The Role Of CTRP9 In Efferocytosis And Its Molecular Mechanisms In Macrophages

BackgroundEfferocytosis refers to the process in which apoptotic cells(ACs)are removed before further necrosis and inflammatory components release.No matter during the normal growth and development of human tissues and organs,or under pathological conditions such as inflammation and aging,hundreds of millions of apoptotic cells will be produced.The membrane structure of apoptotic cells remains intact and will not cause inflammation to surrounding tissues.Under normal circumstances,professional and non-professional phagocytes such as macrophages,dendritic cells and endothelial cells engulf them in time.After phagocytosis,macrophages can produce anti-inflammatory cytokines such as TGF-β,which plays an immune regulatory function.However,if ACs are not removed in time,ACs will undergo secondary necrosis,releasing cell contents and aggravating inflammatory reactions.Therefore,timely and effiective removal of apoptotic cells is of great significance for maintaining the homeostasis of the body’s internal environment.Macrophages’ recognition of apoptotic cells or phagocytosis dysfunction will lead to the occurrence of chronic inflammatory diseases,such as atherosclerosis(AS),which is a lipid metabolism disorder and inflammatory vascular disease.Abundant ACs can be seen in vulnerable plaques of AS.Studies have shown that the disorder of the clearance of ACs is one of the important reasons for the instability of AS plaque,and enhancing the efferocytosis in plaque can delay the occurrence and development of AS.Systemic lupus erythematosus(SLE)is a chronic autoimmune disease involving skin,kidney,cardiovascular and nerve tissues and organs.There are a large number of autoimmune antibodies against autoantigens,such as antinuclear antibodies(ANA),in the peripheral blood of SLE patients,some of which come from the rupture and release of apoptotic cells.Studies have shown that obstruction of phagocytosis and clearance of apoptotic cells is one of the important causes of SLE.Therefore,enhancing the effect of efferocytosis is of great significance to inhibit the occurrence of chronic inflammatory diseases.It is urgent for us to carry out relevant basic research work to explore the physiological mechanism and intervention drugs of the efferocytosis and to find effective prevention and treatment targets at the molecular level.The mechanism of efferocytosis is complex,with various mechanisms and signal pathways.After macrophages engulf apoptotic cells,their cell contents(such as lipids,sugars,proteins,etc.)will increase dramatically.By changing and/or strengthening the basic metabolic pattern,macrophages can cope with huge metabolic pressure and maintain their "normal" function,thus preparing for the next round of efferocytosis.Recent research shows that,after phagocytosis of ACs,macrophages will increase the expression of dynamic related protein 1(drp l),which induces mitochondrial fission,eventually increase’s the concentration of Ca+in cytoplasm.Ca+will promote the maturation of phagocytosome to promote the digestion and degradation of ACs,so mitochondrial fission plays a vital role in tefferocytosis.Apoptotic cell-derived molecular substances released into cytoplasm by phagocytosome digestion will change their metabolic state.For example,macrophages will change from oxidative phosphorylation to glycolysis,glucose transporter 1(GLUT1)expression will increase and secrete a large amount of lactic acid,which will react on macrophages to promote their secretion of anti-inflammatory cytokines.Studies have shown that phagocytosis of apoptotic cells will activate PPAR and LXR in macrophages and increase the expression of ABCA1,which will induce reverse transport of cholesterol to relieve lipid-metabolic pressure.At the same time,activation of PPAR will increase the expression of phagocytic receptors and the secretion of optonins on the surface of macrophages and enhance the phagocytosis of macrophages.Clq/TNF-related protein 9(CTRP9)is a novel adipocytokine and a member of Clq/TNF-related protein(CTRP).Studies have shown that CTRP9 plays an important role in regulating glycolipid metabolism,protecting myocardium,relaxing blood vessels and improving endothelial relaxation function.Previous studies of our research group have shown that CTRP9 can increase plaque stability by inhibiting inflammatory reaction of macrophages in plaque,but the specific mechanism is still unclear,suggesting that CTRP9 may stabilize plaque by enhancing macrophage cell burial.Studies also have shown that CTRP9 can reduce local inflammation and improve cardiac dysfunction by promoting macrophage polarization towards M2 during myocardial infarction.When macrophages are polarized towards M2,their ability to phagocyte apoptotic cells is enhanced,suggesting that CTRP9 may improve cardiac function after myocardial infarction by enhancing macrophage cell burial.However,there is no research on the effect of CTRP9 on macrophage efferocytosis.Therefore,this study will further explore the effect and mechanism of CTRP9 on efferocytosis ofmacrophage.CTRP9 enhances the efferocytosis of macrophage by promoting mitochondrialfission and immune metabolism.1 Research Purpose:(1)To investigate the effect of CTRP9 on efferocytosis of macrophage;(2)To explore the molecular mechanism of CTRP9 affecting the efferocytosis ofmacrophage.2.Materials and Methods2.1 Preparation of Apoptotic Cells and Apoptotic Cell Culture Medium(ACCM)(1)Thymocytes of wild-type C57BL/6 mice aged 4-6 weeks were extracted and stimulated with 1mM dexamethasone for 6 hours in vitro to induce apoptosis.Flowcytometry was used when the apoptosis rate was above 80%.(2)The apoptotic cells were cultured for 6-8 hours,centrifuged at 12000rpm/min to obtain culture medium,and then the apoptotic bodies were filtered out with a 0.22umfilter to obtain ACCM.2.2 Cell culture and treatment(1)In this study,the primary macrophages in mouse abdominal cavity were cultured in a cell incubator based on 37℃ and 5%CO2 with DMEM+10%fetal bovine serum+1%double antibody.(2)First round of efferocytosis experiment:In the experiment,the cells were pretreated with CTRP9 globular domain recombinant protein.Under the stimulation of ACs co-culture,the co-culture was established according to the ratio of apoptotic cells to macrophages of 10:1.After 1 hour of culture,the apoptotic cells were discarded and washed with PBS to detect the efferocytosis of macrophages.(3)Second round of efferocytosis experiment:co-culture was established according to the ratio of apoptotic cells to macrophages of 10:1.After 1 hour of culture,apoptotic cells were sucked and discarded and washed with PBS,and replaced with complete culture medium.CTRP9 was added or not to continue culture for 12 hours.At this time.the groups were divided into ACs-Ist group and ACs-1st+CTRP9 group.After 12 hours,total protein was extracted to detect relevant indexes or apoptotic cells were added after 12 hours,co-culture was established according to the ratio of apoptotic cells to macrophages of 10:1,apoptotic cells were sucked and discarded after 1 hour of culture,and macrophages were collected to detect efferocytosis.2.3 Flow cytometry(1)The apoptotic cells were labeled with Annexin V,and the apoptosis induction rate of thymocytes induced by dexamethasone was detected.(2)After the apoptotic cells labeled with fluorescent dye TAMRA were co-cultured with macrophages,macrophages in each group were collected and labeled F4/80,and flow detection of double positive cell percentage,namely efferocytosis of macrophage.2.4 Laser Confocal Microscope(1)Cell slides of each group were prepared and Mito-Tracker Green labeled mitochondria.Mitochondrial morphology of macrophages in each group was observed under laser confocal microscope.(2)After co-culture of CFSE labeled apoptotic cells and macrophages,the macrophages of each group were collected and labeled with F4/80.The positional relationship between apoptotic cells and macrophages of each group was observed under laser confocal microscope and the phagocytosis differences among each group were compared.2.5 Western BlotThe total protein and/or mitochondrial protein of each group of cells were extracted.The expression changes of mitochondrial kinetics related indexes(p-drp1(s616),drp1,Mff,Mfn2,OPA1),MAPK pathway related indexes(p-erk,erk,p-p38,p-jnk,jnk)and immune metabolism related indexes(ABCA1,GLUT1,UCP2,AdipoR1,par-y,hif-la)were detected.2.6 AdipoR1 siRNA transfectionAdipoR1 siRNA sequence with the best inhibitory effect was screened out.Macrophages were transfected with RNAiMAX and used for subsequent experiments.2.7 Tunel stainWT mice and CTRP9-KO mice were selected to induce thymocyte apoptosis by intraperitoneal injection of dexamethasone.Thymus was extracted and paraffin-embedded sections were stained with Tunel to compare the difference in the number of apoptotic cells among the groups.2.8 Detection of lactic acid concentration in cell culture medium by kit2.9 Cellular immunofluorescencePreparation of cell climbing slices of each group.The expression difference of p-drpl(s616)was observed under fluorescence microscope.2.10 Statistical analysisSPSS 18.0 was used for statistical analysis of the data,and the measurement data were expressed by±standard error.T-test,Chi-square test,ONE-WAY ANOVA and other methods were selected for statistical analysis when comparing the data among each group.P<0.05 indicates that the data has statistically significant.3.Research Results3.1 CTRP9 can promote the efferocytosis of macrophagesThe results of flow cytometry showed that CTRP9 could increase the cell efferocytosis of macrophages in a concentration-dependent manner compared with the control group(p<0.05).At the same time,the results of laser confocal imaging showed that CTRP9 could significantly increase the phagocytosis of apoptotic cells by macrophages(p<0.05).Tunel staining results showed that the number of apoptotic cells in CTRP-KO mice injected with dexamethasone was significantly higher than that in the control group(p<0.05),that is,the number of apoptotic cells that were not phagocytized increased,indicating that CTRP9 could promote macrophages to eliminate apoptotic cells.3.2 CTRP9 enhances the efferocytosis of macrophage by promoting mitochondrial fissionThe results of Western Blot showed that CTRP9 can promote the phosphorylation of drp1 and the expression of Mff in a time-dependent manner compared with the control group(p<0.05);Compared with the control group,CTRP9 can reduce the expression of Mfn-2 and OPA1 in a time-dependent manner(p<0.05).Cell immunofluorescence showed that CTRP9 could significantly promote the phosphorylation level of drp1 compared with the control group.By comparing mitochondrial proteins,CTRP9 can promote drpl translocation from cytoplasm to mitochondrial mediated fission(p<0.05).At the same time,the morphology of mitochondria was observed by laser confocal imaging.Compared with the control group,CTRP9 could significantly increase the fragmentation of mitochondria in macrophages(p<0.05).3.3 CTRP9 promotes mitochondrial fission depending on activation of p38 and jnk signalsThe results of Western Blot showed that CTRP9 can promote the activation of erk,p38,jnk phosphorylation,i.e.the activation of MAPK signaling pathway in a time-dependent manner compared with the control group(p<0.05).Erk inhibitor U0126,p38 inhibitor SB203580 and jnk inhibitor SP600125 significantly inhibited phosphorylation of erk,p38 and jnk induced by CTRP9(p<0.05).Western Blot results showed that compared with ctrp9 group,SB203580 and SP600125 abolished the activation of p-drpl by CTRP9(P<0.05).3.4 CTRP9 promotes the ability of efferocytosis of macrophages by enhancing their immune metabolism.Western Blot and assay of lactic acid concentration in cell culture medium showed that,compared with the control group,the expression of GLUT1 and ABCA1 and lactic acid secretion increased in ACs co-culture group(p<0.05).Compared with the control group and ACs co-culture group,the expression of GLUT1 and ABCA1 and lactic acid secretion increased further in CTRP9+ACs group(p<0.05).Similarly,flow cytometry results showed that in the second round of cell efferocytosis experiment,the phagocytosis efficiency of CTRP9+ACs-lst group was higher than that of ACs-1st co-culture group(p<0.05).The results of Western Blot showed that CTRP9 could promote the expression of par-y,ABCA1,Hif-1a and GLUT1 in a time-dependent manner compared with the control group(p<0.05).3.5 AdipoR1 mediates immune metabolism of CTRP9 in macrophagesThe results of Western Blot showed that ACCM and ACs can increase the expression of AdipoR1 in macrophages compared with the control group(p<0.05);Western Blot showed that AdipoR1 siRNA inhibited the up-regulation of par-y,ABCA1,Hif-1a and GLUT1 expression induced by CTRP9 compared with the control group(p<0.05).4 Research Conclusions(1)CTRP9 can enhance the efferocytosis of macrophages;(2)CTRP9 can promote efferocytosis through p38/jnk-drp1-mediated mitochondrial fission;(3)CTRP9 can enhance immune metabolism and promote efferocytosis through AdipoR1.