A Study Of Androgen Receptor In Experimental Autoimmune Myocarditis

Part 1Androgen receptor expression and location in experimental autoimmune myocarditisObjective:To explore the expression level and location of androgen receptor(AR)in experimental autoimmune myocarditis(EAM),and to indentify the relation between AR expression and inflammation response.Methods:The male Balb/c mice weightedl8-20g,were selected and EAM model was conducted by-immunizing murine a-cardiac myosin heavy chain sequence polypeptides.The changes of body weight,heart weight/body weight ratio(HW/BW)and total number of spleen lymphocytes in mice were observed on day 21 after immunization.H&E and Masson staining were used to detect pathological changes in myocardium.The expression of IL-6,TNF-a,IL-1p in myocardium was detected by QRT-PCR.The expression of AR in myocardium was measured on day 14,day 21 and day 62 after immunizationn by using Western blot.The location of AR in myocardium was detected by immunohistochemistry.And the cell specificity of AR was determined by immunofluorescence double staining.Results:Compared with the control group,the LVEF and LVFS of the EAM mice were significantly decreased.The body weight of EAM mice was significantly reduced,the heart and spleen size were apparently increased in EAM mice,the ratio of heart weight and body weight significantly was increased,and the total number of spleen lymphocytes was increased.H&E staining showed significant infiltration of inflammatory cell and myocardial necrosis in myocardium of EAM mice.Masson staining showed significant collagen deposition in the myocardium of EAM mice.The expression of IL-6,TNF-?,IL-1? in myocardium of EAM mice was significantly increased by using QRT-PCR.The results of Western Blot showed AR was increased in the begining of day 14 and lasted on day 62,in which the AR was reached to the peak on day 21.Immunohistochemical staining showed that the expression of androgen receptor located in the inflammatory cell accumulation area.Using CD68 to label macrophage.Immunofluorescence double staining showed that the AR in the myocardium of the myocardium were mainly expressed in monocyte/macrophages.Conclusion:The expression of AR in myocardial tissue of EAM mice was increased and correlated with the inflammation response.The increased AR in the myocardium of EAM mice is mainly expressed in macrophages.Part 2The effect androgen receptor in experimental autoimmune myocarditisObjective:To study the effect of androgen receptor(AR)on experimental autoimmune myocarditis and the potential mechanism,we treated EAM mice with androgen receptor degradation enhancer ASC-J9.Methods:The male Balb/c mice weighted 18-20g,were randomly divided into four groups:control group,EAM group,EAM + vehicle group,EAM + ASC-J9 treatment group.The EAM model were constructed by using mouse a-myosin heavy chain polypeptide.The mice from EAM + ASC-J9 treated group were addminstrated with androgen receptor degradation enhancer ASC-J9 through intraperitoneal injection every otherday from day 8 to day 20.EAM+vehicle mice were injected with saline that contain 0.5%DMSO.Echocardiacgraphy was used to evaluate the cardiac structure and function of these four groups on day 21.The changes of the heart appearance,heart weight/body weight ratio(HW/BW)and total account of splenic lymphocytes were observed.H&E and Masson histopathologic staining were used to examine the degree of inflammatory infiltration,cell necrosis and fibrosis in the myocardium.Immunohistochemistry staining was used to determine the expression of CD3+ T cells and CD68+ monocytes/macrophages in the myocardium of mice in each group.TUNEL was used to detect the apoptosis of cardiac tissue.QRT-PCR or Western Blot were used to measure the expression of inflammatory cytokines(IL-6,TNF-?,IL-1?,TGF-?),Thl7-related cytokines(ROR?t,IL-17A),M1 macrophage related factor(iNOS)and M2 macrophage-associated factors(Arg-1,MMR).Immunofluorescence was used to observe the percentage of M1-like macrophages(F4/80+/iNOS+)in myocardium.Results:Compared with the vehicle group,LVEF and LVFS were significantly increased in the EAM + ASC-J9 group.The weight of the heart was significantly reduced,and the cardiac mass and body weight ratio were significantly decreased in EAM + ASC-J9 group.The infiltration of inflammatory cells in the myocardium of EAM + ASC-J9 treated group was significantly reduced,the degree of myocardial fibrosis was significantly reduced,the apoptosis was decreased.The number of CD3+ T cells and CD68+ macrophages that infiltrated in myocardium was decreased in EAM + ASC-J9 group.Meanwhile,IL-6,TNF-?,IL-1?,TGF-(?,Th17 cell related factors(ROR?t,IL-17A)and type Ml macrophage-associated factor(iNOS)were significantly decreased in myocardium of EAM + ASC-J9.However,M2-macrophage-related factors were not changed.The results of immunofluorescence double staining showed that the proportion of F4/80+/iNOS+ Ml macrophages in the myocardium of EAM + ASC-J9 treated mice was significantly decreased.Conclusion:The AR degradation enhancer ASC-J9 can improve the cardiac structure and function of EAM mice,reduce the inflammation,fibrosis and the cell apoptosis in myocardium.Treated EAM mice with ASC-J9 can significantly reduce the M1-like macrophages infiltrated in myocardium of EAM mice,which suggests that AR can promote the inflammatory response and suppressing the expression of AR can improve EAM by reducing the infiltration of M1-like macrophages.Part 3The machenism of androgen receptor in regulating M1-like macrophage polarizationObjective:To further study the effects of androgen receptor(AR)on Ml macrophage polarization in vitro by AR overexpression or AR degradation.Methods:Raw264.7 cells were divided into:the androgen receptor degradation enhancer ASC-J9 group,vehicle control(DMEM containing 0.0005%DMSO),AR over-expression plasmid(LV5-AR)and its negative control(LV5-AR-NC).LPS was used to stimuli macrophage polarizated to M1 phenotype.The TNF-a secretion in each group were measured by ELISA.The expression of iNOS,IL-6 and IL-6 were detected by QRT-PCR.The expression of JNK,SOCS1,STAT5 and NF-?B were detected by Western blot.To detect whether ASC-J9 exert its function by regualting androgen receptor,the physiological dose of testosterone were added in ASC-J9 treated cells.The expression levels of TNF-a and iNOS were detected QRT-PCR.Results:The ASC-J9 significantly restricted LPS-stimulated macrophages polarize to M1 phenotype.ASC-J9 reduced the levels of TNF-a which were secreted by M1 macrophage,and decreased the expression of M1 macrophage associated factor iNOS.In addition,ASC-J9 reduced the activation of JNK that was activated by LPS,and decreased the phosphorylation of STAT5 and NF-?B in Raw264.7 cells.Meanwhile,ASC-J9 treatment increased the expression of SOCS1 in macrophages compared with vechile group.On the other hand,AR over-expression plasmid treatment significantly increased iNOS and reduced the expression of M1-macrophage polarization-related factor SOCS-1 in LPS-stimualted Raw264.7 cells compared with corresponding control group.Conclusion:The AR play an important role in Ml-like macrophage polarization.Suppressing AR expression can restrict macrophage polarize to Ml phenotype by up-regualting SOCS1 expression.