Reasearch On The Mechanism Of Angiotensin? Regulating Monocytes/Macrophages To Participate In EAM Progression

Myocarditis is an inflammatory injury of myocardium caused by infection,physical and chemical factors.Myocardial injury leads to myocardial cell necrosis and myosin release,which leads to autoimmune myocarditis.Autoimmune myocarditis is an important cause of inflammatory dilated cardiomyopathy?iDCM?,myocardial infarction,cardiogenic shock,heart failure and even sudden cardiac death.Therefore,it is of great significance to clarify the mechanism of myocardial inflammation and fibrosis to prevent cardiovascular diseases and improve people's quality of life.Macrophage?M??is a highly heterogeneous population,widely distributed in various tissues and organs of the body,which plays a role in regulating immune response,maintaining tissue and organ homeostasis,and participating in inflammatory injury and tissue repair of target organs.AngiotensinII?ANG??is an important active component of Renin angiotensin aldosterone system?RAAS?and widely involved in the occurrence and development of cardiovascular diseases such as myocarditis and cardiomyopathy.Whether ANG? is related to monocytes/macrophage infiltration,functional remodeling,cardiac inflammatory regression,and functional remodeling in damaged heart tissue?Objective:Discussion on the occurrence and development of myocardial inflammatory injury:?1?Effects of ANG? on the chemotactic function of monocytes/macrophages;?2?Effects of ANG? on functional remodeling of damaged myocardial infiltrating monocytes/macrophages and its potential mechanism;?3?Effects of ANG? on inflammatory regression in the late stage of myocardial inflammatory injury and its possible mechanism.Methods:1.ANG? promotes monocytes/macrophages infiltration in EAM miceThe experimental autoimmune myocarditis?EAM?mouse model was constructed with myocardial myosin.Enzyme-linked immunosorbent assay?ELISA?was used to detect the expression of ANG? in peripheral blood serum and heart tissue;After EAM mice were treated with Losartan by gavage,H?5?E staining was used to observe myocardial inflammation and infiltration.qRT-PCR was used to detect the inflammatory mediators IL-1?,IL-6,TNF-?,and chemokine receptors CCR2,CCR5;Sirius-red staining was used to detect cardiac fibrosis,qRT-PCR was used to detect the expression of type ? and type ? collagen?Collagen ?/??;Transwell system was used to detect the chemotactic function of ANG? on macrophages.2.To explore the mechanism of ANG? regulating infiltrated monocytes/macrophages function remodeling and myocardial inflammationIBased on the EAM mouse model,qRT-PCR was used to detect the expression of inducible nitric oxide synthase?iNOS?,and immunofluorescent staining?IF?observed the expression of F4/80 and CD86 double-positive cells in heart tissue of different treatment groups,Sirius-red staining to detect cardiac fibrosis;Flow cytometry?FCM?was used to analyze the expression of CD11b⁺Ly6C^hi in spleen and heart tissue;CD11b⁺Ly6C^hi and CD11b⁺Ly6C^int cells were sorted by flow cytometer and the expression of F4/80 and CD86 were detected.Western-blot was used to detect the expression of iNOS after ANG? stimulated CD11b⁺Ly6C^hi.ELISA was used to detect inflammatory mediators IL-1?,IL-6 and TNF-?in the supernatant after ANG? stimulated CD11b⁺Ly6C^hii and CD11b⁺Ly6Cⁱntnt cells;the expression of iNOS and the phosphorylation levels of P38,Erk1/2,Stat3 were detected by western-blot after RAW264.7 stimulated by ANG?.After phosphorylation inhibitor treatment,the phosphorylation levels of P38,Erk1/2 and Stat3 as well as the expressions of iNOS and Arg-1 were detected by western-blot;the expression of inflammatory mediators IL-1?,IL-6 and TNF-?in the supernatant were detected by ELISA;After intravenous injection of phosphorylation inhibitors,H?5?E staining was used to observe myocardial inflammation infiltration,Sirius-red staining was used to detect cardiac fibrosis,and qRT-PCR was used to detect iNOS and inflammatory mediators IL-1?,IL-6,TNF-?in heart tissues;After intravenous injection of Agtr1 siRNA,H?5?E staining was used to observe myocardial inflammatory infiltration and pathological scores.qRT-PCR was used to detect the expression of inflammatory mediators IL-1?,IL-6,TNF-?in heart tissue,and IF was used to observe the F4/80 and CD86 double positive cells in heart tissues of different treatment groups,and Sirius-red staining was used to detect cardiac fibrosis.3.ANG?-activated fibroblasts induce cardiac M1 cells transdifferentiation and participate in the mechanism of myocardial inflammation regressionMCFs were activated into MFB by ANG? induction.After MFB had co-cultured with macrophages for 24h,western-blot was used to detect the expression of iNOS and apoptosis-related molecules Bax,Bcl-2,and Cleaved-Caspase3 in direct or indirect co-culture system,FCM detected the expression of CD86,MHCII and the proportion of AnnexinV⁺PI⁺double positive cells on M?;ELISA was used to detect the expression of TNF-?in the culture supernatant of MFB,FCM detected the expression of Fas/FasL on the surface of MFB cells,and western-blot was used to detect the TNF-?-mediated apoptotic pathway downstream molecules TNF-R1,TRADD,FADD,and Cleaved-Caspase8 expression;after treated with TNF-?release inhibitor C87,FCM detected the proportion of apoptosis,and western-blot detected the expression of TNF-R1,TRAD,FADD,and Cleaved-Caspase8;After using TNF-R1 neutralizing antibody,western-blot was used to detect the expression of TNF-R1,TRAD,FADD,and Cleaved-Caspase8;After intraperitoneal injection of C87 in EAM mice,H?5?E staining was used to observe myocardial inflammatory infiltration and pathological scores.qRT-PCR was used to detect the inflammatory mediators IL-1?,IL-6 and TNF-?in heart tissue,and IF detected the number of F4/80and CD86 double positive cells in the heart tissue;After co-culturing for 36 hours,western-blot was used to detect the expression of Arg-1 in direct or indirect co-culture system,ELISA detected the expression of IL-4,IL-10,TGF-?1,and qRT-PCR was used to detect Arg-1,Retnla,Chil3 and Mrc1,FCM detected the expression of CX3CR1 and the proportion of AnnexinV⁺PI⁺double positive cells on the surface of M?;the expression of Leptin was detected by ELISA in the cell supernatant,western-blot was used to analyze the phosphorylation level of PI3K/AKT and Arg-1after treating by Leptin;After using the Leptr^-/-M?to co-culture with MFB,western-blot detected the expression of Arg-1,and ELISA measured the expression of IL-10 and TGF-?1 in the supernatant;In vivo,IF was used to observe the number of AnnexinV⁺F4/80⁺and CX3CR1⁺F4/80⁺double positive cells in heart tissue of EAM at different stages;and adoptive transfer was used to verify the proportion of CD45.2⁺CCR2⁺CX3CR1⁺cells settled in spleen and heart tissue.Results:1.The expression of ANG? in peripheral blood serum and cardiac tissue cell suspension of EAM mice was significantly higher than control group?P<0.05?;Compared with the EAM group,the losartan treatment group significantly reduced myocardial inflammation infiltration,the inflammation score was significantly down-regulated?P<0.05?,and the expressions of inflammatory mediators in the cardiac tissue and the chemokine CCR2/5 were also down-regulated?P<0.05?;After treatment with CCR2/5 receptor blocker in vivo,the expression of inflammatory mediators in the heart tissue decreased?P<0.05?,fibrosis was significantly relieved,and collagen expression was also down-regulated?P<0.05?;Chemotactic function was significantly decreased after CCR2/5 receptor blocker treatment in vitro compared with ANG? treatment group?P<0.01?.2.After the treatment of EAM mice with losartan,the expression levels of iNOS and MCP-1 in the heart tissues were significantly decreased compared with the EAM group?P<0.05?.Immunofluorescence showed that the number of infiltrating inflammatory macrophages F4/80⁺CD86⁺?M1?was decreased?P<0.05?and the degree of fibrosis was alleviated?P<0.05?;FCM results showed that compared with the EAM group,the proportion of CD11b⁺Ly6C^hi in the spleen of losartan treated group was down-regulated?P<0.05?,with the same effect in the heart tissue?P<0.05?.After treatment of CD11b⁺Ly6^hi with ANG?,the results showed that the antigen presenting function was enhanced,the iNOS expression was increased,and the secretion of inflammatory mediators of the supernatant were also increased?P<0.05?;Western-blot results showed that ANG? activated macrophages through the P38/Erk1/2/Stat3 pathway,and the phosphorylation inhibitor treatment significantly blocked the activation pathway and decreased the expression of inflammatory mediators in the supernatant?P<0.05?;After treatment with phosphorylation inhibitors in vivo,myocardial inflammatory infiltration was weakened and the score was decreased?P<0.05?,fibrosis was alleviated,and the expression of inflammatory mediators was down-regulated?P<0.05?;After the injection of Agtr1 siRNA,the myocardial inflammatory infiltration was also ameliorated?P<0.001?,inflammatory mediators levels,infiltrated M1 cells and fibrosis were decreased?P<0.05?.3.MCFs were activated into MFB by ANG? induction and co-cultured with macrophages:After co-culturing for 24 hours,M?was induced to transform into M1cells,and its antigen presenting function was enhanced and accompanied by M1 cells apoptosis,the levels of apoptosis-related proteins Bax,Bcl-2,and Cleaved-Caspase3were increased?P<0.05?;MFB-induced M1 cells apoptosis depends on the TNF/TNF-R1 pathway rather than Fas/FasL,and its downstream related proteins TNF-R1,TRAD,FADD,and Cleaved-Caspase8 were also up-regulated?P<0.05?.The expression of apoptotic protein was significantly down-regulated after treated with TNF-?release inhibitor C87,Using TNF-R1 neutralizing antibody had the same phenomenon;After co-culturing for 36 hours,M?was transformed into M2 cells,M2-related mediators were highly expressed and CX3CR1 was up-regulated on M2cells without the occurrence of apoptosis;Leptin was up-regulated in M1,M2 and MFB,Leptin activated the PI3K/AKT signaling pathway to up-regulate the expression of Arg-1;MFB did not induce Leptr^-/-M?expressing Arg-1,and M2-related factors IL-10 and TGF-?1 were not upregulated;In vivo,the number of AnnexinV⁺F4/80⁺cells was increased in the heart of EAM on 21d,and CX3CR1⁺F4/80⁺cells were increased on 35d.The adoptive transfer results showed that the proportion of CD45.2⁺CCR2⁺CX3CR1⁺cells in the spleen and heart was increased.Conclusion:ANG? contributed to EAM inflammatory development by recruiting monocytes/macrophages and by promoting infiltrated their reprogramming through Erk1/2 and P38/Stat3 pathways;furthermore,ANG? also benefited the inflammatory macrophages conversion?M1/M2?by inducing fibroblast transdifferentiation into myofibroblast.