自噬对氧化低密度脂蛋白损伤内皮细胞的保护作用

目的 探讨自噬对氧化低密度脂蛋白(ox-LDL)诱导人脐静脉内皮细胞(HUVEC)损伤的作用.方法 将培养的HUVEC分为正常对照组,ox-LDL组,ox-LDL加雷帕霉素组和ox-LDL加3-甲基腺嘌呤(3-MA)组,分别收集细胞和上清,其细胞用免疫印迹技术检查自噬标记蛋白LC3-Ⅱ/LC-Ⅰ的变化,四氮唑蓝(MTT)法和流式细胞技术检测细胞的增殖及凋亡;上清用于检测乳酸脱氢酶(LDH)活力和内皮素-1(ET-1)的含量.结果 ox-LDL作用能上调HUVEC的LC3-Ⅱ/LC-Ⅰ表达(P＜0.01),增加培养上清中LDH活力(P＜0.01)及ET-1含量(P＜0.05),诱导细胞发生增殖(P=0.028)、凋亡(P＜0.05).雷帕霉素能增加ox-LDL诱导的自噬水平上调,减少其培养上清中LDH活力及ET-1含量的增加(P＜0.05),抑制ox-LDL诱导的细胞增殖.相反,3-MA在下调ox-LDL诱导自噬水平(P＜0.01)的同时,会增加细胞的凋亡及死亡.结论 ox-LDL能通过增加HUVEC LDH漏出率及ET-1分泌水平,促进细胞增殖、凋亡,对内皮细胞产生损伤作用,上调自噬能够减少这种损伤,从而对细胞起到保护作用,反之亦然.

Protective effects of autophagy against oxidized LDL-induced injury in endothelial cells

Objective To investigate the role of autophagy in oxidized low density lipoprotein(ox-LDL)induced injury of human umbilical vein endothelial cells(HUVEC).Methods The cultured HUVECs were randomly divided into four groups of control, ox-LDL, ox-LDL + rapamycin and ox + 3-methyladenine(3-MA).The cells were used to detect the ratio of LC3-Ⅱ/LC3- Ⅰ by Western blot while the proliferation and apoptosis of cells measured by MTT and flow cytometry.The lactate dehydrogenase(LDH)activity and endothelin-1(ET-1)content in the supernatant were detected with enzyme linked immunosorbent assay.Results The Ox-LDL treatment up-regulated the ratio of LC3-Ⅱ/LC3-Ⅰ in HUVEC (P ＜ 0.01).It increased the activity of LDH(P ＜ 0.01)and content of ET-1(P ＜ 0.05)in the supernatant.Also it induced the proliferation(P = 0.028)and apoptosis(P ＜ 0.05)of cells.The autophagic inducer rapamycin increased the up-regulation of autophagic level induced by ox-LDL, decreased the activity of LDH and content of ET-1(P ＜ 0.05)and inhibited the ox-LDL-induced proliferation of cells.Conversely, the autophagic inhibitor 3-MA decreased the elevation of LC3 - Ⅱ/LC3 - Ⅰ induced by ox-LDL(P ＜ 0.01)and increased the cell apoptosis and death.Conclusion Ox-LDL exposure can increase the secretion of LDH and ET-1 and induce the proliferation and apoptosis of cells so as to cause a harmful effect in the survival of HUVEC.The injury may be reduced by rapamycin, an autophagic inducer, and elevated by the autophagic inhibitor 3-MA.