Activation of activin/Smad2 and 3 signaling pathway and the potential involvement of endothelial‑mesenchymal transition in the valvular damage due to rheumatic heart disease

Figures

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  Figure 1.. H&E and Sirius red staining of valve tissues. (A) H&E staining. RHD-induced valvular damage was observed in the RHD group. Magnification, ×400. Scale bar =50 µm. Arrows represent inflammatory cells and inflammatory exudate. (B) Sirius red staining. RHD-induced fibrosis was observed in the RHD group. Magnification, ×100. Scale bar=200 µm. Arrows represent COL3 (green fibers with weak birefringence). (C) Inflammatory cell density of the valves in RHD group was significantly higher compared with the control group. (D) COL3/1 ratio valves in the RHD group were higher compared with the control group. The data are presented as the means ± standard deviation. *P<0.05 vs. the control group. H&E, hematoxylin and eosin staining; RHD, rheumatic heart disease; H&E, hematoxylin and eosin; RHD, rheumatic heart disease; COL3, collagen fiber type 3; COL1, collagen fiber type 1.

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  Figure 2.. RT-qPCR results for the relative expression of (A) activin A, Smad2 and Smad3 in the two groups, (B) LEF-1, Snail1, TWIST, ZEB1 and ZEB2, (C) α-SMA and COL1A1, (D) COL3A1 and FSP1 and (E) BAX in the two groups. The results show that expression of activin A, LEF-1, Snail1, TWIST, ZEB1, ZEB2, α-SMA, COL1A1, COL3A1, FSP1 and BAX was increased in the RHD group. Data are presented as the means ± standard deviation. *P<0.05 vs. respective control group. RT-qPCR, reverse transcription-quantitative PCR; LEF-1, lymphoid enhancer factor-1; ZEB, zinc finger E-box binding homeobox; α-SMA, α smooth muscle actin; COL1A1, type I collagen; COL3A1, collagen type III α1 chain; FSP1, fibroblast-specific protein 1; RHD, rheumatic heart disease.

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  Figure 3.. Western blot analysis for (A) activin A, Smad2, Smad3, p-Smad2 and p-Smad3 levels in the two groups and (B) quantified protein levels. Protein levels of activin A, p-Smad2 and p-Smad3 were increased in RHD group. Data are presented as the means ± standard deviation. *P<0.05 vs. respective control. p-, phosphorylated; RHD, rheumatic heart disease.

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  Figure 4.. Western blot analysis of (A) LEF-1, Snail1, TWIST, ZEB1, ZEB2, α-SMA and COL1A1 levels in the two groups and (B) quantified protein expression levels. (C) Relative protein expression of α-SMA and COL1A1. Protein expression of LEF-1, Snail1, TWIST, ZEB1, ZEB2, α-SMA and COL1A1 was increased in RHD group. The data are presented as the means ± standard deviation. *P<0.05 vs. respective control. LEF-1, lymphoid enhancer factor-1; ZEB, zinc finger E-box binding homeobox; α-SMA, α smooth muscle actin; COL1A1, type I collagen; RHD, rheumatic heart disease.

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  Figure 5.. Analysis of valvular inflammation markers, apoptosis-related markers, cytokines and rheumatoid factor. (A) Western blot analysis of NF-κB, p-NF-κB, caspase-3 and cleaved caspase-3 levels in the two groups and (B) quantified relative protein expression levels. (C) Relative protein expression of caspase-3 and cleaved caspase-3. (D) Concentrations of IL-6, IL-17, TNF-α and rheumatoid factor in serum detected using ELISA. Results show that the ratio of p-NF-κB/t-NF-κB and the expression of p-NF-κB, cleaved caspase-3, IL-6, IL-17, TNF-α and rheumatoid factor was increased in the RHD group. Data are presented as the means ± standard deviation. *P<0.05 vs. respective control. p-, phosphorylated; RHD, rheumatic heart disease.