Study On The Mechanism Of Bazedoxifene For Autoimmune Myocarditis Treatment

The treatment of myocarditis is mainly to control the systemic acute inflammatory response.It is still the difficulty and focus of clinical treatment to find a treatment method that can not only reduce the inflammatory cardiac injury,but also slow down the cardiac remodeling.Bazedoxifene(BAZ)is the third generation estrogen receptor modulator approved by the FDA for the clinical prevention and treatment of osteoporosis in menopausal women,which shows good long-term safety and tolerability.The purpose of this study was to observe the efficacy of Bazedoxifene in treatment of autoimmune myocarditis,and to explore the mechanism of BAZ on the Th17 cell immune response.The study includes the following three parts:Part 1 Effect of Bazedoxifene on autoimmune myocarditis in miceObjective:To investigate the effects of Bazedoxifene on inflammatory cardiac injury and cardiac function in mice with experimental autoimmune myocarditis.Methods:BALB/c male inbred mice were randomly divided into three groups:control group,experimental autoimmune myocarditis model group(EAM group)and Bazedoxifene intervention group(BAZ group).A model of experimental autoimmune myocarditis(EAM)was established by injecting α-myosin heavy chain derived polypeptide(MyHC-α614-629)into lymph nodes subcutaneously on day 0 and day 7.The BAZ mice were treated with Bazedoxifene(5mg/Kg/day,po.)daily from 7 to 21 days after modeling.At the end of the experiment on day 21,echocardiography was performed to observe the changes of heart morphology and function in each group.Blood pressure was measured and compared in each group.The body weight of mice was recorded,and the general heart performance of mice in each group was observed.The ratio of heart weight to body weight and the ratio of heart weight to tibia length in each group were measured and compared.HE staining of heart sections was used to compare the number of inflammatory cell infiltration and inflammatory score in myocardium.Masson staining shows the degree of cardiac fibrosis.CD45 immunohistochemical staining was used to compare the number of lymphocytes in myocardial tissue,and IL-6 immunohistochemical staining was used to compare the inflammation level in myocardial tissue of each group.The TUNEL assay of myocardial tissue and cTnI ELISA test of serum were used to compare the degree of myocardial inflammatory injury.Results:Compared with the control mice,the body weight of mice in EAM group decreased,and the ratio of heart weight to body weight and heart weight to tibia length increased.Systolic function of EAM mice was decreased,but there was no significant change in blood pressure in each group.The results of HE and Masson staining showed that the inflammatory score of myocardial tissue was increased,and the myocardial fibrosis was obvious.Immunohistochemical results showed that lymphocyte infiltration and IL-6 expression were increased in EAM mice.TUNEL assay and serum cTnI showed severe myocardial injury in EAM mice.Compared with the mice in the EAM group,the weight loss of mice in the BAZ group was not significant,the ratio of heart weight to body weight and heart weight to tibia length were reduced,and the abnormal cardiac systolic function was relieved.HE and Masson staining results showed that the inflammatory score of myocardial tissue was decreased,and the degree of myocardial fibrosis was reduced.Immunohistochemical results showed that lymphocyte infiltration was reduced in EAM mice.The TUNEL assay and the serum cTnI showed that the myocardial injury was relieved in EAM mice.Conclusions:Bazedoxifene reduced lymphocyte infiltration in myocardium,reduced myocardial inflammatory injury,improved cardiac hypertrophy and fibrosis,and relieved cardiac systolic insufficiency caused by experimental autoimmune myocarditis.Part 2 Study on the therapeutic mechanism of Bazedoxifene in autoimmune myocarditis miceObjective:To investigate the effect of Bazedoxifene on Th17 cell differentiation and function in EAM mice.Methods:Ditto for mouse modeling and drug intervention.Cardiac lymphocytes were isolated and counted on day 21.The ratio of Th17 cells and Treg cells in cardiac lymphocytes was detected by flow cytometry.The expression levels of p-STAT3,STAT3,RORyt and Foxp3 in heart lymphocytes of mice in each group were detected by Western blot.The mRNA levels of RORyt,Foxp3,IL-6 and IL-17 in heart lymphocytes of mice were detected by RT-PCR.Spleen of mice at 14 and 21 days were taken and separated into single-cell suspensions.The proportion of Th17 cells and Treg cells in splenocytes was compared by flow cytometry in each group.The expression levels of p-STAT3,STAT3,RORyt and Foxp3 in splenocytes of each group were detected by Western blot.The mRNA levels of RORyt and Th17 cell-related inflammatory cytokines IL-17A,IL-21 and IL-22 in spleens of each group were detected by RT-PCR.The mRNA levels of Foxp3 and Treg cell-related inflammatory cytokines IL-35,TGF-β and IL-10 in spleens of each group were detected by RT-PCR.Finally,the proportion of antigen-presenting cells(DCs)in spleens of mice in each group was detected and the levels of inflammatory cytokines IL-23 and IL-6 released by DCs were detected by ELISA.Results:EAM increased the number of lymphocytes in the heart tissues of mice.The proportion of Th17 cells increased while the proportion of Treg cells decreased.The mRNA transcription and protein expression levels of p-STAT3 and RORyt were increased.Transcriptional levels of IL-6 and IL-17A were elevated.While the mRNA transcription and protein expression levels of Foxp3 transcription level decreased.Compared with the EAM mice,the proportion of Th17 cells were decreased with Bazedoxifene administration,but the proportion of Treg cells did not change significantly.The mRNA transcription and protein expression levels of p-STAT3 and RORyt were decreased.The mRNA of Foxp3 was increased;The mRNA levels of IL-6 and IL-17A were reduced.On day 14 and 21,the spleen of EAM mice became enlarged.The Th 17 cell proportion,the mRNA and protein expression levels of p-STAT3 and RORyt were increased.The expression levels of Th17 related factors(IL-17A,IL-21 and IL-22)were increased.On day 14,the proportion of Treg and expression level of Foxp3 decreased.The expression levels of Treg related cytokines IL-35,TGF-β and IL-10 were increased.However,there was no significant change in the proportion of Treg cells on day 21.The expression levels of Treg related factors(IL-35,TGF-β and IL-10)were decreased to varying degrees.Bazedoxifene decreased proportion of Th17 cells in the spleen of mice on day 14 and 21.The expression levels of p-STAT3 and RORyt were down-regulated by Bazedoxifene.The expression levels of Th17 related factors(IL-17A,IL-21 and IL-22)were also reduced.However,the effects of Bazedoxifene on Treg cells were not consistent.On day 14,Bazedoxifene increased the proportion of Treg cells,up-regulated the expression levels of Foxp3 and related cytokines IL-35,TGF-β and IL-10.On day 21,Bazedoxifene down-regulated the expression levels of Foxp3 and related cytokines IL-35,TGF-β and IL-10.At last,Bazedoxifene reduced the elevated CD11c+positive DCs in EAM and inhibit the secreting levels of IL-23 and IL-6.Conclusions:Bazedoxifene regulate the differentiation and function of Th17 cells in myocarditis and spleen and reduced secretion of IL-17A,IL-21 and IL-22 by inhibiting STAT3/RORy signaling pathway in EAM mice.The effects of Bazedoxifene on Treg cell were inconsistent in different stages.The differentiation and function of Treg cells were up-regulated at the initial stage of immunity,but the immune response of Treg cells was not significantly affected at the peak of immunity.Bazedoxifene decreased the ratio of CD11c+DC and its ability to secrete IL-6 and IL-23,suggesting that Bazedoxifene could also inhibit Th17 cell differentiation by inhibiting the ability of DCs to secrete inflammatory cytokines.Part 3 In vitro analysis of the effect of Bazedoxifene on Th17 cellObjective:The effect of Bazedoxifene on differentiation and function of Th17 cells was further studied by polarization Th17 cells in vitro.Methods:Naive CD4+T cells from spleen of healthy Balb/c mice were separated by magnetic beads and differentiated into Th 17 and Treg cells under the stimulation of multi-cytokines in vitro.The percentage of Th17 cells and the proliferation ratio of CFSE assay were detected by flow cytometry.The effect of Bazedoxifene on the proportion of Treg cells in vitro was examined.The formation of autophagosomes and the co-expression of autophagy maker proteins with IL-17 in polarized Th17 cell were detected and compared with BAZ group by transmission electron microscopy and immunofluorescence localization experiments.Meanwhile,the expression levels of p-STAT3 and autophagy maker proteins(LC3,P62 and Beclin-1)were detected by Western blot.The Stattic group(STAT3-targeted inhibitor)was set up as positive control.In order to discuss whether the effect of Bazedoxifene on Th17 is related to estrogen signaling pathway,the splenic naive CD4+T cells of female and male mice were separated for Th17 polarization in vitro with Bazedoxifene(5 μM)or estradiol(100 nM).The differentiation ratio of Th17 cells under different treatments was compared by flow cytometry.Results:Flow cytometry showed that compared with the control group,the proportion and the proliferation rate of Th17 cells induced by polarized stimulation in vitro was decreased gradually with the increase of the concentration gradient of Bazedoxifene.However,Bazedoxifene had no significant effect on the differentiation of Treg cells.Western blot showed that after Bazedoxifene treatment,STAT3 phosphorylation and RORyt expression levels were decreased with the decreased proportion of Th17 cells.At the same time,the expression of LC3Ⅱ/Ⅰ was increased,the expression of P62 and Beclin-1 was decreased was decreased with Bazedoxifene treatment.Western blot showed that Stattic inhibited the expression of RORyt,increased the expression of LC3Ⅱ/Ⅰ,decreased the expression of P62 and Beclin-1,which enhanced the autophagy of Th17 cells.Transmission electron microscopy showed that more bilayer autophagosomes were formed in naive CD4+T cells treated with Bazedoxifene under Th17 polarization.Immunofluorescence showed that both expression of IL-17 and phosphorylated STAT3 were decreased in the same cell with Bazedoxifene.The expression of IL-17 and LC3 were opposite.While the expression of IL-17 and Beclin-1 were decreased simultaneously,and the expression of IL-17 and P62 were decreased similarly.Finally,there was no significant difference on Th17 cell differentiation between female and male mice by Bazedoxifene.Compared with the control group,estrogen could not increase the proportion of Th17 cells in vitro,but Stattic could significantly inhibit the differentiation of Th17 cells.Conclusions:Bazedoxifene reduced differentiation of Th17 cells induced in vitro,which did not affect differentiation of Treg cells.Bazedoxifene affects the formation of autophagosomes in Th17 by inhibiting STAT3 activation.The inhibitory effect of Bazedoxifene on differentiation and function on Th17 cells was independent of estrogen signaling pathway.Bazedoxifene had no obvious effect on Treg cells.