The Effect Of Endoplasmic Reticulum Stress-Wnt/β-catenin Cycle On Endothelial Dysfunction

Objective:Atherosclerosis（AS）is closely related to cardiovascular disease,and endothelial dysfunction（ED）is the beginning of AS.In addition,endoplasmic reticulum stress（ER-Stress）and Wnt/β-catenin signaling pathway can promote ED.Previous studies in our laboratory have shown that treatment of human umbilical vein endothelial cells（HUVECs）with H₂O₂ or direct addition of cholesterol can cause cholesterol accumulation,activate ER-Stress and Wnt/β-catenin signaling pathways,and ultimately lead to ED.However,the specific relationship between ER-Stress and Wnt/β-catenin signaling pathway remains to be studied.In order to further explore the relationship between them,this article activated ER-Stress by tunicamycin,salinomycin or shRNA technology to knock down LRP6 to inhibit Wnt/β-catenin signaling pathway,and detect the expression and content of related molecules and the biological behavior of cells.Method:1、The effects of ER-Stress,Wnt/β-catenin signaling pathway and cholesterol efflux on endothelial dysfunction were studied by activating ER-Stress by tunicamycin and inhibiting Wnt/β-catenin signaling pathway by salinomycin.To study the relationship between ER-Stress,Wnt/β-catenin signaling pathway and cholesterol efflux.In this article,HUVECs were divided into the following four groups:C group（control group）,Tun group（tunicamycin group）,Sal group（salinomycin group）and Tun+Sal group.Western blot was used to detect the expression of ER-Stress,Wnt/β-catenin signaling pathway,cholesterol efflux and ED-related protein molecules.The content of cholesterol and ROS was detected by immunofluorescence.The contents of LDH,SOD and NOS were detected by kits.THP-1 cells were co-cultured with HUVECs for cell adhesion experiments.2、The effects of ER-Stress,Wnt/β-catenin signaling pathway and cholesterol efflux on endothelial dysfunction were further verified by knocking down LRP6.To further determine the relationship between ER-Stress,Wnt/β-catenin signaling pathway and cholesterol efflux.In this article,HUVECs were divided into the following four groups:C group,Tun group,sh LRP6 group and Tun+sh LRP6 group.Western blot was used to detect the expression of ER-Stress,Wnt/β-catenin signaling pathway,cholesterol efflux and ED-related protein molecules.The content of cholesterol and ROS was detected by immunofluorescence.The contents of LDH,SOD and NOS were detected by kits.THP-1 cells were co-cultured with HUVECs for cell adhesion experiments.Result:1、ER-Stress and Wnt/β-catenin signaling pathways regulate the occurrence of ED through mutual influence.（1）The interaction between ER-Stress and Wnt/β-catenin signaling pathway:Tunicamycin not only activates ER-Stress,but also activates Wnt/β-catenin signaling pathway.Salinomycin not only inhibits Wnt/β-catenin signaling pathway,but also inhibits ER-Stress.（2）ER-Stress and Wnt/β-catenin signaling pathways can regulate cholesterol efflux:Tunicamycin can activate ER-Stress and Wnt/β-catenin signaling pathway,inhibit the expression of cholesterol efflux related molecules ABCA1 and ABCG1 and promote cholesterol accumulation,while salinomycin can reverse the above process and promote cholesterol efflux.（3）ER-Stress and Wnt/β-catenin signaling pathways regulated ED through interaction:Tunicamycin can increase the content of LDH and ROS in HUVECs,but decrease the content of SOD and NOS,and enhance the adhesion ability,while salinomycin can reverse the above process.2、Knockdown of LRP6 alleviated the effects of ER-Stress on cholesterol efflux inhibition and ED promotion.（1）Knockdown of LRP6 can inhibit ER-Stress:Knockdown of LRP6 can inhibit Wnt/β-catenin signaling pathway and ER-Stress.（2）Knockdown of LRP6 promotes cholesterol efflux:Knockdown of LRP6promoted the expression of cholesterol efflux-related molecules ABCA1 and ABCG1and reduced intracellular cholesterol accumulation,and alleviated the inhibition of ABCA1 and ABCG1 expression and cholesterol accumulation by ER-Stress.（3）Knockdown of LRP6 can reduce ED level:Knockdown of LRP6 can alleviate the effect of ER-Stress on ED of HUVECs cells,which can alleviate the effects of ER-Stress on the increase of LDH and ROS content,the decrease of SOD and NOS and the enhancement of adhesion ability.Conclusion:ER-Stress and Wnt/β-catenin signaling pathways can regulate the occurrence of ED through interaction,and this process is related to cholesterol efflux metabolism.