The Effect Of ASC-J9 On Regulating M2-type Macrophage Polarization In Experimental Autoimmune Myocarditis And The Mechanism

Part1The effect of ASC-J9 on anti-inflammatory factors expression in experimental autoimmune myocarditisBackgroundMyocarditis is defined as myocardial localized or diffused inflammatory lesions caused by multiple pathological factors,such as infection,autoimmunity,toxicity/drug,which could result in loss of myocardial systolic and diastolic function and arrhythmias.Myocarditis can present an extreme wide range of clinical manifestations,from asymptomatic to life-threatening,even including sudden death.Viral myocarditis is the most frequent type.Experimental autoimmune myocarditis(EAM)can be induced by immunization with anti-heart auto-antibodies,with the participation of innate and adaptive self-specific immune response,EAM has immunologic features resembling the viral myocarditis,making it suitable to research the pathophysiologic insights on myocarditis.The current studies show that myocarditis demonstrates a gender bias,characterized with a higher incidence and more serious symptom in males comparing with females,which is attributed to androgen and androgen receptor(AR).AR acting as a nuclear transcription factor can regulate the transcription of target genes through binding to androgen,exerting an important function in cardiovascular disease.Our previous research showed that the expression of AR in myocardium of EAM mice was increased and correlated with the inflammatory response.AR coiuld aggravate the myocardial damage of EAM via promoting M1-type macrophage polarization.ASC-J9 as an androgen receptor-degradation enhancer could degrade AR via the process of degradation specially.ASC-J9 develops anti-inflammatory via regulating macrophage without inhibiting testosterone level.Our previous research found that the expression of IL-6?TNF-? IL-1? and IL-17A decreased in EAM with ASC-J9 treatment.It indicated that ASC-J9 could ameliorate myocardial inflammatory response by increasing pro-inflammatory factors,and ACS-J9 could also improve inflammatory response via inhibiting M1-type macrophage polarization.However,some anti-inflammatory cytokines such as IL-10 and IL-35 also involve in the inflammatory progress which play a protective role in myocarditis.The influence of ASC-J9 on anti-inflammatory cytokines in EAM,including IL-10 and IL-35,has not been investigated.Our research aimed to elucidate the effect of ASC-J9 on the inflammatory response and the anti-inflammatory cytokines expression in EAM.ObjectiveTo explore the effect of ASC-J9 on the inflammatory response and the expression level of anti-inflammatory factors in EAM.Methods1.Animal model and experimental groupsThe male Balb/c mice weighted 18-20g,were divided into 4 groups randomly:1)control group,2)EAM group,3)EAM+DMSO group(vehicle group)and 4)EAM+ASC-J9 treatment group.At first,EAM model was induced by immunization with murine a-cardiac myosin heavy chain polypeptides on day 0 and 7.Then,the mice from EAM+ASC-J9 treatment group were intraperitoneally injected with ASC-J9 every other day between day 8 and 20 of EAM.In parallel,the vehicle group received normal saline containing 0.5%DMSO at the same time.At the end of 21 days,animal models were obtained.2.Identification of EAM modelThe changes of the heart appearance,HW/BW(heart weight/body weight ratio)and total number of splenic mononuclear cells were compared between EAM and control group to verify the modeling.HE and Masson staining were used to detect the degree of inflammatory infiltration and fibrosis in myocardium of mice.3.Inflammatory response after ASC-J9 treatment.The appearance of heart,HW/BW and splenic mononuclear cells was examined in EAM+DMSO and EAM+ASC-J9.The degree of inflammatory infiltration in myocardium was detected by HE staining,and the myocardial fibrosis was examined with Masson staining.4.The expression of anti-inflammatory factors after ASC-J9 treatment.QRT-PCR was used to examine the expression of anti-inflammatory cytokines IL-10 and IL-35 in myocardium of mice in each group.Results1.Identification of EAM model(1)Compared with control group,the heart of EAM mice were obviously enlarged,and the appearance was mildly white,and HW/BW significantly increased(p<0.001),and the total number of splenic mononuclear cells increased.(p<0.001).(2)HE staining revealed that the infiltration of inflammatory cells in myocardium of EAM mice was more significant,compared with the control group.(3)Masson staining showed that the degree of fibrosis and collagen deposition was significantly increased in the myocardial tissue of EAM mice.2.ASC-J9 improve EAM myocardial inflammation(1)Compared with the vehicle group,after ASC-J9 treatment,the heart size of mice,HW/BW(P<0.01)and total account of splenic mononuclear cells(P<0.001)were obviously decreased.(2)After ASC-J9 treatment,the degree of inflammatory cell invasion in myocardium of mice was reduced,and the inflammatory score was significantly decreased(P<0.01).(3)Masson staining showed that the area of myocardial fibrosis and collagen deposition was significantly reduced(P<0.005)..3.ASC-J9 increased anti-inflammatory factors expressionCompared with the vehicle group,the expression of IL-10(P<0.01)and IL-35(P<0.05)in myocardium of EAM + ASC-J9 treated group increased obviously.ConclusionThe androgen receptor degradation enhancer ASC-J9 could increase the expression of anti-inflammatory factors(IL-10 and IL-35)and ameliorate myocardial inflammation of EAM mice.Therefore,ASC-J9 might become a novel therapeutic option for male myocarditis patients.Part 2The effect of ASC-J9 on regulating M2-type macrophage polarization in EAM miceBackgroundMacrophages are important mediators of inflammatory cardiovascular disease.Several studies have shown that macrophages play an important role in the inflammatory response of myocarditis.Macrophages are heterogeneous and can be differentiated into M1(classic-activated)and M2(alternative-activated)macrophages depending on various stimulators.Macrophages with different phenotypes may play opposite roles.Macrophage polarization exerts a pivotal function in modulating heart inflammatory response.Studies have suggested that Ml macrophages are increased in myocardial tissue of myocarditis and that they aggravate inflammation in the development of myocarditis.By contrast,M2 macrophages show an anti-inflammatory character associated with tissue repairment,and polarization to M2 macrophage significantly alleviates the inflammatory response of the myocardium in myocarditis.Therefore,regulating macrophage polarization may be a novel therapeutic target for the treatment of myocarditis.Our previous study proved that AR exacerbated inflammation in experimental autoimmune myocarditis by promoting Ml macrophage polarization,and conversely,ASC-J9 alleviated myocardial inflammation by reducing M1 macrophage polarization.Nevertheless,it remains unknown that whether M2 macrophages regarded as anti-inflammatory cells could take part in the development of myocarditis,and whether ASC-J9 influences M2 polarization.ObjectiveTo study the effect of ASC-J9 on promoting macrophages polarization towards M2 macrophages in vitro and vivo.Methods1.Animal experiment1.1 The male Balb/c mice weighted 18-20g,were divided into 3 groups:control group,EAM+DMSO(vehicle group)and EAM+ASC-J9 treatment group.EAM model was induced by immunization with murine ?-cardiac myosin heavy chain polypeptides on day 0 and 7.The mice from EAM+ASC-J9 treatment group were intraperitoneally injected with ASC-J9 every other day between day 8 and 20 of EAM.In parallel,the vehicle group received saline containing 0.5%DMSO at the same time.At the end of 21 days,animal models were obtained.1.2 Immunofluorescence staining was used to determine the percentage of F4/80+/Arg-1+ M2 macrophages in the myocardial tissue of mice in each group.1.3 QRT-PCR was used to detect the expression of M2 macrophage-associated factors(Arg-1,MMR)in the myocardial tissue of mice in each group.2.Experiment in vitro2.1 Cell culture and groups:The Raw264.7 cell line was cultured in DMEM medium supplemented with 10%fetal bovine serum.The cells were divided into 4 groups:1)control group,2)IL-4 stimulating group,3)IL-4 +DMSO group(vehicle control group,containing 0.0005%DMSO),4)1L-4 + ASC-J9 group.In the vehicle control group and ASC-J9 treatment group,the cells were pretreated with DMSO and ASC-J9 with appropriate concentration for 2 hs,and then were stimulated with IL-4(20 ng/ml)for 48 hours to induce M2 macrophages polarization.2.2 QRT-PCR was used to measure the expression of M2 macrophage-associated factors(Arg-1,MMR)in each group.2.3 The expression of cytokine IL-10 secreted by M2 macrophages was detected with QRT-PCR in each groupResults1.ACS-J9 increased M2 macrophages in EAM1.1 Compared with the vehicle group,double immunofluorescence staining revealed that the proportion of M2 macrophages witih double-positive F4/80 +/Arg-1+ in myocardial tissue increased after ASC-J9 treatment,but there was no statistical significance.(P=0.053)1.2 Compared with the vehicle group,the expression of M2-specific gene Argl and the specific surface marker MMR were significantly elevated in the ASC-J9 group.2.ACS-J9 promoted M2 macrophage polarization in vitro2.1 Using QRT-PCR to detect the expression of M2 macrophage-related molecules in Raw264.7 cells,the results showed that the expression levels of Argl and MMR were significantly higher after IL-4 treatment comparing with control group,which suggested M2-type macrophages were successfully induced by IL-4,and ASC-J9 stimulation further upregulated Argl and MMR compared with the vehicle group.2.2 Compared with the vehicle group,the expression level of IL-10 secreted by M2 macrophages in Raw264.7 cells treated with ASC-J9 was significantly increased(P<0.05).ConclusionASC-J9 promoted macrophages polarization to M2 phenotype in EAM mice,and significantly increased the expression of anti-inflammatory cytokines secreted by M2 macrophages.AS a result,ASC-J9 ameliorated myocardial inflammatory response and improved EAM.Part 3The mechanism of ASC-J9 in regulating M2-type macrophage polarizationBackgroundMacrophages are characterized by heterogeneity and functional plasticity.Studies have suggested that macrophages can be polarized into M2 macrophages in response to the cytokines such as IL-4,IL-13 or IL-10.There are various molecular mechanisms involved in regulating the polarization of M2 macrophages.It has been proved activating JAK/STAT signaling pathway can facilitate M2 macrophages polarization.Suppressors of cytokine signaling(SOCS)are a family of cytokine-inducible negative feedback regulators,consisting of CIS and SOCS1-SOCS7,which can suppress the JAK/STAT signaling pathway and control both innate and adaptive immune responses.It has been proved that SOCS is a pivotal factor and relevant to the polarization of macrophages.Studies have confirmed that activating the JAK1/STAT3/SOCS3 signaling pathway contributes to M2 macrophage polarization.Enhanced STAT3 phosphorylation and decreased SOCS3 expression are correlated with M2 polarization.We found that there were predicted androgen-response-element on the promoter region of SOCS3 using the ALGGEN PROMO system,by binding to which androgen receptor(AR)exerted effect.Our previous study confirmed that ASC-J9 increased IL-10 expression via degrading AR.Moreover,SOCS3 downregulation led to permissive IL10/STAT3 signaling that promoted the polarization to M2 macrophages.Therefore,ASC-J9 may regulate the M2 macrophages reprogramming through the STAT3/SOCS3 signaling pathway.ObjectiveTo study the mechanism of ASC-J9 in promoting the polarization of M2 macrophage via EAM mice in vivo and macrophages in vitro.Results1.Experiment in vivoThe male Balb/c mice were divided into control group,EAM+DMSO(vehicle group)and EAM+ASC-J9 treatment group.EAM model was induced by immunization with murine a-cardiac myosin heavy chain polypeptides.ASC-J9 or normal saline containing 0.5%DMSO was intraperitoneally injected every other day between day 8 and 20 of EAM.At the end of 21 days,animal models were obtained.M2-type macrophage polarization-related factor SOCS3 in myocardial tissue was detected by QRT-PCR.2.Experiment in vitroThe Raw264.7 cell line was cultured in DMEM medium supplemented with 10%fetal bovine serum,divided into 4 groups:1)control group,2)IL-4 stimulating group,3)IL-4 +DMSO group(vehicle control group,containing 0.0005%DMSO),4)IL-4 + ASC-J9 group.In the vehicle control group and ASC-J9 treatment group,the cells were pretreated with DMSO and ASC-J9 with appropriate concentration for 2 hs respectively,and then were stimulated with IL-4(20 ng/ml)for 48 hours to induce M2 macrophages polarization.The expression of SOCS3 and.phosphorylation of STAT3 which were related to the polarization to M2 macrophages were detected with Western Blot after IL-4 stimulation.Results1.ASC-J9 suppressed the expression of SOCS3 in vivoThe result of QRT-PCR indicated that compared with the vehicle group,the expression of SOCS3,an inhibitory factor related to the polarization to M2 macrophages,was significantly reduced in the ASC-J9 treatment group(P<0.01).2.ASC-J9 reduced SOCS3 and enhanced STAT3 activation in vitroWestern Blot analysis showed that,compared with the vehicle group,the expression of SOCS3,an inhibitory factor related to the polarization to M2 macrophages,was significantly reduced in Raw264.7 cells treated with ASC-J9(P<0.05).Meanwhile,the phosphorylation level of STAT3 was significantly increased(P<0.05).ConclusionASC-J9 suppressed SOCS3 expression and enhanced STAT3 phosphorylation,which suggested ASC-J9 promoted macrophages polarization to M2 macrophages via the STAT3/SOCS3 signaling pathway.