The Effect Of Apolipoprotein A1 Binding Protein Mediated-mitophagy On The Macrophage M1/M2 Polarization And The Progression Of Atherosclerosis

[Background and Objective]:Atherosclerosis is a chronic inflammatory disease.Immune/inflammatory activation of vascular wall is considered as one of the key factor for the occurrence and development of atherosclerosis.As the main cells of the immune/inflammatory system,macrophage infiltrated into the blood vessel wall,uptake lipids and secrete a variety of inflammatory factors,thus participating in the whole progress of atherosclerosis.Further study on the regulatory mechanism of macrophage-mediated inflammation during the formation of atherosclerosis plaque is of great significance for the prevention and treatment of atherosclerotic cardiovascular diseases.Macrophages is a kind of immune cells with heterogeneity and plasticity.Under the stimulation of different induction factors,macrophage can form subgroups with different functions（namely,macrophage polarization）,which are mainly divided into two categories:classic activated macrophages（M1）and alternative activated macrophages（M2）.M1 macrophages are mainly induced by lipopolysaccharide（LPS）and interferon-gamma（IFN-γ）,secreting tumor necrosis factor alpha（TNF-α）,interleukin-1 beta（IL-1β）and other inflammatory factors,which play a pro-inflammatory effect.M2macrophages are mainly induced by IL-4 and highly express arginase1（Arg1）and IL-10,etc.,exerting anti-inflammatory and tissue repair effects.There are different types of macrophages in the plaques of atherosclerosis,M1 macrophages mainly exist in the progression and unstable plaques and promote the rupture of plaques,and M2macrophages mainly exist in the stable plaques,which can alleviate the inflammatory environment of plaque formation.AIBP,Apolipoprotein A-I binding protein,has been found to combind apoA-I（apolipoprotein A-I）in recent years,and its expression is closely related to the risk of cardiovascular disease.It has been found previously that treatment of AIBP can promote apoA-I/ABCA1-mediated cholesterol efflux,reduce cholesterol accumulation and inflammation in macrophages,and inhibit the occurrence and development of atherosclerosis.Studies have suggested that AIBP also exists in mitochondria,but whether it have a role in the inflammation mediated by macrophages and the progress of atherosclerosis and relative mechanism is still unclear.Therefore,the purpose of this study is to explore the influence of mitochondrial AIBP on macrophage polarization and atherosclerosis and its potential mechanism.This study will clarify the role of mitochondrial proteins represented by AIBP in regulating macrophage inflammation and atherosclerosis,and provides theoretical basis and new targets for the development of drugs for the prevention and treatment of atherosclerosis.[Methods]:Mitochondrial AIBP deficient macrophages were established in AIBP^-/-bone marrow-derived macrophages（BMDMs）by over-expressing mutational AIBP which lack mitochondrial localization sequence（MLS）.The expression of inflammatory cytokines such as IL-1β,IL-6,TNF-α,IL-4 and IL-10 was detected by q-PCR after overexpression of full-length AIBP or mutant AIBP.Subsequently,LPS and IFN-γwas used to induce M1macrophage,and IL-4 was used to induce M2 macrophage.The expressions of M1 and M2 macrophage markers were detected by q-PCR.The effect of mitochondrial oxidative phosphorylation and glycolysis after mitochondrial AIBP deficiency was detected by real-time seahorse cell energy metabolism assay.Mitochondrial ROS（mtROS）and mitochondrial membrane potential in macrophages were detected by fluorescent dye labeling.QPCR and Western blot were used to detect the effect of mitochondrial AIBP deletion on the expression of PINK1 and Parkin genes related to mitochondrial autophagy.Bafilomycin A1 was used to inhibit autophagy process,and the effect of mitochondrial AIBP deficiency on macrophage M1/M2 polarization was detected.Bone marrow transplantation was used to construct LDLR^-/-mice without AIBP in macrophages,and the expression of AIBP was detected by Western Blot.The following experiments were performed.First,Oil red O and HE staining were used to evaluate the formation of atherosclerosis plaque in mice.Aorta and blood of mice were collected,and the expression levels of inflammatory factors and M1/M2 marker genes in serum,aorta and atherosclerotic plaques of mice were detected by ELISA,qPCR,Western Blot and immunofluorescence.[Results]:Western blot show that the virus after transfection AIBP expression in mitochondria significantly increased,and the expression of AIBP（ΔMLS）is disappear in mitochondria,illustrates the cells model that express span AIBP and AIBP（ΔMLS）are established.Q-PCR results showed that the expression of pro-inflammatory cytokines in macrophages increased and the expression of anti-inflammatory cytokines decreased after the absence of mitochondrial AIBP.Meanwhile,after the deficiency of mitochondrial AIBP,the expressions of iNOS and COX2,markers of M1 macrophages,were increased,and the expressions of MRC1 and Arg1,markers of M2macrophages were decreased.Further results revealed that mitochondrial oxidative phosphorylation was impaired and glycolysis level increased as AIBP deletion.The mechanism of mitochondrial AIBP deficiency was found to reduce the levels of PINK1 and Parkin protein related to mitophagy,suggesting that mitochondrial AIBP deficiency may inhibit the occurrence of PINK1-mediated mitophagy.Meanwhile bafilomycin A1 treatment significantly affected the regulation of AIBP on macrophage M1/M2 polarization.Western blot and qPCR results showed that the expression of AIBP protein in macrophages was significantly reduced after bone marrow transplantation,indicating the successful establishment of the model of macrophages AIBP deficiency in LDLR^-/-mice.Frozen section staining of aortic sinus in mouse heart showed that the absence of macrophage AIBP could significantly promote the formation of atherosclerotic plaques.Meanwhile,the level of inflammatory cytokines in serum were increased.Immunofluorescence showed that the absence of AIBP promoted the infiltration of macrophages in the plaque.QPCR detection of the expression of M1/M2 marker gene in aorta root and LPS+IFN-γor IL-4 induced peritoneal macrophages also found that AIBP deletion promoted M1 phenotype and inhibited M2 phenotype.[Conclusion]:1.Mitochondrial AIBP increased mitochondrial autophagy mediated by PINK1,inhibits M1 macrophage polarization,promotes M2 macrophage polarization,and reduces macrophage inflammation.2.AIBP alleviates the occurrence and development of atherosclerosis by regulating the polarization of macrophages and reducing inflammatory responses.