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Salvianolic acid B prevents epithelial-to-mesenchymal transition through the TGF-beta1 signal transduction pathway in vivo and in vitro.

Wang QL, Tao YY, Yuan JL, Shen L, Liu CH - BMC Cell Biol. (2010)

Bottom Line: Sal B also has potential protective effects on renal diseases.In contrast, Sal B and vitamin E significantly attenuated the expression of alpha-SMA, TGF-beta1, TbetaR-I, p-Smad2/3, and MMP-2 activity, but increased E-cadherin expression.Furthermore, Sal B and SB-431542 profoundly down-regulated the expressions of TbetaR-I and p-Smad2/3 but prevented the decreased expression of Smad7 in TGF-beta1 stimulated HK-2 cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Liver Diseases, Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, 528 Zhangheng Road, Shanghai, China.

ABSTRACT

Background: Salvianolic Acid B (Sal B) is a water-soluble component from Danshen (a traditional Chinese herb widely used for chronic renal diseases) with anti-oxidative and cell protective properties. Sal B also has potential protective effects on renal diseases. Tubular epithelial cells can undergo epithelial-to-mesenchymal transition (EMT), which plays an important role in the pathogenesis of renal interstitial fibrosis (RIF) and is mainly regulated by TGF-beta1/Smads pathway. The aims of the study are to investigate the effect of Sal B on tubular EMT in vivo and in vitro, and to elucidate its underlying mechanism against EMT related to TGF-beta1/Smads pathway.

Results: For in vivo experiments, RIF was induced in rats by oral administration of HgCl2 and prophylaxised with Sal B and vitamin E. The protein expression of E-cadherin was down-regulated, while the expression of alpha-SMA, TGF-beta1, TbetaR-I, p-Smad2/3 and the activity of matrix metalloproteinase-2 (MMP-2) were up-regulated in kidneys of model rats when compared with those of normal rats. In contrast, Sal B and vitamin E significantly attenuated the expression of alpha-SMA, TGF-beta1, TbetaR-I, p-Smad2/3, and MMP-2 activity, but increased E-cadherin expression. For in vitro experiments, HK-2 cells were incubated with TGF-beta1 to induce EMT, and the cells were co-cultured with 1 and 10 microM Sal B or SB-431542 (a specific inhibitor of TbetaR-I kinase). TGF-beta1 induced a typical EMT in HK-2 cells, while it was blocked by Sal B and SB-431542, as evidenced by blocking morphologic transformation, restoring E-cadherin and CK-18 expression, inhibiting alpha-SMA expression and F-actin reorganization, and down-regulating MMP-2/9 activities in TGF-beta1 mediated HK-2 cells. Furthermore, Sal B and SB-431542 profoundly down-regulated the expressions of TbetaR-I and p-Smad2/3 but prevented the decreased expression of Smad7 in TGF-beta1 stimulated HK-2 cells.

Conclusions: Sal B can prevent tubular EMT in the fibrotic kidney induced by HgCl2 as well as HK-2 cells triggered by TGF-beta1, the mechanism of Sal B is closely related to the regulation of TGF-beta1/Smads pathway, manifested as the inhibition of TGF-beta1 expression, suppression of TbetaR-I expression and function, down-regulation of Smad2/3 phosphorylation, and restoration of the down-regulation of Smad7, as well as inhibition of MMP-2 activity.

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Sal B depressed CK-18 expression induced by TGF-β1 in HK-2 cells. HK-2 cells were cultured in complete medium containing 5% FBS for 18 h. Thereafter, the cells were kept in serum-free medium or treated with (1) 2.5 ng/ml TGF-β1, (2) 2.5 ng/ml TGF-β1 plus 1 μM of Sal B, (3) 2.5 ng/ml TGF-β1 plus 10 μM of Sal B, (4) 2.5 ng/ml TGF-β1 plus 10 μM of SB-431542 for 24 h. (a) Immunofluorescence staining (×200) showed decreased immunolabeling intensity of CK-18 after TGF-β1 treatment. This effect was depressed by 1 μM and 10 μM of Sal B as well as SB-431542 treatments. The blue-colored stain is nuclear counterstaining with Hoechst 33258. (B) Western blot analysis for CK-18. CK-18 expression was decreased when cells were exposed to TGF-β1, whereas 1 μM and 10 μM of Sal B and SB-431542 significantly attenuated the TGF-β1-induced up-regulation of α-SMA. (C) Graphic presentation of the relative expression of CK-18. The values are represented as 100% vs. control. **P < 0.01 vs. control; #P < 0.05 vs. TGF-β1, # #P < 0.01 vs. TGF-β1.
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Figure 8: Sal B depressed CK-18 expression induced by TGF-β1 in HK-2 cells. HK-2 cells were cultured in complete medium containing 5% FBS for 18 h. Thereafter, the cells were kept in serum-free medium or treated with (1) 2.5 ng/ml TGF-β1, (2) 2.5 ng/ml TGF-β1 plus 1 μM of Sal B, (3) 2.5 ng/ml TGF-β1 plus 10 μM of Sal B, (4) 2.5 ng/ml TGF-β1 plus 10 μM of SB-431542 for 24 h. (a) Immunofluorescence staining (×200) showed decreased immunolabeling intensity of CK-18 after TGF-β1 treatment. This effect was depressed by 1 μM and 10 μM of Sal B as well as SB-431542 treatments. The blue-colored stain is nuclear counterstaining with Hoechst 33258. (B) Western blot analysis for CK-18. CK-18 expression was decreased when cells were exposed to TGF-β1, whereas 1 μM and 10 μM of Sal B and SB-431542 significantly attenuated the TGF-β1-induced up-regulation of α-SMA. (C) Graphic presentation of the relative expression of CK-18. The values are represented as 100% vs. control. **P < 0.01 vs. control; #P < 0.05 vs. TGF-β1, # #P < 0.01 vs. TGF-β1.

Mentions: Consistent with the alteration in E-cadherin mRNA expression, both immunofluorescence and Western blot analyses showed that CK-18, an epithelial cell marker, was significantly down-regulated after TGF-β1 treatment and was then reversed by 1 μM and 10 μM of Sal B treatments (Figure 8).


Salvianolic acid B prevents epithelial-to-mesenchymal transition through the TGF-beta1 signal transduction pathway in vivo and in vitro.

Wang QL, Tao YY, Yuan JL, Shen L, Liu CH - BMC Cell Biol. (2010)

Sal B depressed CK-18 expression induced by TGF-β1 in HK-2 cells. HK-2 cells were cultured in complete medium containing 5% FBS for 18 h. Thereafter, the cells were kept in serum-free medium or treated with (1) 2.5 ng/ml TGF-β1, (2) 2.5 ng/ml TGF-β1 plus 1 μM of Sal B, (3) 2.5 ng/ml TGF-β1 plus 10 μM of Sal B, (4) 2.5 ng/ml TGF-β1 plus 10 μM of SB-431542 for 24 h. (a) Immunofluorescence staining (×200) showed decreased immunolabeling intensity of CK-18 after TGF-β1 treatment. This effect was depressed by 1 μM and 10 μM of Sal B as well as SB-431542 treatments. The blue-colored stain is nuclear counterstaining with Hoechst 33258. (B) Western blot analysis for CK-18. CK-18 expression was decreased when cells were exposed to TGF-β1, whereas 1 μM and 10 μM of Sal B and SB-431542 significantly attenuated the TGF-β1-induced up-regulation of α-SMA. (C) Graphic presentation of the relative expression of CK-18. The values are represented as 100% vs. control. **P < 0.01 vs. control; #P < 0.05 vs. TGF-β1, # #P < 0.01 vs. TGF-β1.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 8: Sal B depressed CK-18 expression induced by TGF-β1 in HK-2 cells. HK-2 cells were cultured in complete medium containing 5% FBS for 18 h. Thereafter, the cells were kept in serum-free medium or treated with (1) 2.5 ng/ml TGF-β1, (2) 2.5 ng/ml TGF-β1 plus 1 μM of Sal B, (3) 2.5 ng/ml TGF-β1 plus 10 μM of Sal B, (4) 2.5 ng/ml TGF-β1 plus 10 μM of SB-431542 for 24 h. (a) Immunofluorescence staining (×200) showed decreased immunolabeling intensity of CK-18 after TGF-β1 treatment. This effect was depressed by 1 μM and 10 μM of Sal B as well as SB-431542 treatments. The blue-colored stain is nuclear counterstaining with Hoechst 33258. (B) Western blot analysis for CK-18. CK-18 expression was decreased when cells were exposed to TGF-β1, whereas 1 μM and 10 μM of Sal B and SB-431542 significantly attenuated the TGF-β1-induced up-regulation of α-SMA. (C) Graphic presentation of the relative expression of CK-18. The values are represented as 100% vs. control. **P < 0.01 vs. control; #P < 0.05 vs. TGF-β1, # #P < 0.01 vs. TGF-β1.
Mentions: Consistent with the alteration in E-cadherin mRNA expression, both immunofluorescence and Western blot analyses showed that CK-18, an epithelial cell marker, was significantly down-regulated after TGF-β1 treatment and was then reversed by 1 μM and 10 μM of Sal B treatments (Figure 8).

Bottom Line: Sal B also has potential protective effects on renal diseases.In contrast, Sal B and vitamin E significantly attenuated the expression of alpha-SMA, TGF-beta1, TbetaR-I, p-Smad2/3, and MMP-2 activity, but increased E-cadherin expression.Furthermore, Sal B and SB-431542 profoundly down-regulated the expressions of TbetaR-I and p-Smad2/3 but prevented the decreased expression of Smad7 in TGF-beta1 stimulated HK-2 cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Liver Diseases, Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, 528 Zhangheng Road, Shanghai, China.

ABSTRACT

Background: Salvianolic Acid B (Sal B) is a water-soluble component from Danshen (a traditional Chinese herb widely used for chronic renal diseases) with anti-oxidative and cell protective properties. Sal B also has potential protective effects on renal diseases. Tubular epithelial cells can undergo epithelial-to-mesenchymal transition (EMT), which plays an important role in the pathogenesis of renal interstitial fibrosis (RIF) and is mainly regulated by TGF-beta1/Smads pathway. The aims of the study are to investigate the effect of Sal B on tubular EMT in vivo and in vitro, and to elucidate its underlying mechanism against EMT related to TGF-beta1/Smads pathway.

Results: For in vivo experiments, RIF was induced in rats by oral administration of HgCl2 and prophylaxised with Sal B and vitamin E. The protein expression of E-cadherin was down-regulated, while the expression of alpha-SMA, TGF-beta1, TbetaR-I, p-Smad2/3 and the activity of matrix metalloproteinase-2 (MMP-2) were up-regulated in kidneys of model rats when compared with those of normal rats. In contrast, Sal B and vitamin E significantly attenuated the expression of alpha-SMA, TGF-beta1, TbetaR-I, p-Smad2/3, and MMP-2 activity, but increased E-cadherin expression. For in vitro experiments, HK-2 cells were incubated with TGF-beta1 to induce EMT, and the cells were co-cultured with 1 and 10 microM Sal B or SB-431542 (a specific inhibitor of TbetaR-I kinase). TGF-beta1 induced a typical EMT in HK-2 cells, while it was blocked by Sal B and SB-431542, as evidenced by blocking morphologic transformation, restoring E-cadherin and CK-18 expression, inhibiting alpha-SMA expression and F-actin reorganization, and down-regulating MMP-2/9 activities in TGF-beta1 mediated HK-2 cells. Furthermore, Sal B and SB-431542 profoundly down-regulated the expressions of TbetaR-I and p-Smad2/3 but prevented the decreased expression of Smad7 in TGF-beta1 stimulated HK-2 cells.

Conclusions: Sal B can prevent tubular EMT in the fibrotic kidney induced by HgCl2 as well as HK-2 cells triggered by TGF-beta1, the mechanism of Sal B is closely related to the regulation of TGF-beta1/Smads pathway, manifested as the inhibition of TGF-beta1 expression, suppression of TbetaR-I expression and function, down-regulation of Smad2/3 phosphorylation, and restoration of the down-regulation of Smad7, as well as inhibition of MMP-2 activity.

Show MeSH
Related in: MedlinePlus