The Role Of CPLA2 In MGO-induced Apoptosis Of HUVECs

Methylglyoxal(MGO),a highly reactive dicarbonyl compound,is mainly formed as a toxic byproduct of glycolysis and it is widely distributed in human body.Under physiological conditions,only little glucose would change into MGO.However,under disease states or metabolic abnormalities,an exception occurred during the glycolysis,thus resulting in a accumulated excess of MGO.As an intermediate of nonenzymatic glycation,MGO could modify various proteins to generate advanced glycation end products(AGEs).Meanwhile,both MGO and AGEs could stimulate cells to generate reactive oxygen species(ROS)and ultimately induce apoptosis of cells.Studies demonstrated that MGO and AGEs are intimately implicated with the development of diabetes mellitus,blood vessel lesion and neurodegenerative diseases.In the present study,we investigated the role of MGO in apoptosis of vascular endothelial cells.The experiments were carried out with human umbilical vein endothelial cells(HUVECs).The apoptosis rate of HUVECs stimulated by MGO was detected by flow cytometry and the results showed that MGO significantly increased apoptosis of HUVECs in a concentration-dependent manner.MGO(1.0mM)significantly increased apoptosis(14.05%)compared with the control group(3.11%),which is consistent with the results of MTT,WB and q-PCR.Recently,researchers found that abnormal expression of cPLA2 is related to the apoptosis of cells.To determine whether cPLA2 was involved in MGO-induced apoptosis,cPLA2 inhibitor as well as cPLA2-targeted siRNA was used to detect the apoptosis rate.WB results showed that treatment with the cPLA2 inhibitor PYR significantly decreased the ratio of Bcl-2 and Bax compared with the MGO group,which indicated a dramatic decrease of apoptosis.In accordance with the usage of PYR,MGO lost the power of pro-apoptosis when cPLA2 was interfered by a small interference RNA(si-cPLA2).Taken together,inhibition or silencing of cPLA2 attenuated MGO-induced apoptosis,indicating that cPLA2 played an important role in MGO-induced cell apoptosis.It is reported that p38 MAPK is the upstream of cPLA2.This may suggest that cPLA2 mediated MGO-induced apoptosis in HUVECs is due to the activation of p38 MAPK.The data revealed that MGO increased cPLA2 expression in a concentration-and time-dependent manner.During this process,p3 8 MAPK was activated and the time of activation of p38 MAPK was coincided with the activation of cPLA2.To our surprise,pretreatment with the inhibitor of p38(SB202190),failed to attenuate MGO-induced apoptosis of HUVECs,suggesting that p38 MAPK was not directly involved in cPLA2 mediated MGO-induced apoptosis in HUVECs.NF-κB signaling pathway is well known to participate in apoptosis processes.The present results showed that MGO significantly decreased the phosphorylation of NF-κB.Pretreatment with the inhibitor of NF-κB further increased apoptosis.However,the desensitization of NF-κB could be reversed in the presence of si-cPLA2 or PYR.These results revealed that NF-κB played an anti-apoptotic role in the presence of MGO,which is modulated by cPLA2.Additionally,treatment with antioxidant,NAC significantly decreased cell apoptosis.What’s more,NAC decreased the phosphorylation of cPLA2 and enhanced the activation of NF-κB.In summary,the results demonstrated that MGO increased the phosphorylation and expression of cPLA2,accompanied by desensitization of NF-κB and decrease of Bcl-2 through activating redox signaling pathway in the process of HUVECs apoptosis.Additionally,all of the changes could be reversed in the presence of the antioxidant NAC.