Roles Of Endoplasmic Reticulum Stress And Autophagy On OX-LDL-induced Apoptosis In HUVECs

Backgroud and purpose:Cardiovascular disease(CVD)as a leading cause of global death seriously endangers human health.Atherosclerosis is a critical pathological factor in the development of CVDs.It refers to a chronic inflammatory disease resulted from lipid deposition in the arterial wall.And endothelial cell dysfunction is commonly believed to be the first stage of atherosclerosis.Vascular smooth muscle cells(VSMCs),local inflammation and immune cells also play a role in the formation of atherosclerotic plaques.The ER has been identified as an important organelle for protein synthesis,calcium homoeostasis,lipid biosynthesis and several other cellular functions.Due to its vital role in maintaining normal cell activities.Any internal or external perturbation that impairs ER homeostasis triggers ER stress leading to misfolded or unfolded protein accumulation.ER stress then activates a series of signal pathways aiming at restoring normal ER functions known as the unfolded protein response(UPR).Accumulating evidence suggests that UPR-activated ER-resident chaperones can be detected in atherosclerotic lesions.It is therefore likely that ER stress has been implicated in the pathogenesis of atherosclerosis by causing cell dysfunction or inducing cell apoptosis.Autophagy as a cellular self-digestion process plays a vital role in maintaining cell homostasis.It’s in charge of degrading damaged organelles,misfolded proteins etc.Autophagy is therefore one of the important defense mechanisms by which cells maintain homeostasis and survival.While the effect produced by autophagy also can lead to cell death.Excessive autophagy can destroy lysosomes and autophagosomes,which lead to cell damage.It may depend on the different stage of the pathogenesis.Autophagy is a crucial mechanism in cardiac homeostasis.And recent studies show that autophagy can be triggered after ER stress suggesting their Synergistic effects in cell function.ER stress interacts with autophagy to orchestrate cell survival or apoptosis.The purpose of this study was to investigate the interaction between endoplasmic reticulum stress and autophagy and their roles in endothelial cell apoptosis.In this study,we established an OX-LDL inducing human umbilical vein endothelial cells(HUVEC)apoptosis model to investigate the cross effects of endoplasmic reticulum stress and autophagy and try to figure out the effect on cell apoptosis.Methods:HUVECs treated with OX-LDL in different concentrations for different time,and the cell activity was detected by CCK8 method to determine the appropriate concentration and treatment time.OX-LDL induced endothelial cell apoptosis model was established,and blank group,OX-LDL treatment group,ER stress stimulation group,ER stress inhibition group,autophagy stimulation group,and autophagy inhibition group were set.The level of apoptosis was assessed by flow cytometry,apoptosis-related proteins were detected by Western blot,the mechanism of ER stress and autophagy on endothelial cell apoptosis and the regulation of apoptosis were investigated,and ER stress and autophagy-related proteins were detected by Western blot to analyze the effects of ER stress and autophagy on each other.Results:1.OX-LDL treatment induced endothelial cell apoptosis: Compared with the control group without drugs treatment,OX-LDL treatment significantly reduced the survival rate of endothelial cells,and with the increase of OX-LDL content,the survival rate of cells gradually decreased with the extension of treatment time.2.Endoplasmic reticulum stress regulates OX-LDL induced endothelial cell apoptosis:In the endothelial cell apoptosis model induced by OX-LDL,If ER stress inhibitors were added,the expression levels of ER stress-related proteins such as GRP78 and apoptosis-related protein CHOP were down-regulated by Western blot,and apoptotic rate was reduced measured by flow cytometry;while related protein level and apoptotic rate were increased under ER stress agonists treatment.3.Autophagy is activated in endothelial cells treated with OX-LDL: in OX-LDLtreated cells,increased expression of autophagy-related proteins can be measured by western blot.4.ER stress affects autophagy in OX-LDL treated endothelial cells: autophagy related proteins measured by western blot were significantly decreased in the experimental group using ER stress inhibitors.5.Activation of autophagy inhibits CHOP expression and reduces endothelial cell apoptosis: p-PERK and p-IRE1 which are downstream proteins of ER stress have no significant change in protein level compared with the control group,while the expression of downstream protein CHOP is significantly decreased(p < 0.05),and the apoptosis level measured by flow cytometry analysis is also significantly decreased.Conclusion:Inhibition of ER stress and activation of autophagy play a protective role in endothelial cells,ER regulates autophagy,and autophagy has a certain regulatory effect on CHOP proteins downstream of ER stress signaling pathway,and the complex regulation between them together determines cell survival and apoptosis..