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Methylglyoxal (MGO), an active metabolite of glucose, can cause cellular injury which has an affinity for the progression of diabetes-associated atherosclerosis. Phosphocreatine (PCr) is a well-known high-energy phosphate compound. However, its protective effects and mechanism in the formation of a diabetes-associated atherosclerosis have not been clarified. In the present study, we investigated whether PCr could prevent MGO-induced apoptosis in human umbilical vascular endothelial cells (HUVECs) and explored the possible mechanisms. Cells were pre-treated with PCr and then stimulated with MGO. Cell morphology, cytotoxicity and apoptosis were assessed by light microscopy, MTT assay, and Annexin V-FITC respectively. Apoptotic-related proteins were evaluated by Western blotting. Reactive oxygen species (ROS) generation, intracellular calcium and mitochondrial membrane potential (MMP) were measured with fluorescent probes. Our results showed that PCr dose-dependently prevented MGO associated HUVEC cytotoxicity and suppressed MGO activated ROS generation as well as apoptotic biochemical changes such as lactate dehydrogenase, malondialdehyde leakage, loss of MMP, decreased Bcl-2/Bax protein ratio, levels of caspase-3 and 9. In addition, the antiapoptotic effect of PCr enhanced p-Akt/Akt protein ratio, NO synthase (eNOS) activation, NO production and cGMP levels and also was partially suppressed by a PI3K inhibitor (LY294002). Furthermore, PCr also inhibited MGO-induced transcriptional activity of Nuclear factor kappa B (NFκB). In conclusion, our data described that PCr exerts an antiapoptotic effect in HUVECs exposed to oxidative stress by MGO through the mitochondrial pathway and the modulation of PI3K/Akt/eNOS and NF-κB signaling pathway. Thus, it might be a candidate therapeutic agent for diabetic-associated cardiovascular diseases., (Copyright © 2016. Published by Elsevier Inc.)

Endothelial apoptosis triggered by oxidized low-density lipoprotein (oxLDL) can accelerate the progression of endothelial dysfunction atherosclerosis. Phosphocreatine (PCr) is a natural compound, which has been used in cardiac disease and cardiopulmonary resuscitation. However, its protective effects on atherosclerosis and its mechanism have not been clarified. In the present study, we investigated the anti-apoptotic effect of phosphocreatine in human umbilical vein endothelial cells (HUVECs) exposed to oxLDL and explored the possible mechanisms. HUVECs were pre-treated with 10-30 mM PCr and then stimulated with oxLDL. Cell morphology, cytotoxicity and apoptosis were evaluated by light microscopy, CCK assay, and flow cytometry respectively. Levels of Bax, Bcl-2, protein expression of protein kinase B (Akt), eNOS and caspase activities were assessed by Western blotting. Reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) were measured with fluorescent probes. Lactate dehydrogenase (LDH), malondialdehyde (MDA), nitric oxide (NO) and superoxide dismutase (SOD) contents were determined by spectrophotometer. Our results showed that PCr dose-dependently prevented oxLDL associated HUVEC cytotoxicity and apoptotic biochemical changes such as loss of MMP, LDH and MDA leakage and loss of SOD, decrease of Bcl-2/Bax protein ratio, activation of caspase-3 and 9, and ROS generation. In addition, the antiapoptotic effect of PCr was partially inhibited by a PI3K inhibitor (LY294002) and also enhanced p-Akt/Akt protein ratio, eNOS activation and NO production. In conclusion, our data show that the inhibition of oxLDL-induced endothelial apoptosis by PCr is due, at least in part to its anti-oxidant activity and its ability to modulate the PI3K/Akt/eNOS signaling pathway.