Bazedoxifene Regulates Th17 Immune Response to Ameliorate Experimental Autoimmune myocarditis via Inhibition of STAT3 Activation

Figures

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  FIGURE 1. Bazedoxifene ameliorated cardiac inflammation and injury induced by EAM and improved EAM-induced left ventricular dysfunction (A). Representative heart images of the indicated groups (B). Change in body weight, HW/BW ratio, and HW/TL ratio of the indicated groups (C). Representative echocardiography M-mode images and analysis of left ventricular function. Representative images of immunohistochemistry of inflammatory cells stained with anti-CD45 and anti-IL-6 (D) in each heart section. Scale bars: 50 μm. E and F. Representative H and E-stained and Masson staining images of left ventricular sections of indicated groups. Bar graphs on the right show quantitation of data (E) (n = 5). Scale bars: left 0.5 mm, inset 100 μm. Scale bars: left 0.5 mm, inset 100 μm *p < 0.05, **p < 0.01, ***p < 0.001.

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  FIGURE 2. Bazedoxifene reduced Th17 cell infiltration and inhibited STAT3 phosphorylation in EAM hearts at day 21 (A). The percentages of Th17 cells and Treg cells in isolated heart-infiltrating cells of each group (n = 6) (B). Representative images of Western blots for RORγt, FOXP3 p-STAT3, and STAT3 protein from heart-infiltrating cells (n = 6). (C). The mRNA expression levels of RORγt, Foxp3, and inflammatory factors (IL-6 and IL-17A) in isolated heart-infiltrating cells of the indicated groups (n = 5). *p < 0.05, **p < 0.01, ***p < 0.001.

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  FIGURE 3. Bazedoxifene attenuated the EAM-induced differentiation of Th17 and Treg cells in EAM splenic CD4+ T cells at 14 and 21 days (A). Representative spleen images of each group at days 14 and 21, respectively. The percentage of Th17 and Treg cells from spleen were determined at days 14 (B) and 21 (C) by flow cytometry. Representative FACS pictures of each group are shown. Averages are presented graphically on the right. *p < 0.05, **p < 0.01, ***p < 0.001.

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  FIGURE 4. Bazedoxifene affected expression of the Th17/Treg cell ratio and related inflammatory factors in EAM splenic CD4+ T cells at 14 and 21 days. Western blots are shown for p-STAT3 (Tyr 705), STAT3, RORγt, and Foxp3 protein in splenic CD4+ T cells of each group at days 14 (A) and 21 (B). mRNA expression is shown for Th17 cell-related factors and Treg cell-related factors in splenic CD4+ T cells from each group at days 14 (C) and 21 (D) (n = 4 per control group). *p < 0.05, **p < 0.01, ***p < 0.001.

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  FIGURE 5. Bazedoxifene inhibited Th17 cell differentiation and proliferation in vitro. (A) Naïve T cells were labeled with CFSE and generated under Th17-polarizing conditions with different Bazedoxifene concentrations. (B) Naïve T cells sorted from healthy mice were cultured under Th17-polarizing conditions in the presence or absence of Bazedoxifene treatment for 72 h (n = 3 in each group). *p < 0.05, **p < 0.01, ***p < 0.001.

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  FIGURE 6. Bazedoxifene regulated autophagy in polarized Th17 cells (A). Representative TEMs revealing the ultrastructure of CD4+ T cells cultured under Th17-polarizing conditions with or without Bazedoxifene treatment (B). p-STAT3, STAT3, RORγt, LC3-Ⅱ/LC3-Ⅰ, P62, and beclin-1 protein levels were analyzed by western blotting (C). Isolated naïve CD4+ T cells were cultured under Th17-polarizing condition in the presence or absence of Bazedoxifene. The cells were stained with anti-p-STAT3, anti-IL-17, anti-LC3, anti-beclin-1, and anti-P62 antibodies, and visualized using confocal microscopy. *p < 0.05, **p < 0.01, ***p < 0.001.