eNOS参与铁过载诱导的血管内皮细胞线粒体损伤

摘要

Aim To investigate the damage of mitochondria in HUVECs cells by iron overload and the role of ADMA/eNOS/DDAHⅡ in it.Methods HUVECs cells were cultured and randomly divided into normal control (Ctrl) group, dextran iron (Iron) group and L-arginine (L-Arg) group.After 48 h, the survival rate of cells was detected by MTT assay;ADMA content and DDAHⅡactivity were measured by HPLC method;the expression of eNOS was determined by Western blot;LDH activity, MDA and NO content, and mitochondrial permeability transition pores(mPTP) openness were determined by colorimetric assay;ROS generation, mitochondrial membrane potential and apoptosis were determined by flow cytometry.Results After 48 h treatment with iron, the survival rate of HUVECs significantly decreased, while the activity of LDH in culture medium increased.The results showed that ADMA and MDA content significantly increased, NO content, DDAHⅡactivity, and the expression of eNOS markedly decreased, the generation of ROS was evidently elevated, mitochondrial membrane potential was lost apparently, mPTP openness was obvious, and the apoptosis of the HUVECs were worsened.However, as ADMA physiological antagonist, L-Arg significantly attenuated the above effects of iron.Conclusion Iron overload could damage mitochondrial function by eNOS and induce the apoptosis of HUVECs, in which ADMA/DDAHⅡ mechanism may also be engaged.%目的 探讨eNOS及ADMA/DDAHⅡ介导的铁过载对HUVECs细胞线粒体的损伤作用.方法 常规培养HUVECs细胞,随机分为正常对照(Ctrl)组、右旋糖酐铁(Iron) 组、L-精氨酸(L-Arg)组.48 h后,MTT法检测细胞存活率;HPLC法检测ADMA含量及DDAHⅡ活性;Western blot法检测eNOS表达;比色法检测培养液LDH活性、NO含量、细胞MDA含量以及mPTP开放;流式细胞仪检测心肌细胞ROS含量、线粒体膜电位及细胞凋亡.结果 Iron处理48 h后,HUVECs细胞存活率明显降低,培养液ADMA及LDH活性升高,NO含量减少;细胞eNOS表达下调、DDAHⅡ活性降低;MDA含量与ROS生成明显增加,线粒体膜电位减小,mPTP大量开放,细胞凋亡增加;ADMA生理性对抗剂L-Arg则可明显减弱Iron的上述损伤作用.结论 eNOS参与铁过载诱导的HUVECs细胞线粒体损伤,ADMA/DDAHⅡ机制也可能发挥了作用.