sRAGE inhibits myocardial ischemia/reperfusion injuries via regulating Treg cells

Figures

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  Fig. 1. sRAGE increased Tregs in myocardial tissue during I/R. A Representative images of CD4 and Foxp3 double staining. CD4 was shown as brown, Foxp3 was shown as red. The scale bars are 50 μm–20 μm. B Quantitative data of CD4 and Foxp3 immunohistochemistry double staining in heart tissue, n = 6 per group. C Representative flow analysis images of Tregs in the mice heart, n = 6 per group. D Quantitative data of CD4+ T cells in heart by flow cytometry. E Quantitative data of CD4+CD25+Foxp3 Tregs in heart by flow cytometry. The data are expressed as the mean ± SEM. **p < 0.01; ns, p ≥ 0.05

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  Fig. 2. Tregs involved in the protective effects of sRAGE on cardiac function. A The representative M-mode echocardiography images of long-axis. B Quantitative data of LVEF for each group. C Quantitative data of FS for each group. D Quantification data of CO for each group. E Quantitative data of LVEDV for each group. F Quantitative data of LVESV for each group. G Quantitative data of HR for each group. The data are expressed as the mean ± SEM. **p < 0.01; ns, p ≥ 0.05

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  Fig. 3. Tregs involved in the inhibiting effects of sRAGE on I/R-induced cell death. A The representative images of TTC staining. The scale bars are 1 cm. B Quantitative data of the infarct area/total left ventricular area, n = 6 per group. C Representative images of TUNEL staining. The red spots indicated TUNEL-positive cells. The green fluorescence indicated α-actinin. The blue fluorescence indicated the nuclei. Arrows indicated TUNEL-positive cells. The scale bars are 200 μm. D Quantitative data of TUNEL-positive cells, n = 6 per group. E Representative images of Cleaved caspase-3 immunochemistry staining. The target protein was shown as brown. The scale bars are 50 μm. F Quantitative data of Cleaved caspase-3 immunochemistry staining, n = 6 per group. The data are expressed as the mean ± SEM. **p < 0.01

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  Fig. 4. Tregs involved in the inhibiting effects of sRAGE on myocardial fibrosis. A Representative images of Masson and Sirius red staining of cardiac tissue. Scale bars are 50 μm. B Quantitative data of Masson staining. C Quantitative data of Sirius red staining, n = 6 per group. The data are expressed as the mean ± SEM. **p < 0.01

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  Fig. 5. Tregs involved in the inhibiting effects of sRAGE on inflammatory response. A Representative images of HE staining of heart sections. The scale bars are 100 μm. B Representative images of CD68, iNOS and CD206 staining. The scale bars are 50 μm. C Quantitative data of CD68 (macrophage marker) positive cells. D Quantitative data of iNOS (M1 macrophage marker) positive cells. E Quantitative data of CD206 (M2 macrophage marker) positive cells. F Quantitative data of plasma IL-4 level, n = 6 per group. G Quantitative data of plasma IL-10 level, n = 6 per group. H Quantitative data of plasma IL-17 level, n = 6 per group. The data are expressed as the mean ± SEM, *p < 0.05, **p < 0.01

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  Fig. 6. sRAGE increased the expression of PD-L1 in cardiomyocytes. A Representative images of PD-L1 immunohistochemical staining. The scale bars are 50 μm–20 μm. B Quantitative data of PD-L1 immunohistochemical staining, n = 6 per group. C Representative images of Western blotting for PD-L1 in cardiomyocytes. D Quantitative data of the Western blotting results for PD-L1, n = 3 per group. The data are expressed as the mean ± SEM, *p < 0.05, **p < 0.01

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  Fig. 7. Inhibited PD-L1 decreases the effect of sRAGE on Tregs. A Representative images of Western blot for Foxp3. B Quantitative data of the Western blotting results for Foxp3, n = 3 per group. C Representative images of CD4 and Foxp3 double staining. CD4 was shown as brown, Foxp3 was shown as red. The scale bars are 50 μm–20 μm. D Quantitative data of CD4 and Foxp3 immunohistochemistry double staining, n = 6 per group. E The proportions of CD4+T cells and Tregs in heart tissue were detected by flow cytometry. n = 6 per group. F The proportions of CD4+T cells and Tregs in the spleen tissue by flow cytometry, n = 6 per group. The data are expressed as the mean ± SEM, *p < 0.05; **p < 0.01; ns, p ≥ 0.05

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  Fig. 8. sRAGE promoted the differentiation of CD4+ T cells into Tregs during I/R. A Representative flow analysis images of Tregs. B Quantitative data of Tregs by flow cytometry, n = 3 per group. The data are expressed as the mean ± SEM. **p < 0.01

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  Fig. 9. sRAGE regulated the expression of PD-L1 through JAK2-STAT3 pathway. A Representative images of Western blotting for p-STAT3, STAT3, PD-L1 of each group. B Quantitative data of the Western blotting results for p-STAT3/STAT3. C Quantitative data of the Western blotting results for PD-L1. D Representative images of Western blotting for p-JAK2, JAK2, p-STAT3, STAT3 of each group. E Quantitative analysis of the Western blotting results for p-JAK2/JAK2. F Quantitative analysis of the Western blotting results for p-STAT3/STAT3. n = 3 per group. The data are expressed as the mean ± SEM. **p < 0.01