不同程度炎性血管内皮细胞损伤时线粒体功能及过氧化物酶体增殖物活化受体γ共激活因子-1α、活化T细胞核因子的变化

Changes of Mitochondrial Function, PGC-1α and NFAT in Different Degree of Inflammatory Vascular Endothelial Cell Injury

目的 观察肿瘤坏死因子（TNF-α）诱导的离体血管内皮细胞损伤后线粒体功能、过氧化物酶体增殖物活化受体γ共激活因子-1α（PGC-1α）、活化T细胞核因子1（NFAT1）、活化T细胞核因子2（NFAT2）的变化.方法 （1）不同浓度的TNF-α（10、20、40、80 ng/mL）刺激人脐静脉内皮细胞（HUVECs）12、24、36 h制备不同程度的血管内皮细胞损伤模型,CCK-8法检测细胞活性、AnnexinⅤ-FITC/PI双染法检测细胞凋亡,倒置光学显微镜下观察细胞形态,以此确定模型是否建立成功.（2）采用Jc-1免疫荧光染色结合荧光显微镜检测线粒体膜电位,MitoSOX？Red染色结合荧光显微镜检测线粒体活性氧（mtROS）.（3）采用RT-qPCR、Western blot方法分别检测PGC-1α、NFAT1、NFAT2 mRNA及蛋白水平.结果 （1）随着TNF-α刺激浓度及作用时间的增加,HUVECs活性逐渐下降,在浓度为40 ng/mL,刺激时间为24 h,细胞活性下降约49％,接近TNF-α诱导HUVECs损伤的IC50,差异有统计学意义（P〈0.05）.20、40、80 ng/mL TNF-α刺激HUVECs 24 h后,细胞凋亡率逐渐升高,差异具有统计学意义（P〈0.05）.（2）随着血管内皮细胞损伤程度的增加,线粒体膜电位逐渐下降,mtROS逐渐升高（均P〈0.05）.（3）PGC-1αmRNA及蛋白表达水平逐渐下降,核转录因子NFAT1、NFAT2 mRNA及蛋白表达水平逐渐升高（均P〈0.05）.结论 随着血管内皮细胞损伤程度的增加,线粒体功能障碍逐渐加剧,NFAT表达量逐渐增加.

Objective To observe the changes of mitochondrial function, peroxisome proliferator-activated receptor-γ coactivator-1α （PGC-1α） and nuclear factor of activated T cell （NFAT） in TNF-α-induced injury of vascular endothelial cells. Methods （1）Different concentrations of tumor necrosis factor TNF-α （10, 20, 40, 80 ng/mL） stimulated human umbilical vein endothelial cells （HUVECs） 12, 24, 36 hours to prepare different degrees of vascular endothelial cell injury model, and CCK-8 method was used to detect the cell viability. Apoptosis was detected by AnnexinⅤ-FITC/PI double staining method. Morphological changes were observed under inverted optical microscope to determine whether the model was established successfully. （2）The mitochondrial membrane potential was detected by JC-1 immunofluorescence staining and fluorescence microscopy, MitoSOX？ Red staining combined with fluorescence microscopy was used to detect mitochondrial reactive oxygen species. （3）RT-qPCR, Western blot method to detect mRNA and protein levels of PGC-1α, NFAT1 and NFAT2. Results （1）With the increase of TNF-α concentration and the time of action, the activity of HUVECs decreased gradually. When the concentration was 40 ng/mL and the stimulation time was 24 hours, the cell viability decreased by about 49%, which was close to the IC50 of HUVECs injury induced by TNF-α. The difference was statistically significant （P〈0.05）. After treatment with TNF-α at the concentration of 20, 40 and 80 ng/mL, the apoptotic rate of HUVECs increased gradually, and the difference was statistically significant （P〈0.05）. （2）With the increase of vascular endothelial cell injury, the mitochondrial membrane potential was decreased gradually, and the level of mtROS was increased gradually （all P〈0.05）. （3）The mRNA and protein levels of PGC-1α were decreased gradually, and the mRNA and protein levels of NFAT1, NFAT2 were increased gradually （all P〈0.05）.Conclusion With the increase of vascular endothelial cell injury, mitochondrial dysfunction gradually increased, NFAT expression increased gradually.