The Role And Mechanisms Of Peli1 Involved In Macrophage Polarization Following Myocardial Ischemia-Reperfusion Injury

BackgroundMyocardial ischemia-reperfusion（I/R）injury is the main problem that hinders the optimal therapeutic effect of ischemic myocardium from coronary revascularization.There is still no effective method to interfere with myocardial I/R injury.Therefore,deep-going study of its mechanism and the search for effective intervention targets is one of the major issues that need to be solved in basic research and clinical practice.Excessive activation of inflammatory response,especially macrophage infiltration polarization in myocardial tissue,is an important pathogenesis following myocardial I/R injury.Macrophages can be transformed to M1 or M2 phenotype respectively,under different local microenvironmental stimuli.M1 macrophages mainly secrete pro-inflammatory factors,which can amplify the inflammatory cascade and hydrolyze the extracellular matrix and result in cardiac dysfunction;while M2 macrophages can phagocytose cell debris and necrotic tissue,promote angiogenesis and collagen synthesis and accelerate the recovery of damage.During the development of myocardial I/R injury,apoptotic or necrotic cardiomyocytes and endothelial cells in myocardial tissue can release a large number of danger associated molecular patterns（DAMPs）,such as heat shock proteins（Heat shock proteins,HSPs）,High mobility group protein B1（HMGB1）,and so on.DAMPs act as ligands to activate corresponding receptors,the most interestingly,leading to the activation of NF-κB,MAPK and IRFs by intracellular signal transduction mediated by Toll-like receptors（TLRs）.The downstream signals,in turn,promote the infiltration of inflammatory cells such as macrophages in myocardial tissue,and induce macrophage M1 polarization and aggravate myocardial tissue damage and cardiac dysfunction.Our previous study suggested that Pellino1（Peli1）protein can affect the development of myocardial infarction（MI）and ventricular remodeling after myocardial infarction by regulating TLRs/NF-κB signaling.However,the mechanism of ventricular remodeling after myocardial infarction and myocardial infarction can not be extended to explain the mechanism of myocardial I/R injury.Moreover,myocardial tissue includes cardiomyocytes,cardiac fibroblasts,endothelial cells and other intrinsic cells in the heart and various inflammatory cells in the blood vessels,and in which cells Peli1 can play a regulatory role remains to be elucidated.This study investigated whether Peli1 in inflammatory cells,especially macrophage,play a important role in myocardial I/R injury.Does its regulatory mechanism be related to the infiltration of macrophages to I/R-damaged myocardial tissue and the induction of macrophage to M1-type polarization?Furthermore,on the basis of solving the above problems,the signal transduction mechanism of Peli1 regulating macrophage infiltration and polarization need to be further explored.Aims1.To clarify the regulation of Peli1 in inflammatory cells,especially in macrophages,on myocardial I/R injury.2.To elucidate the regulation of macrophage Peli1 on macrophage polarization and its molecular mechanism in the development of myocardial I/R injury.3.To clarify the regulation of macrophage Peli1 in cardiomyocyte apoptosis induced by hypoxia/reoxygenation.Method and test index1.Human peripheral blood testIn order to understand the correlation between Peli1 in leukocytes and myocardial I/R in clinical patients,the peripheral blood samples from patients with ST-segment elevation suffering from myocardial infarction who were performed with PCI one day ago were collected and correlated.Since blood-derived macrophages mainly originate from monocytes,human Peripheral Blood Mononuclear Cells（PBMCs）were extracted and the expression of Peli1 in peripheral blood PBMCs was detected by RT-PCR and Western blotting.Flow cytometry was used to detect the ratio of mononuclear cell surface marker CD14 positive and Peli1^high monocytes.2.Animal in vivo experimentThe mouse myocardial I/R injury model was replicated by ligating the left anterior descending coronary artery for 45 minutes and then restoring blood flow and reperfusion.At different time points of reperfusion,the expression level of Peli1protein in myocardial tissue was detected to determine the correlation between myocardial Peli1 and myocardial I/R injury.To confirm the regulatory function of Peli1 on myocardial I/R injury in inflammatory cells,the bone marrow of Peli1knockout（Peli1KO）mice was transplanted into recipient wild type（WT）mice irradiated with bone marrow completely through bone marrow transplantion operation,and then the bone marrow-derived Peli1 knockout chimeric mice were constructed.At 24 hours after I/R,left ventricular myocardium was taken and detected by HE staining,immunohistochemical staining,immunoblotting,etc.,to detect inflammatory cells in the myocardium,CD68-positive cells（macrophage surface markers）,and infiltration of Ly6G-positive cells（central granulocyte surface markers）;detection of macrophage polarization（M1/M2）in myocardial tissue by flow cytometry;detection of myocardial infarction in mice by TTC and Evans blue double staining Area;using small animal heart color Doppler ultrasound to detect cardiac function in mice.To further clarify the role of Peli1 in macrophages,macrophage Peli1 knockout(Peli1^{c KO})mice were constructed,and Peli1f/f mice were used as control mice to replicate the myocardial I/R injury model.24 hours after I/R,the polarization of macrophages in mouse myocardial tissue was detected by flow cytometry.TUNEL staining was used to detect apoptosis in myocardial tissue.The same method was used to detect myocardial infarct size and cardiac function as chimeric mice.3.In vitro cell experimentsBecause the damaged cardiomyocytes in myocardial I/R are the main source of DAMPs,DAMPs can promote aseptic inflammation,induce macrophage infiltration by activating pattern-receptor-mediated intracellular signaling and change the macrophage polarization phenotypes.Therefore,in this study,myocardial hypoxia/reoxygenation（H/R）conditioned medium was used as a stimulating factor to investigate its effect on macrophage polarization.The primary cardiomyocytes cultured in vitro were subjected to H/R stimulation or normoxia culture,and the culture supernatant was collected to prepare cardiomyocyte H/R conditioned medium CM（H/R）and the corresponding normoxia culture control conditioned medium CM（N）.Bone marrow-derived macrophages（BMDMs）from wild-type and Peli1knockout mice were stimulated with CM（H/R）and CM（N）,respectively,and real-time PCR and immunoblotting were used to detect markers related to macrophage M1 and M2 polarization.Transwell assay was used to detect the effect of conditioned medium of cardiomyocytes on macrophage migration.Co-immunoprecipitation experiments were performed to verify whether Peli1 and IRF5 could bind to each other,and ubiquitination and nuclear import of IRF5;Overexpression of Peli1 by knockout macrophages by adenovirus and knockdown of IRF5 by si RNA to verify whether Peli1 affects macrophage polarization via IRF5signaling;Co-culture with macrophages,TUNEL staining and immunoblotting were used to detect the expression of apoptosis-inducing molecule Bax and anti-apoptosis-related molecule Bcl2,and to investigate the effect of macrophages on cardiomyocyte apoptosis.Results1.Increased expression of Peli1 in peripheral blood mononuclear cells after myocardial ischemia-reperfusion in patients with clinical myocardial infarctionAfter myocardial I/R in patients with acute myocardial infarction,the expression of Peli1 m RNA and protein in PBMCs increased significantly.Further the percentage of monocyte surface markers CD14 positive and Peli1^high increased significantly in PBMCs.2.Peli1 knockout of bone marrow-derived inflammatory cells can inhibit myocardial tissue inflammatory cell infiltration and improve myocardial structural damage and cardiac dysfunction after myocardial I/R1).In the development of myocardial I/R injury in mice,the expression level of Peli1protein in myocardial tissue increased gradually from 6h after reperfusion,reached the peak at 24h,and was maintained until 72h after reperfusion.2).The bone marrow of Peli1KO mice was transplanted into bone marrow-derived recipient WT mice by bone marrow（BM）transplantation to construct a Peli1knockout of BM-derived inflammatory cells（WT/Peli1KO BM）mice.The myocardial I/R injury model was replicated 6 weeks after bone marrow transplantation.HE staining showed that compared with the WT/WT BM mouse sham operation group,the infiltration of inflammatory cells in the myocardial tissue of the WT/WT BM mice I/R group was significantly increased,while the WT/Peli1KO BM mice were caused by I/R.The inflammatory cell infiltration of myocardial tissue was significantly less than that of the WT/WT BM mouse I/R group.Immunohistochemistry and immunoblotting of CD68 and Ly6G also showed that Peli1 knockout of bone marrow-derived inflammatory cells significantly inhibited I/R-induced myocardial infarction and risk zone macrophages and central granulocyte infiltration.Flow cytometry analysis showed that Peli1 knockout of bone marrow-derived inflammatory cells inhibited M1 type polarization of macrophages induced by myocardial I/R injury and increased M2-type polarization.3).Myocardial I/R injury model was replicated 6 weeks after bone marrow transplantation.At 24 hours after operation,myocardial infarction in WT/WT BM mice was significantly associated with cardiac dysfunction.Peli1 knockout of bone marrow-derived inflammatory cells can significantly reduce infarct size,and can also significantly improve cardiac ejection fraction and shortening score reduction caused by myocardial I/R injury.3.Macrophage Peli1 conditinonal knockout can inhibit its polarization to M1and reduce myocardial I/R injury.1).At 24 h after myocardial I/R,the proportion of M1 macrophages in total macrophages（M1/M）in Peli1f/f mice was significantly increased,while the proportion of M2 macrophages in total macrophages was increased.（M2/M）was significantly reduced;whereas Peli1c KO mice could inhibit the change of macrophage polarization characteristics induced by myocardial I/R due to the conditional knockout of Peli1 by macrophages,causing the giant lesion induced by I/R injury.The phagocyte M1 type is down-regulated while the M2 type is up-regulated.2).Myocardial I/R injury was observed for 24 h,and myocardial infarction in Peli1f/f mice was significantly associated with cardiac dysfunction.Peli1c KO mice with macrophage Peli1 conditional knockout significantly reduced infarct size and significantly improved cardiac ejection fraction and shortened fractional reduction due to myocardial I/R injury.3).Myocardial I/R injury can cause a large number of Tunel staining apoptotic cells in the risk zone and infarct area of Peli1f/f mice,while macrophage Peli1 knockout can significantly reduce the number of apoptotic cells.4.Effects of Peli1 knockout on CM（H/R）-induced macrophage polarization and migration and its signal regulation mechanism1).CM（H/R）stimulation can induce a significant increase in m RNA and protein levels of macrophage Peli1.2).CM（H/R）stimulation can significantly increase macrophage M1 type polarization markers and reduce M2 type polarization markers can also increase macrophage migration ability.Compared with wild-type macrophages,Peli1 knockout reverses the changes in M1 and M2 polarization markers and migration abilities of macrophages induced by CM（H/R）.3).Macrophages Peli1 and IRF5 have mutual binding,and CM（H/R）stimulation significantly increases the mutual binding of the two.CM（H/R）stimulation causes IRF5 in the cytoplasm of macrophages to enter the nucleus in a time-dependent manner.CM（H/R）stimulation significantly increased the ubiquitination of the K63locus in macrophage IRF5.Compared with wild type,Peli1 knockout inhibited the increase of Peli1 and IRF5 binding in macrophages induced by CM（H/R）,ubiquitination of K63 in IRF5,and IRF5 entry into the nucleus.4).Macrophage IRF5 knockdown reverses the increase of M1 marker Nos2 protein and the decrease of M2 marker CD206 protein induced by overexpression of Peli1.5.Macrophage Peli1 knockout can reduce H/R-induced cardiomyocyte apoptosis in co-culture system1).H/R can induce cardiomyocyte apoptosis,and the addition of macrophage co-culture during reoxygenation can further increase myocardial cell apoptosis.Compared with the wild-type macrophage group when reoxygenation,the addition of Peli1 knockout macrophages reduced the degree of apoptosis of cardiomyocytes.2).H/R can lead to an increase in the expression of the pro-apoptotic signaling molecule BAX and a decrease in the expression of the anti-apoptotic signaling molecule Bcl2 in cardiomyocytes.The addition of macrophage co-culture during cardiomyocyte reoxygenation resulted in a further increase in BAX and a further decrease in Bcl2 in cardiomyocytes,whereas macrophage Peli1 knockout significantly reversed these changes.Conclusions:Peripheral blood mononuclear cell Peli1 expression may be associated with myocardial I/R in patients with myocardial infarction;in the development of myocardial I/R injury,bone marrow-derived inflammatory cell Peli1 can promote inflammatory cells in infarcted and dangerous areas of myocardial tissue.Infiltration and macrophage M1 polarization increases myocardial infarct size and aggravates cardiac dysfunction.Macrophage Peli1 can promote the polarization of macrophages in the infarcted and dangerous areas of myocardial tissue to M1 type,increase apoptosis and myocardial infarct size,and reduce cardiac function;in the treatment environment of myocardial cell H/R injury conditioned medium Peli1 in macrophages can promote its polarization to M1 and increase its migration ability.Its regulatory mechanism may be related to CM（H/R）-induced binding of Peli1 to IRF5in macrophages and increase of K63 of IRF5.Ubiquitination and promotion of IRF5into the nucleus.Moreover,macrophage Peli1 knockout can reduce H/R-induced cardiomyocyte apoptosis in the co-culture system.