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

Abstract:

Objective：To investigate the effect of lipopolysaccharide（LPS） on vascular endothelial cell apoptosis and the role of mito－chondrial 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 deter－mine 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 mito－chondrial membrane potential（F=92.286，P=0.000），and signif－icantly increased production of ROS of mitochondrial origin（t=5.324，P=0.006）. Treatment with the mitochondrial ROS scav－enger 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 vascu－lar endothelial cell apoptosis.