eNOS参与铁过载诱导的血管内皮细胞线粒体损伤

Mitochondrial damage induced by iron overload via eNOS in vascular endothelial cells

目的探讨e NOS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e NOS表达;比色法检测培养液LDH活性、NO含量、细胞MDA含量以及m PTP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e NOS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,m PTP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 e NOS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。

Aim To investigate the damage of mitochondria in HUVECs cells by iron overload and the role of ADMA/e NOS/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 DDAH Ⅱ activity were measured by HPLC method; the expression of e NOS was determined by Western blot; LDH activity,MDA and NO content,and mitochondrial permeability transition pores（ m PTP） 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,DDAH Ⅱ activity,and the expression of e NOS markedly decreased,the generation of ROS was evidently elevated,mitochondrial membrane potential was lost apparently,m PTP 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 e NOS and induce the apoptosis of HUVECs,in which ADMA/DDAH Ⅱ mechanism may also be engaged.