated with Hey. Cell viability, the activity of superoxide dismutase (SOD), lactate dehydrogenase (LDH), and the content of malondialdehyde (MDA) in the medium were determined and the concentrations of sICAM-1 (by ELISA), TNF-a(by ELISA) in the conditioned medium. The activity of NF-KB was determined by fluorescence microscopy and the expression of I- KBa protein were measured via Western-blot. Result: Incubation of Eahy926 with different concentrations of the serumcontaining Buyang Huanwu decoction (5% , 10% , 20% ) enhanced cell viability. Pretreatment with the serum containing Buyang Huanwu decoction 10% , 20% group improved activity of SOD E which was ( 15.87 ± 0.46), (16.31 ±0.96) U .mL-1] compared with the model group (11.44 ± 1.00) U .mL-1, P 〈0.01;decreased the levels of LDH, MDA (1.62 ±0.06), (1.47 ±0. 18) nmol -L-1 , compared with the model group ( 1.89 ± 0. 25) nmol -L-1, P 〈 0.01 ; down-regulated the content of sICAM-1 (0.93 ± 0. 11 ), ( 1.00 ± 0, 14) μg .L-1 and TNF-a (63.71 ±2.64), (66.22 ±4.43) ng .L-1, compared with the model group (2. 32 ±0. 23) μg.L-1, (125.67 ± 2.69) ng .L-1, P 〈0. 05; as well as markedly inhibited the increased activity of NF-KB. Conclusion: The serum containing Buyang Huanwu decoction could prevent endothelial dysfunction induced by Hcy and inhibition the activity of NF-KB may be its mechanism of preventing.