Melatonin Ameliorates Coxsackievirus B3-Induced Myocarditis by Regulating Apoptosis and Autophagy

Figures

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  FIGURE 1. The survival rates of three groups of mice followed for 14 days, with HE staining of the myocardial tissue to evaluate the histological changes on days 7 and 14 (n = 5 in each group, three sections per heart were examined), and the expression of proinflammatory cytokines in the myocardium (n = 5 in each group). (A) Compared to the CVB3 group, the survival rate of the CVB3 + MLT group was increased. (B,C) Histological analysis of the infected hearts on days 7 and 14 (original magnification × 200, × 400). (D,F) Representative levels of proinflammatory cytokines on days 7 and 14. (E,G) Density ratio of the proinflammatory cytokines to Gapdh. ∗P < 0.05 versus the normal group, #P < 0.05 versus the CVB3 group.

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  FIGURE 2. Transmission electron microscopy showing the myocardial microstructure and autophagosomes (red arrowheads) on days 7 and 14 (scale bars = 0.5 μm). (A) Representative transmission electron microscopy image. (B) The results of the statistical analysis of the numbers of autophagosomes from 20 fields (n = 5 hearts per group). ∗P < 0.05 versus the normal group, #P < 0.05 versus the CVB3 group.

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  FIGURE 3. Levels of autophagy in the heart on day 7 (n = 5 in each group). (A,B) Expression of autophagy-related proteins in the myocardial tissues of mice in four groups on day 7. (C,D) Immunohistochemistry for LC3 on day 7. ∗P < 0.05 versus the normal group, #P < 0.05 versus the CVB3 group, ∆P < 0.05 versus the CVB3 + MLT group.

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  FIGURE 4. Levels of autophagy in the heart on day 14 (n = 5 in each group). (A,B) Protein expression levels of LC3II/I, Beclin1, and p62 in myocardial tissue of mice in four groups on day 14. (C,D) Immunohistochemistry for LC3 on day 14. ∗P < 0.05 versus the normal group, #P < 0.05 versus the CVB3 group, ∆P < 0.05 versus the CVB3 + MLT group.

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  FIGURE 5. The effects of melatonin on inflammation and apoptosis on day 7. (A) Representative myocardial pathological changes on day 7 (n = 5 in each group, three sections per heart were examined). (B–D) Expression of proinflammatory cytokines, apoptosis-related protein Bax, Bcl-2, cleaved caspase-3, and cleaved caspase 9 in the myocardium on day 7 (n = 5 in each group). (E) Double staining for DAPI (blue) and TUNEL (green) of sections from myocardium on day 7, scale bar = 50 μm (n = 5 in each group). ∗P < 0.05 versus the normal group, #P < 0.05 versus the CVB3 group, ∆P < 0.05 versus the CVB3 + MLT group.

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  FIGURE 6. The effects of melatonin on inflammation and apoptosis on day 14. (A) Representative myocardial pathological changes on day 14 (n = 5 in each group, three sections per heart were examined). (B–D) Expression of proinflammatory cytokines, apoptosis-related protein Bax, Bcl-2, cleaved caspase-3, and cleaved caspase 9 in the heart on day 14 (n = 5 in each group). (E) Double staining for DAPI (blue) and TUNEL (green) of sections from myocardium on day 14, scale bar = 50 μm (n = 5 in each group). ∗P < 0.05 versus the normal group, #P < 0.05 versus the CVB3 group, ∆P < 0.05 versus the CVB3 + MLT group.

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  FIGURE 7. The echocardiography results in each group on day 14 (n = 5 in each group). (A) Representative M-type cardiac ultrasound image. (B–E) The changes of LVEF, LVEDd, LVESd, and LVFS in five groups. LVEF, left ventricular ejection fraction; LVEDd, left ventricular end-diastolic dimension; LVESd, left ventricular end-systolic dimension; LVFS, left ventricular shortening fraction. ∗P < 0.05 versus the normal group, #P < 0.05 versus the CVB3 group.