Mitochondrial Ido1-derived Singlet Oxygen Inducing Nucleophagy Mediates Apelin-13-Induced Monocytes-Vascular Endothelial Cells Adhesion

Objective:Our group previously reported that reactive oxygen species(ROS)autophagy pathway mediates apelin-13,an endogenous ligand of APJ receptor,to promote the adhesion of monocytes(MCs)and vascular endothelial cells(ECs).Study reveals that the reduction of Lamin B1,a marker of nucleophagy,induces vascular senescence and promotes the occurrence and development of atherosclerosis(AS).Singlet oxygen oxidize nuclear DNA,causing nuclear damage.Mitochondrial singlet oxygen can trigger autophagy.Indoleamine 2,3 dioxygenase 1 in ECs generates singlet oxygen.Accordingly,we propose scientific hypothesis that mitochondrial IDO 1-derived singlet oxygen inducing nucleophagy mediates apelin-13-induced monocytes-vascular endothelial cells adhesion.This project will apply Western Blot,immunofluorescence to clarify whether mitochondrial IDO 1-derived singlet oxygen and nucleophagy mediate apelin-13/APJ to promote MCs-ECs adhesion.Based on ROS-autophagy pathway,we further explore mitochondrial IDO1 derived singlet oxygennucleophagy pathway,and uncovers the molecular mechanism of MCs-ECs adhesion induced by apelin-13,providing strong evidence for the screening of new drugs for AS that target APJ receptor.Methods:1.Western Blot:detected the expression of IDO1、Lamin B1、LC3、ICAM1、VCAM1 in HUVECs.2.The level of nucleophagy marker Lamin B1 was detected by immunoeletron microscopy.3.RNA interference:designed small interfering RNA chain to interfere with the expression of IDO1,Lamin B1 in HUVECs.4.Mitochondrial singlet oxygen fluorescent probe Si-DMA detected the level of mitochondrial singlet oxygen in HUVECs.5.Immunoeletron microscopy was used to detect the positive number of Lamin B1-related colloidal gold in HUVECs.6.The colocalization of Lamin B1 and LC3 was detected by immunofluorescenc in HUVECs.7.Monocytes was labeled by Calcein AM to detect the adhesion of MCs and ECs by fluorescence microscope.Results:1.Apelin-13 activates nucleophagy in HUVECs.2.Apelin-13 inhibits the expression of nucleophagy marker Lamin B1 in HUVECs.3.Apelin-13 promotes the colocalization of Lamin B1 and LC3 in HUVECs.4.Apelin-13 induces the production of mitochondrial singlet oxygen in HUVECs.5.Singlet oxygen scavenger 1-histidine inhibits apelin-13-induced mitochondrial singlet oxygen generation in HUVECs.6.Singlet oxygen scavenger 1-histidine reverses apelin-13-inhibited Lamin B1 expression in HUVECs.7.Singlet oxygen scavenger 1-histidine blocks apelin-13-facilitated the colocalization of Lamin B1 and LC3 in HUVECs.8.Apelin-13 promotes the expression of IDO1 in HUVEC.9.IDO1 siRNA impedes apelin-13-triggered mitochondrial singlet oxygen generation in HUVECs.10.IDO1 siRNA reverses apelin-13-suppressed Lamin B1 expression in HUVECs.11.IDO1 siRNA represses apelin-13-promoted the colocalization of Lamin B1 and LC3 in HUVECs.12.IDO1 siRNA inhibits apelin-13-stimulated the expression of ICAM1,VCAM1 in HUVECs.13.IDO1 siRNA impedes apelin-13-engendered MCs-ECs adhesion.14.Singlet oxygen scavenger 1-histidine suppresses apelin-13-induced the expression of ICAM1,VCAM1 in HUVECs.15.Singlet oxygen scavenger 1-histidine restrains apelin-13-triggered MCs-ECs adhesion.16.Lamin B1 siRNA inhibits apelin-13-stimulated the expression of ICAM1,VCAM1 in HUVECs.17.IDO1 siRNA prevents apelin-13-induced MCs-ECs adhesion.Conclusion:Mitochondrial IDO1-derived singlet oxygen inducing nucleophagy mediates apelin-13-induced monocytes-vascular endothelial cells adhesion...