RESUMEN
Lipopolysaccharide (LPS) displays a robust immunostimulatory ability upon Toll-like receptor 4 (TLR4) recognition. N-methyl-D-aspartate receptors (NMDARs) are highly compartmentalized in most cells and implicated in various inflammatory disorders. However, the relationship between TLR4 and NMDARs has not been explored deeply. This study aimed to examine the role of NMDARs and its specific inhibitor MK801 in LPS-treated endothelial cell dysfunction and the related mechanism in vivo and in vitro. The results showed that pre-treatment with MK801 significantly decreased LPS-induced cell death, cellular Ca2+, cellular reactive oxygen species, and glutamate efflux. Moreover, MK801 restrained LPS-induced mitochondrial dysfunction by regulating mitochondrial membrane potential and mitochondrial Ca2+ uptake. The oxygen consumption, basal and maximal respiration rate, and ATP production in LPS-treated HUVECs were reversed by MK801 via regulating ATP synthesis-related protein SDHB2, MTCO1, and ATP5A. The molecular pathway involved in MK801-regulated LPS injury was mediated by phosphorylation of CaMKII and ERK and the expression of MCU, MCUR1, and TLR4. LPS-decreased permeability in HUVECs was improved by MK801 via the Erk/ZO-1/occluding/Cx43 axis. Co-immunoprecipitation assay and western blotting showed three subtypes of NMDARs, NMDAζ1, NMDAε2, and NMDAε4 were bound explicitly to TLR4, suppressed by LPS, and promoted by MK801. Deficiency of NMDAζ1, NMDAε2, or NMDAε4 induced cell apoptosis, Ca2+ uptake, ROS production, and decreased basal and maximal respiration rate, and ATP production, suggesting that NMDARs integrity is vital for cell and mitochondrial function. In vivo investigation showed MK801 improved impairment of vascular permeability, especially in the lung and mesentery in LPS-injured mice. Our study displayed a novel mechanism and utilization of MK801 in LPS-induced ECs injury and permeability.
RESUMEN
The strategy of decreasing atherosclerotic cardiovascular disorder is imperative for reducing premature death and improving quality of life in patients with diabetes mellitus. The aim of this study was to investigate whether the natural flavone acacetin could protect against endothelial injury induced by high glucose and attenuate diabetes-accelerated atherosclerosis in streptozotocin-(STZ) induced diabetic ApoE-/- mice model. It was found that in human umbilical vein endothelial cells (HUVECs) cultured with normal 5.5 mM or high 33 mM glucose, acacetin (0.3-3 µM) exerted strong cytoprotective effects by reversing high glucose-induced viability reduction and reducing apoptosis and excess production of intracellular reactive oxygen species (ROS) and malondialdehyde in a concentration-dependent manner. Acacetin countered high glucose-induced depolarization of mitochondrial membrane potential and reduction of ATP product and mitoBcl-2/mitoBax ratio. Silencing Sirt3 abolished the beneficial effects of acacetin. Further analysis revealed that these effects of acacetin rely on Sirt1 activation by increasing NAD+ followed by increasing Sirt3, pAMPK and PGC-1α. In STZ-diabetic mice, acacetin significantly upregulated the decreased signaling molecules (i.e. SOD, Bcl-2, PGC-1α, pAMPK, Sirt3 and Sirt1) in aorta tissue and attenuated atherosclerosis. These results indicate that vascular endothelial protection of acacetin by activating Sirt1/Sirt3/AMPK signals is likely involved in alleviating diabetes-accelerated atherosclerosis by preserving mitochondrial function, which suggests that acacetin may be a drug candidate for treating cardiovascular disorder in patients with diabetes.