香烟提取物诱导牛冠状动脉内皮细胞损伤的机制

Mechanism of Bovine Coronary Artery Endothelial Cells Damage Induced by Cigarette Smoke Extract

目的通过观察香烟提取物对牛冠状动脉内皮细胞(BCAEC)的损伤作用,为研究吸烟与心血管疾病之间的关系提供依据。方法以吸烟者体内通常的尼古丁浓度为基准,分别用尼古丁、香烟主流烟雾提取物(MSW)和侧流烟雾提取物(SSW)对 BCAEC 进行染毒(尼古丁染毒终浓度分别为1×10^(-5)、0.8×10^(-5)、0.9×10^(-5)mol/L),用显微数码成像系统记录细胞形态变化,检测凋亡细胞与坏死细胞的发生比例,测定 caspase 酶活力。结果经尼古丁、MSW 处理的 BCAEC 形态上发生凋亡样变化,处理24 h 后凋亡细胞发生率为5.89%和11.94%。而 SSW 处理后的 BCAEC 呈坏死样变化.处理24 h 后62.84%的细胞发生坏死。尼古丁和 MSW 可诱导 caspase-3活力增高。结论香烟提取物可诱导牛冠状动脉内皮细胞损伤,MSW 可引起细胞凋亡,SSW 导致细胞坏死,caspase-3的激活可能是香烟提取物诱导牛冠状动脉内皮细胞凋亡的机制。

Objective To study the bovine coronary artery endothelial cells （BCAEC） damage induced by cigarette abstracts and further clarify the relationship between smoking and cardiovascular diseases. Methods BCAEC were treated with nicotine, mainstream smoke extract （MSW） and sidestream smoke extract （SSW） which had the normal concentration （1.0× 10^-5,0.8× 10^-5, 0.9×10^-5 mol/L） of nicotine in smoker. The morphological changes of BCAEC were recorded by microscope digital image system. The quantification of apoptotic BCAEC cells was performed by visualization of nuclei stained with 4, 6 ＇-diamidino-2-phenylindole and trypan blue exclusion assay was used to examine the percentage of necrotic BCAEC. The caspase activity assay was employed to discuss the mechanism of BCAEC apoptosis. Results BCAEC exposed to nicotine and MSW appeared the typical morphological alteration of apoptosis and necrotic morphological alteration were observed after BCAWC were treated with SSW. 5.89% and 11.94% apoptotic cells were found after BCAEC were exposed to nicotine and MSW for 24 hours. The level of BCAEC necrosis after treated with SSW was 62.84%. Caspase-3 activity was induced by nicotine and MSW. Conclusion Cigarette smoke extract can induce the cell death of BCAEC. Nicotine and MSW can induce caspase-3 activity increase. Even in the presence of a non-cytotoxic concentration of nicotine and mainstream smoke solution, protease-induced apoptosis of BCAEC can be significantly increased. Sidestream smoke solution may cause BCAEC necrosis instead of apoptosis. Caspase-3 activation is probably the mechanism of BCAEC apoptosis.