Protection Effects And Mechanisms Of The Mitochondrial Na⁺/Ca²⁺ Exchanger ?NCLX? In Endothelial Cell

Background:Endothelial dysfunction is the basis of diabetic vascular injury. One of the main mechanisms of vascular endothelial injury is mitochondrial dysfunction induced by hyperglycemia stimulating. Mitochondrial Na+/Ca2+ exchanger, NCLX is the newest discovered sodium calcium exchanger protein. NCLX protein was first officially named in 2010, and later found expression in heart, pancreas, neurons and other organizations. NCLX played an important role in these organizations. NCLX localizes in the mitochondria cristae, and acts as mitochondrial calcium efflux regulator. But it is still unclear about the expression, localization, and function of NCLX in endothelial cells.Methods:MitoTracker(?) was used to mark mitochondria,; PECAM-1(CD31) was used to mark the endothelial level. Pearson's co-localization coefficient was used to determine the coincidence rate of MitoTracker(?) and NCLX. DM rats were induced by streptozotocin injection. The primary rat aortic endothelial cell REACs were cultured in PRMI1640. siRNA interference was used to down-express NCLX expression. NCLX protein overexpression was constructed in rat aortic endothelial cell line by Lentiviral-NCLX infection. Cells were stimulated by high glucose or hydrogen peroxide. Application of high glucose (35mmol/L) triggered a robust mitochondrial Ca2+ transient using laser confocal microscopy for measurement. Confocal microscopy, Luminometer chemiluminescence analyzer, Western Blot and ELISA were used to test ROS, ATP,8-OHdG, eNOS, NLRP3 inflammasome and its related factors changes.Results:1) We first identified NCLX was expressed in rat aortic endothelia and located in mitochondria 2) We found that NCLX expression was significantly increased in DM rat aortic endothelia. This was consistent with RAECs in vitro, which were stimulated by high glucose (35mmol/L) culturing (P<0.05).3) Suppression of NCLX expression enhanced mitochondrial Ca2+ influx and blocked efflux induced by glucose. Comparison of the peak amplitude of mitochondrial Ca2+ signals demonstrated an increase in cells in siNCLX group. And AROS production was markedly increased by high glucose stimulation in the siNCLX group (P<0.05).4) RAEC cells whitch were treated with siNCLX or siControl were cultured in high glucose (35mmol/L) for 24 h. Total intracellular ROS production and NLRP3 expression were significantly increased in siNCLX vs. siControl cells. We next determined caspasel expression and IL-1? release, consistent with NLRP3 expression(P<0.05).5) NCLX protein overexpression was constructed in rat aortic endothelial cell line by Lentiviral-NCLX infection, Lentiviral-vector infection as control group. The cells were cultured in hydrogen peroxide (100umol/L) for 24 hours. The concentration of ATP was significantly increased,8-OHdG expression was significantly decreased and eNOS expression was significantly increased in LV-NCLX vs. LV-vector cells (P<0.05).Conclusions:NCLX was expressed in rat aortic endothelia and located in the mitochondria. NCLX may affect hyperglycemia-induced mitochondrial function by regulation of mitochondrial calcium efflux.And NCLX played an important role on regulating of ROS generation and the NLRP3 inflammasome. We Speculated NCLX expression was significantly increased in early stage DM rat contribute to protect mitochondrial stabilization and function.