sST2: A Bridge Between Sirt1/p53/p21 Signal-Induced Senescence and TGF-β1/Smad2/3 Regulation of Cardiac Fibrosis in Mouse Viral Myocarditis

Figures

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  Fig. 1. sST2 increased and promoted inflammatory damage in the viral myocarditis. A The generation of sST2 was determined using ELISA. B Western blot analyses of protein expression of sST2. C H&E stain was used to evaluate the degree and grade of cardiac inflammation. D LDH, CK-MB, and AST levels in the serum were determined using a fully automatic biochemical analyzer. E RT-PCR analyses of the mRNA levels of IL-1β, IL-6, and TNF-α. Data were represented as the mean ± S.D. Compared with the control group, *p < 0.5; **p < 0.01; ***p < 0.001.

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  Fig. 2. sST2 promoted the activation of MCF and inhibits its senescence in vitro. A Western blot analyses of protein expression of Collagen-I, Collagen-III, and α-SMA. B RT-PCR analyses of the mRNA levels of Collagen-I, Collagen-III, and α-SMA. C CCK8 assay was used to detect proliferation ability of MCFs. D Western Blot analyses of protein expression of p16, p21, and p53. E RT-PCR analyses of the mRNA levels of p21, p53, and Sirt1. Data were represented as the mean ± S.D. Compared with the control group, *p < 0.5; **p < 0.01; ***p < 0.001.

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  Fig. 3. sST2 inhibits MCF senescence through Sirt1/p53/p21 signaling pathway. (A) CCK8 assay was used to detect proliferation ability of MCFs. B SA-β-gal stain was used to detect the senescence of MCFs. C Western blot analyses of protein expression of p21, p53, and Sirt1. D RT-PCR analyses of the mRNA levels of p21, p53, and Sirt1. Data were represented as the mean ± S.D. Compared with the control group, *p < 0.5; **p < 0.01; ***p < 0.001.

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  Fig. 4. sST2 promotes MCF activation by inhibiting senescence. A Western blot analyses of protein expression of Collagen-I, Collagen-III, and α-SMA. B Western blot analyses of protein expression of Collagen-I, Collagen-III, and α-SMA. C RT-PCR analyses of the mRNA levels of Collagen-I, Collagen-III, and α-SMA. D RT-PCR analyses of the mRNA levels of TGF-β1, Smad2, and Smad3. E Western blot analyses of protein expression of TGF-β1, p-Smad2, and p-Smad3. Data were represented as the mean ± S.D. Compared with the control group, *p < 0.5; **p < 0.01; ***p < 0.001.

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  Fig. 5. sST2 inhibits MCFs senescence and Sirt1/p53/p21 signaling, accelerating cardiac fibrosis in VMC mice. The heart tissues were collected on day 15 for pathological examination. A Representative micrographs of paraffin-embedded heart stained histochemically for Masson’s trichrome (Masson). B, C Immunofluorescence images showing the fibroblast activation in heart tissue; n = 5 per group. Collagen-I staining is showed in green; α-SMA is shown in red (Scale bar = 20 μm). D RT-PCR analysis for Collagen-I/III and α-SMA mRNA in the heart at 15d after VMC. The gene expression level was normalized with that of GAPDH (n = 5). E Western blotting for Sirt1, Collagen-I/III, p53, α-SMA, p21, and p16. GAPDH was used as the loading control. Protein expression relative to β-actin was assessed by densitometric analysis. F RT-PCR analysis for Sirt1/p53 mRNA in the heart at 15d after VMC. The gene expression level was normalized with that of GAPDH (n = 5). G Representative micrographs of cell immunofluorescently stained for p21 and α-SMA, with DAPI staining the nucleus. Data were represented as the mean ± S.D. Compared with the control group, *p < 0.5; **p < 0.01; ***p < 0.001.

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  Fig. 6. Elevated sST2 in the VMC could have a deleterious effect on the heart by inhibiting senescence of MCF, leading to cardiac fibrosis.