Roles And Mechanisms Of Macrophage ?-arrestin-1 In Atherosclerosis And Multiple Sclerosis

Nowadays,the mobility of inflammation-related diseases increases greatly which largely harm the public health.Among all of them,atherosclerosis and multiple sclerosis belong to the most serious ones.Atherosclerosis has been regarded as one of the largest killers of people's health.It has been reported so far that advanced atherosclerosis might lead to several kinds of acute cardiovascular emergent events,such as myocardial ischemia,myocardial infarction as well as the formation of thrombosis,the dropping of which leads to the the occurrence of emergent events.So far,it is widely acknowledged that the process of pathogenesis and progression of atherosclerosis in pathology is divided into four stages,including lipid stripe stage,fibrous plaque stage,atheromatous plaque stage and the late secondary changes.The formation of foam cells is the pathological basis of atherosclerotic plaque formation.It is generally acknowledged that the major source of foam cell is derived from the endothelial cells as well as the accumulated macrophages and smooth muscle cells located under the arteria intima.Among all of the source cells,macrophages are the major source of foam cells.The processes of the formation of macrophage-derived foam cells are as follows:The blood monocytes accumulate under the arteria intima with the damage of the endothelial cells,which turn into macrophages.Macrophages swallow the abnormal accumulating oxygenized low density lipoproteins?oxLDL?or other modified LDLs under the arteria intima,which in turn transfer to foam cells.Multiple sclerosis is regarded as an inflammation-related neurodegenerative diseases,serving as the main reason of disability among teenagers.So far,the etiology of multiple sclerosis is the chronic inflammatory reaction mainly induced by macroglia,the macrophage in central nervous system.Therefore,inhibiting the inflammatory reaction induced by microglia is vital in the prevention and treatment of multiple sclerosis.Although numerous studies have studied the prevention and treatment of atherosclerosis and multiple sclerosis so far,yet the specific mechanisms of the pathogenesis and progression of atherosclerosis remain unclarified.Furthermore,the effective therapeutic strategies against atherosclerosis are demanded to be developed.?-arrestin-1 is a member of the family of?-arrestins.It is a multi-functional cytoplasm adaptor protein.It is traditionally acknowledged that the major function of?-arrestin-1 is to participate in the regulation of G protein-coupled receptors.It mainly mediates the endocytosis and desensitization of G protein-coupled receptors.However,according to the recent related studies,?-arrestin-1 can act as a scaffold protein,participating in many kinds of G protein-coupled receptor-dependent of independent signaling pathways.There are several studies pointing out that?-arrestin-1plays an important role in various kinds of diseases.In atherosclerosis,it was previously reported that?-arrestin-1 expressing in smooth muscle cells could attenuate neointimal hyperplasia through inhibiting proliferation and migration of smooth muscle cells,thus protecting against atherosclerosis.However,the roles of?-arrestin-1 expressed in macrophages in atherosclerosis and the specific mechanisms are not fully elucidated.For multiple sclerosis,mice with the knockdown of?-arrestin-1 showed less severity in EAE,indicating the role of?-arrestin-1 in multiple sclerosis.However,the role of?-arrestin-1in microglia remains unclear.In this study,we studied the roles and mechanisms of macrophage?-arrestin-1 in atherosclerosis and EAE.The current study was divided into three sections:In Section One,we demonstrated the protective roles of macrophage?-arrestin-1 in atherosclerosis,and the process of autophagy was involved in the protective roles of macrophage?-arrestin-1 in atherosclerosis.In Section Two,we found out that macrophage?-arrestin-1 was involved in the mediation of?7nAChR-induced roles of anti-atherosclerosis.In Section Three,we detected the regulatory role of macrophage?microglia??-arrestin-1 in?7nAChR-induced anti-multiple sclerosis effect.A.Macrophage?-arrestin-1 plays an anti-atherosclerotic role through autophagyAutophagy is an important metabolic mechanism in organisms.The function of autophagy is mainly degrading the cellular long-lived proteins,misfolding proteins and damaged organelles.The function of autophagy mainly relies on lysosomes.It has been reported that autophagy process plays an important role in the pathogenesis and progression of various kinds of diseases.In atherosclerosis,inducing autophagy process is beneficial to attenuate the inflammatory and immune reaction of macrophages in atherosclerotic plaques.In addition,autophagy in macrophages produces an anti-atherosclerotic effect through inhibiting apoptosis and enhancing the levels of cholesterol efflux.For the relationship between?-arrestin-1 and autophagy,a previous study by our lab demonstrated that neuronal?-arrestin-1 could protect against neuronal ischemia through inducing the enhancement of autophagy level.However,the relationship between?-arrestin-1 and autophagy in atherosclerosis is not fully elucidated.In the experiments of this section,we aimed to discuss the roles of macrophage?-arrestin-1 in the attenuation of atherosclerosis and the autophagy mechanisms.The specific experimental results are briefly described as follows:1.Detecting the expression of macrophage?-arrestin-1 in atherosclerotic plaque in the occurrence of atherosclerosisWe detected the expression of?-arrestins in aorta isolated from normal mice and atherosclerosis mice models through Western blot.We found out that compared with the normal mice,the levels of expression of?-arrestin-1 were significantly increased in aorta in atherosclerosis mice models.Furthermore,we found out the highly expression of?-arrestin-1 in the lesional macrophages in atherosclerotic plaque.2.Construction of myeloid?macrophage??-arrestin-1 conditional knockout atherosclerosis mice modelWe constructed the myeloid?macrophage??-arrestin-1 conditional knockout mice through Cre-LoxP system and further created the myeloid?macrophage??-arrestin-1conditional knockout mice on the background of ApoE^-/-.High fat diet was fed to mice for 4 month to create the atherosclerosis mice model.3.The anti-atherosclerotic roles of macrophage?-arrestin-1We ran the oil red O staining,immunohistochemical staining,Masson staining and sirius red staining in atherosclerosis mice.We found out that in the occurrence of atherosclerosis,compared with the wild type mice,the aortic atherosclerosis plaque areas were significantly increased and the aortic atherosclerosis plaque vulnerability in myeloid?macrophage??-arrestin-1 conditional knockout mice.The blood lipid levels were further detected by us.We found out that the levels of total cholesterol and low density lipid were significantly increased in myeloid?macrophage??-arrestin-1conditional knockout mice,while the level of high density lipid was significantly decreased.4.The inhibitory effects of macrophage?-arrestin-1 in the formation of macrophage-derived foam cellWe ran the oil red O staining and Dil-oxLDL swallowing test in murine macrophages and detected the cellular levels of cholesterol to test the formation of macrophage-derived foam cells.We found out that knocking out macrophage?-arrestin-1led to the significant increase of the ratio of foam cell formation.We further ran the real-time PCR test and found out that knocking out macrophage?-arrestin-1 largely increased in the mRNA levels of lipid swallowing-related receptors including scavenger receptor class A1?SR-A1?,lectin-like oxidized LDL receptor-1?LOX-1?and cluster of differentiation?CD?36,while the mRNA levels of cholesterol efflux-related vehicles including ATP-binding cassette?ABC?transporters ABCA1 and ABCG1 were significantly decreased.However,overexpressing macrophage?-arrestin-1 led to the reverse effects.5.The inhibitory effects of macrophage?-arrestin-1 in monocyte/macrophage migrationWe ran the Transwell cell migration test to detect the levels of monocyte/macrophage migration in murine macrophages.We found out that knocking out macrophage?-arrestin-1 played an important role in increasing the levels of inflammatory tendency and migration in murine macrophages.6.The inductive effects of macrophage?-arrestin-1 in autophagy processWe ran the tests of Western blot,transmission electron microscope and laser confocal microscope to detect the levels of autophagy process in murine aorta and atherosclerotic plaque macrophages.We found out that in the occurrence of atherosclerosis,the levels of autophagy process in aorta were significantly increased,while knocking out macrophage?-arrestin-1 significantly inhibited the level of autophagy.We further ran the test of Western blot and found out that the classic signaling pathway of autophagy process,adenosine 5'-monophosphate?AMP?-activated protein kinase?AMPK?-mammalian target of rapamycin?mTOR?-p70 ribosomal protein S6kinase?p70S6K?,was involved in this process.7.The inhibitory effects of macrophage?-arrestin-1-induced autophagy process in the formation of foam cellsWe ran the oil red O staining and detected the levels of cellular cholesterol in murine macrophages.We found out that overexpressing macrophage?-arrestin-1,the ratio of macrophage-derived foam cell formation was significantly decreased,while inhibiting autophagy process through autophagy inhibitors largely attenuated those effects.Conclusion:Macrophage?-arrestin-1 plays an attenuative role in atherosclerosis through the mechanism of autophagyB.Macrophage?-arrestin-1 involves in the?7nAChR-mediated protective roles of atherosclerosis?7 nicotinic acetylcholine receptor??7nAChR?is one of the subtypes of nicotinic acetylcholine receptors.The recent related studies revealed that?7nAChR plays an important role in suppressing inflammatory responses through the mediation of cholinergic anti-inflammatory pathway.It has been already reported that?7nAChR plays an important role in the process of pathogenesis and progression of various kinds of diseases.In atherosclerosis,it is generally acknowledged that activating?7nAChR produces an attenuative effect in the pathogenesis and progression of atherosclerosis.However,the specific mechanisms underlying this effect have not been fully illustrated yet.In this section of study,we aimed to explore whether macrophage?-arrestin-1 was involved in the mediation of?7nAChR-induced anti-atherosclerosis effects.The results in this section of experiments were briefly described as follows:1.Macrophage?-arrestin-1 mediates the?7nAChR-induced attenuative effects on atherosclerosisWe ran the oil red O staining,immunohistochemical staining,Masson staining and sirius red staining in atherosclerosis mice models.We found out that in the occurrence of atherosclerosis,activating?7nAChR could significantly reduce the areas of atherosclerotic plaque in aorta as well as enhancing the stability of atherosclerotic plaque in atherosclerotic mice models,while myeloid?macrophage??-arrestin-1 conditional knockout significantly attenuated those effects led to by activating?7nAChR.We further detected the levels of blood lipid.We found out that activating?7nAChR significantly reduced the levels of total cholesterol and low density lipid,while myeloid?macrophage??-arrestin-1 conditional knockout significantly attenuated those effects led to by activating?7nAChR.2.Macrophage?-arrestin-1 mediates the?7nAChR-induced attenuative effects on macrophage-derived foam cell formationWe ran the oil red O staining and detection of levels of cellular cholesterol levels in murine macrophages.We found out that activating?7nAChR could significantly reduce the proportion of formation of macrophage-derived foam cells,while knocking out myeloid?macrophage??-arrestin-1 significantly attenuated those effects led to by activating?7nAChR.We ran the real-time PCR detection.We found out that activating?7nAChR could enhance the mRNA levels of cholesterol efflux-related vehicles including ABCA1 and ABCG1,while knocking out macrophage?-arrestin-1significantly attenuated those effects led to by activating?7nAChR.3.Macrophage?-arrestin-1 mediates the?7nAChR-induced inhibiting effects on monocyte/macrophage migration in murine macrophagesWe ran the Transwell monocyte/macrophage migration in murine macrophages to detect the level of monocyte/macrophage migration.We found that activating?7nAChR could significantly reduce the level of monocyte/macrophage migration in murine macrophages,while knocking out macrophage?-arrestin-1 significantly attenuated those effects led to by activating?7nAChR.Conclusion:Macrophage?-arrestin-1 is involved in the mediation of?7nAChR-induced anti-atherosclerosis effects.C.Macrophage?-arrestin-1 regulates the protective role of?7nAChR in EAEActivating?7nAChR alleviates the onset of multiple sclerosis through the induction of cholinergic anti-inflammatory pathway.However,the specific mechanisms remain unclear.In this section of study,we aimed to investigate the effect of macrophage?microglia??-arrestin-1 on?7nAChR-induced anti-multiple sclerosis role.The specific experimental results are briefly described as follows:1.Detecting the expression of?-arrestin-1 in microglia in spinal cords plaque in the occurrence of atherosclerosisWe ran the Real-time PCR,Western blot and immunofluorescence and found that on the occurrence of EAE,the level of?-arrestin-1 was significantly increased in spinal cords and microglia in spinal cords.2.?-arrestin-1 regulates the anti-inflammatory effect of?7nAChR in BV2microglia under the stimulation of LPS/ATPWe ran the Western blot test and found that the application of PNU282987 for the activation of?7nAChR significantly inhibited the activation of NLRP3 inflammasome,while overexpressing?-arrestin-1 largely attenuated those effects.3.Activating?7nAChR inhibits the interaction between?-arrestin-1 and NLRP3 protein in BV2 microglia under the stimulation of LPS/ATPWe ran the immunofluorescence and immunoprecipitation and found that in BV2microglia,LPS/ATP significantly enhanced the interaction of?-arrestin-1 and NLRP3protein,while PNU282987 largely attenuated those effects.Conclusion:Macrophage?microglia??-arrestin-1negativelyregulates?7nAChR-induced anti-multiple sclerosis effect.