Study On The Mechanism Of Myocardial Infiltration And M? Polariation Regulated By Intracellular HMGB1 In EAM Mice

ObjectiveHMGB1 was downregulated by Ad-HMGB1-shRNA in macrophages.We observe the process of HMGB1 as a gene construction factor to participate in and regulate the polarization of macrophages and its potential molecular mechanism.Meanwhile,we constructed experimental autoimmune myocarditis mouse models(EAM),and elucidated the regulation mechanism of intracellular HMGB1 on the polarization of macrophages infiltrating myocardium during the development of EAM.Methods(1)To screen an interference RNA fragment with high transfection efficiency:three interference sequences were designed to constructe and repackage the recombinant adenovirus,and then the mouse macrophage RAW264.7 was cultured to carry out transfection in vitro during the logarithmic stage.After that,transfection efficiency of three groups of recombinant adenovirus was determinated through quantitative real-time PCR and Western Blot.(2)To explore the effect of HMGB1 on the polarization and antigen processing of macrophages:western blot was used to determinate iNOS proteins level expression in macrophage RAW264.7 and Bone Marrow-derived Macrophage(BMDM)treated by LPS after successfully transfection.Enzyme linked immunosorbent assay was used to determinate the secretion of inflammatory mediators in cells.We singled dye CD86 and MHC?molecular.Flow cytometry was used to determinate macrophages antigen processing ability.(3)To explore the effect of intracellular HMGB1 on the polarization of macrophages induced by ANG II:HMGB1 in macrophages successfully reduced by Ad-HMGB1-shRNA.Afterwards,the macrophages were treated with ANG II for 12h in vitro,and Western Blot was used to determinate iNOS proteins expression in each group of cells.Flow cytometry was used to determinate the changes of antigen processing.ELISA was used to determinate the secretion of M1-type correlation factors.(4)The molecular mechanism of intracellular HMGB1 affecting macrophage polarization is:after successful downregulation of intracellular HMGB1,ANG II stimulated cell 30min and Western Blot to detect transcription factor NF-?B,protein kinase ERK1/2 and P38 phosphorylation level(5)Construction of EAM mouse models of recombinant AAV infection:the male BALB/c mice aged 3 weeks were divided into the Control group(wild group),EAM+AAV-HMGB1 group(experimental group),EAM+AAV-Ns group(positive control group).The virus suspension was injected into the tail vein of the mice once every 10days,and twice in total.The mice were immunized with the prepared antigen peptide to establish the EAM model.After 41 days,take out mouse bone marrow derived macrophages to observe transfection efficiency by inverted fluorescence microscope.(6)Treatment of myocardium inflammation in EAM mice by AAV-HMGB1:The heart in EAM mouse was taken out to the hospital pathology for HE staining and pathological grading of myocardial tissue section.ELISA showed changes in the secretion of inflammatory cytokines IL-6 in serum.Immunofluorescence was used to detect F4/80~+CD86~+macrophages in myocardial tissue of EAM mice.Results(1)The recombinant adenovirus could obviously dowregulate the level of HMGB1protein and mRNA level of macrophage,and the transfection efficiency was over 90%.(2)The results of Western Blot showed that after the downregulation of intracellular HMGB1,the expression of iNOS protein in the experimental group was significantly less than that of the control group.ELISA showed that the secretion of inflammatory factors decreased in the experimental group and had a statistically significant difference compared with the control group(P<0.05).FCM detection showed that the ability to process macrophage antigen processing was also inhibited in the experimental group.(3)Western Blot,ELISA and FCM showed that the expression of iNOS,inflammatory mediators and CD86 and MHC II molecules could be reduced after downregulation of HMGB1,which could inhibit ANG II induced M1 polarization reaction.(4)After successful downregulation of HMGB1 in macrophages and stimulation with ANG II,the phosphorylation level of NF-?B,ERK1/2 and P38 showed a decreasing trend compared with the control group.The experiment was repeated three times,and all of them had statistical significance.(5)EAM mouse model of recombinant AAV infection was successfully constructed.(6)After treatment with AAV-HMGB1,the myocardial tissue pathological score,serum inflammatory factor secretion and F4/80~+CD86~+M1 macrophage infiltration in mice were significantly lower than those in the control group.Conclusion(1)The downregulation of HMGB1 in macrophage by Ad-HMGB1-shRNA can effectively reverse the proinflammatory M1 polarization process,reduce the secretion of inflammatory mediators,and regulate it through transcription factors NF-?B,protein kinase ERK1/2,P38 signaling pathway.(2)AAV-HMGB1 can significantly reduce the infiltration of F4/80~+CD86~+macrophages in myocardium and decrease the levels of inflammatory factors of experimental autoimmune myocarditis,and play a role in relieving and treating myocarditis.