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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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The effects of Sal B on E-cadherin mRNA expression, cell morphologic transformation and F-actin reorganization induced by TGF-β1 in HK-2 cells. (A) The effects of Sal B on E-cadherin mRNA expression. Real-time PCR analysis showed that TGF-β1 significantly suppressed E-cadherin mRNA expression in HK-2 cells, while 1 μM and 10 μM of Sal B prevented the loss of its expression (B) The effects of Sal B on cell morphology transformation and F-actin reorganization. HK-2 cells were incubated with 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. HK-2 cells changed from a cuboidal to a spindle shape in response to TGF-β1, whereas treatment with 1 μM and 10 μM Sal B and SB-431542 blocked this morphologic transformation (×200). Representative micrographs of FITC-conjugated phalloidin staining showed significanat F-actin reorganization and the formation of abundant long stress fibers in HK-2 cells induced by TGF-β1 were formed. 1 μM and 10 μM of Sal B and SB-431542 significantly blocked these processes.
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Figure 7: The effects of Sal B on E-cadherin mRNA expression, cell morphologic transformation and F-actin reorganization induced by TGF-β1 in HK-2 cells. (A) The effects of Sal B on E-cadherin mRNA expression. Real-time PCR analysis showed that TGF-β1 significantly suppressed E-cadherin mRNA expression in HK-2 cells, while 1 μM and 10 μM of Sal B prevented the loss of its expression (B) The effects of Sal B on cell morphology transformation and F-actin reorganization. HK-2 cells were incubated with 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. HK-2 cells changed from a cuboidal to a spindle shape in response to TGF-β1, whereas treatment with 1 μM and 10 μM Sal B and SB-431542 blocked this morphologic transformation (×200). Representative micrographs of FITC-conjugated phalloidin staining showed significanat F-actin reorganization and the formation of abundant long stress fibers in HK-2 cells induced by TGF-β1 were formed. 1 μM and 10 μM of Sal B and SB-431542 significantly blocked these processes.

Mentions: E-cadherin is a tubular epithelial cell-cell adhesion receptor that is essential for the formation and maintenance of renal epithelial homeostasis and architecture [23,24]. We found that TGF-β1 significantly suppressed E-cadherin mRNA expression in HK-2 cells, while 1 and 10 μM of Sal B prevented the loss of its expression (Figure 7A).


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)

The effects of Sal B on E-cadherin mRNA expression, cell morphologic transformation and F-actin reorganization induced by TGF-β1 in HK-2 cells. (A) The effects of Sal B on E-cadherin mRNA expression. Real-time PCR analysis showed that TGF-β1 significantly suppressed E-cadherin mRNA expression in HK-2 cells, while 1 μM and 10 μM of Sal B prevented the loss of its expression (B) The effects of Sal B on cell morphology transformation and F-actin reorganization. HK-2 cells were incubated with 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. HK-2 cells changed from a cuboidal to a spindle shape in response to TGF-β1, whereas treatment with 1 μM and 10 μM Sal B and SB-431542 blocked this morphologic transformation (×200). Representative micrographs of FITC-conjugated phalloidin staining showed significanat F-actin reorganization and the formation of abundant long stress fibers in HK-2 cells induced by TGF-β1 were formed. 1 μM and 10 μM of Sal B and SB-431542 significantly blocked these processes.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2874764&req=5

Figure 7: The effects of Sal B on E-cadherin mRNA expression, cell morphologic transformation and F-actin reorganization induced by TGF-β1 in HK-2 cells. (A) The effects of Sal B on E-cadherin mRNA expression. Real-time PCR analysis showed that TGF-β1 significantly suppressed E-cadherin mRNA expression in HK-2 cells, while 1 μM and 10 μM of Sal B prevented the loss of its expression (B) The effects of Sal B on cell morphology transformation and F-actin reorganization. HK-2 cells were incubated with 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. HK-2 cells changed from a cuboidal to a spindle shape in response to TGF-β1, whereas treatment with 1 μM and 10 μM Sal B and SB-431542 blocked this morphologic transformation (×200). Representative micrographs of FITC-conjugated phalloidin staining showed significanat F-actin reorganization and the formation of abundant long stress fibers in HK-2 cells induced by TGF-β1 were formed. 1 μM and 10 μM of Sal B and SB-431542 significantly blocked these processes.
Mentions: E-cadherin is a tubular epithelial cell-cell adhesion receptor that is essential for the formation and maintenance of renal epithelial homeostasis and architecture [23,24]. We found that TGF-β1 significantly suppressed E-cadherin mRNA expression in HK-2 cells, while 1 and 10 μM of Sal B prevented the loss of its expression (Figure 7A).

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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Related in: MedlinePlus