The aqueous extract of Gentianella acuta improves isoproterenol‑induced myocardial fibrosis via inhibition of the TGF‑β1/Smads signaling pathway

Figures

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  Figure 1. Analysis of xanthone compounds in G. acuta using high performance liquid chromatography. (A) The aqueous extracts of G. acuta. (B) The mixed standard solutions. The peaks were numbered as 1: Swertianolin, 2: Demethylbellidifolin and 3: Bellidifolin. G. acuta, Gentianella acuta.

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  Figure 2. G. acuta ameliorates ISO-induced myocardial fibrosis and cardiac remodeling in rats. (A) H&E staining to detect morphological changes; Masson trichrome staining to detect myocardial fibrosis; scale bars=200 µm; WGA staining to access cardiomyocyte cross-sectional area; scale bars=50 µm. (B) Bar graphs show the percentage of relative myocardial damage. (C) Bar graphs show the percentage of collagen deposition. (D) Bar graphs show the cardiomyocyte cross-sectional area. Data are shown as the mean ± standard deviation. **P<0.01 vs. the Control, #P<0.05 and ##P<0.01 vs. ISO. ISO, isoproterenol; H&E, hematoxylin and eosin; WGA, wheat germ agglutinin; G. acuta, Gentianella acuta.

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  Figure 3. G. acuta inhibits the expression of collagens I, III and α-SMA induced by ISO. (A) Immunohistochemical staining for collagens I, III and α-SMA, scale bars=100 µm. Bar graphs show the percentage of (B) collagens I, (C) III and (D) α-SMA. (E) Western blotting detection of collagens I, III and (F) α-SMA. (G) Bar graphs show fold-changes for the collagens I and III and (H) α-SMA expression as analyzed by western blotting. GAPDH was used as a loading control (n=3). Data are shown as the mean ± standard error of the mean. **P<0.01 vs. the Control, ##P<0.01 vs. ISO. α-SMA, α-smooth muscle actin; ISO, isoproterenol; G. acuta, Gentianella acuta.

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  Figure 4. G. acuta causes a reduction in ISO-induced TGF-β1 expression. (A) Immunohistochemical staining to detect TGF-β1, scale bars=100 µm. (B) Bar graphs show the percentage of TGF-β1. (C) Western blotting to detect TGF-β1 and bar graphs show fold-changes of TGF-β1 expression as analyzed by western blotting. GAPDH was used as a loading control (n=3). Data are shown as the mean ± standard error of the mean. **P<0.01 vs. the Control, ##P<0.01 vs. ISO. TGF-β1, transforming growth factor; ISO, isoproterenol; G. acuta, Gentianella acuta.

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  Figure 5. G. acuta causes a reduction in ISO-induced TβRI levels and the phosphorylation of TβRI and II. (A) Western blotting analysis of TβRII, phosphory-lated (P)-TβRII, TβRI, P-TβRI. (B) Bar graphs show fold-changes of the TβRII, P-TβRII, TβRI, P-TβRI expression, as analyzed by western blotting. GAPDH was used as a loading control (n=3). (C) Bar graphs show fold-changes of P-TβRII/TβRII and (D) P-TβRI/TβRI. Data are shown as mean ± standard error of the mean. **P<0.01 vs. the Control and ##P<0.01 vs. ISO. ISO, isoproterenol; G. acuta, Gentianella acuta; P-TβR, phosphorylated- TGF-β receptor.

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  Figure 6. G. acuta causes inhibition of ISO-activated TGF-β1/Smads signaling. (A) Immunohistochemistry staining to detect Smads2, 3 and 4; scale bars=100 µm. (B) Western blotting analysis of Smad2, P-Smad2, Smad3, P-Smad3 and Smad4 expression. (C) Bar graphs show fold-changes of Smad2, P-Smad2, Smad3, P-Smad3 and Smad4 expression as analyzed by western blotting. GAPDH was used as a loading control (n=3). (D) Bar graphs show fold-changes of P-Smad2/Smad2 and (E) P-Smad3/Smad3. Data are shown as mean ± standard error of the mean. **P<0.01 vs. control, #P<0.05 and ##P<0.01 vs. ISO. P-Smad, phosphorylated-mothers against decapentaplegic homolog; ISO, isoproterenol; G. acuta, Gentianella acuta.

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  Figure 7. The proposed antifibrotic mechanism of G. acuta in ISO-induced myocardial fibrosis in rats. Myocardial injury activates the TGF-β/Smads pro-fibrotic signaling pathway. G. acuta inhibits TGF-β1 expression, downregulates TβRI expression and TβRI and II phosphorylation levels, suppresses Smad expression and phosphorylation activation, and causes a decrease in ISO-induced α-SMA, collagens I and III expression in heart tissues. The results indicate that G. acuta improves ISO-induced myocardial fibrosis via inhibition of the TGF-β1/Smads signaling pathway. P-Smad, phosphorylated-mothers against decapentaplegic homolog; ISO, isoproter-enol; G. acuta, Gentianella acuta; P-TβR, phosphorylated- TGF-β receptor.