线粒体损伤在脂多糖诱导的血管内皮细胞凋亡中的作用

Role of mitochondrial injury in lipopolysaccharide-induced vascular endothelial cell apoptosis

目的:探讨脂多糖(lipopolysaccharide,LPS)对血管内皮细胞凋亡的影响及线粒体损伤在其中的作用。方法:LPS刺激体外培养人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)后,采用CCK-8检测血管内皮细胞存活率,Western blot检测HUVECs细胞内Bax、Bcl-2、细胞色素C(cytochrome C,Cyt C)和Cleaved-caspase 3蛋白的表达,用流式细胞计数检测细胞的凋亡情况,用荧光探针Mitotracker、JC-1、Mito SOX染色检测细胞内线粒体形态及其膜电位的变化,以及线粒体来源活性氧(reactive oxygen species,ROS)的产生水平;用MitoTEMPOL+LPS干预细胞,检测Bax、Bcl-2、Cyt C蛋白表达以及凋亡。结果:LPS浓度依赖性降低细胞存活率(F=99.31,P=0.000)。与对照组相比,LPS可以明显诱导HUVECs细胞凋亡(t=17.184,P=0.000),并呈浓度依赖性增加Cleaved-caspase 3(F=13.52,P=0.001)、Cyt C(F=21.008,P=0.000)和Bax(F=16.325,P=0.000)蛋白表达,并减少Bcl-2(F=17.287,P=0.000)蛋白的表达。LPS刺激后HUVECs细胞内线粒体发生明显的片段化和颗粒状变、线粒体膜电位下降(F=92.286,P=0.000),线粒体来源的ROS产生明显增加(t=5.324,P=0.006)。采用线粒体ROS清除剂(MitoTEMPOL)处理,可抑制LPS刺激的HUVECs细胞Bax(F=19.854,P=0.002)和Cyt C(F=11.594,P=0.009)蛋白表达,增加Bcl-2(F=8.077,P=0.020)蛋白表达,同时降低Cleaved-caspase 3(F=15.941,P=0.004)蛋白表达和减少细胞凋亡(F=57.482,P=0.000)。结论:LPS可以诱导HUVECs细胞凋亡和线粒体损伤,损伤线粒体所释放的ROS可能在LPS诱导的血管内皮细胞凋亡的过程中发挥了重要的作用。

Objective:To investigate the effect of lipopolysaccharide(LPS) on vascular endothelial cell apoptosis and the role of mitochondrial injury in the process. Methods:The human umbilical vein endothelial cells(HUVECs) cultured in vitro were stimulated with LPS,then Cell Counting Kit-8 was used to determine the viability of vascular endothelial cells;Western blot was used to determine the protein expression of Bax,Bcl-2,cytochrome C(Cyt C),and cleaved caspase-3(CC3);flow cytometry was used to detect cell apoptosis;fluorescent staining with Mitotracker,JC-1,and Mito SOX was employed to determine the changes in mitochondrial morphology in the cells and their membrane potentials as well as the production level of reactive oxygen species(ROS) of mitochondrial origin. The cells were treated with MitoTEMPOL and LPS,and then were tested for cell apoptosis and the protein expression of Bax,Bcl-2,and Cyt C. Results:LPS reduced cell viability in a concentration-dependent manner(F=99.310,P=0.000). Compared with those in the control group,LPS significantly induced the apoptosis of HUVECs(t=17.184,P=0.000),concentration-dependently increased the protein expression of CC3,Cyt C,and Bax(F=13.520,21.008,and 16.325,respectively,P=0.001,0.000,and 0.000,respectively),and reduced the protein expression of Bcl-2(F=17.287,P=0.000). LPS stimulation resulted in significant fragmentation and granular degeneration of the mitochondria in HUVECs,decreased mitochondrial membrane potential(F=92.286,P=0.000),and significantly increased production of ROS of mitochondrial origin(t=5.324,P=0.006). Treatment with the mitochondrial ROS scavenger MitoTEMPOL inhibited the protein expression of Bax and Cyt C(F=19.854 and 11.594,respectively,P =0.002 and 0.009,respectively),increased the protein expression of Bcl-2(F=8.077,P=0.020),reduced the protein expression of CC3(F =15.941,P =0.004),and reduced cell apoptosis(F=57.482,P=0.000) in the LPS-stimulated HUVECs. Conclusion:LPS can induce mitochondrial injury and apoptosis in HUVECs,and the ROS released from injured mitochondria may play an important role in LPS-induced vascular endothelial cell apoptosis.