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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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Effects of Sal B on cell viability and toxicology in HK-2 cells. (A) Cell viability was determined by the alamarBlue assay. The results were expressed as a percentage in the reduction of alamarBlue. 1 μM and 10 μM of Sal B had no significant effect (P > 0.05) on cell viability during each incubation time period, whereas 100 μM of Sal B decreased the viability of HK-2 cells by 11% (P < 0.05) and 20% (P < 0.05), when compared with control cells after 12 h and 24 h of incubation, respectively. (B) Cell toxicology was determined by high content screening (HCS) assay. HK-2 cells were plated in a 96-well plate at a density of 4,000 cells/well. Cells were incubated with 1-100 μM of Sal B for 24 h and then stained with 1 × MPCT1 Fluor Dye. Cell images from the various fluorescent dye stains were taken by HCS (×200). There were no obvious changes in nuclear morphology, membrane permeability, or lysosomal mass/pH after incubation with 1 μM and 10 μM of Sal B when compared with control cells. However, 100 μM of Sal B presented as an obvious cytotoxicity to cells. As shown by the indicator dye, there are fewer nuclei and an increase in membrane permeability.
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Figure 6: Effects of Sal B on cell viability and toxicology in HK-2 cells. (A) Cell viability was determined by the alamarBlue assay. The results were expressed as a percentage in the reduction of alamarBlue. 1 μM and 10 μM of Sal B had no significant effect (P > 0.05) on cell viability during each incubation time period, whereas 100 μM of Sal B decreased the viability of HK-2 cells by 11% (P < 0.05) and 20% (P < 0.05), when compared with control cells after 12 h and 24 h of incubation, respectively. (B) Cell toxicology was determined by high content screening (HCS) assay. HK-2 cells were plated in a 96-well plate at a density of 4,000 cells/well. Cells were incubated with 1-100 μM of Sal B for 24 h and then stained with 1 × MPCT1 Fluor Dye. Cell images from the various fluorescent dye stains were taken by HCS (×200). There were no obvious changes in nuclear morphology, membrane permeability, or lysosomal mass/pH after incubation with 1 μM and 10 μM of Sal B when compared with control cells. However, 100 μM of Sal B presented as an obvious cytotoxicity to cells. As shown by the indicator dye, there are fewer nuclei and an increase in membrane permeability.

Mentions: Cell viability was determined by the alamarBlue assay. The results were expressed as the percentage of reduction in alamarBlue. 1 μM and 10 μM of Sal B had no significant effect (P > 0.05) on cell viability during each incubation period, whereas 100 μM of Sal B decreased the viability of HK-2 cells by 11% (P < 0.05) and 20% (P < 0.05) when compared to control cells after 12 h and 24 h of incubation, respectively (Figure 6A).


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)

Effects of Sal B on cell viability and toxicology in HK-2 cells. (A) Cell viability was determined by the alamarBlue assay. The results were expressed as a percentage in the reduction of alamarBlue. 1 μM and 10 μM of Sal B had no significant effect (P > 0.05) on cell viability during each incubation time period, whereas 100 μM of Sal B decreased the viability of HK-2 cells by 11% (P < 0.05) and 20% (P < 0.05), when compared with control cells after 12 h and 24 h of incubation, respectively. (B) Cell toxicology was determined by high content screening (HCS) assay. HK-2 cells were plated in a 96-well plate at a density of 4,000 cells/well. Cells were incubated with 1-100 μM of Sal B for 24 h and then stained with 1 × MPCT1 Fluor Dye. Cell images from the various fluorescent dye stains were taken by HCS (×200). There were no obvious changes in nuclear morphology, membrane permeability, or lysosomal mass/pH after incubation with 1 μM and 10 μM of Sal B when compared with control cells. However, 100 μM of Sal B presented as an obvious cytotoxicity to cells. As shown by the indicator dye, there are fewer nuclei and an increase in membrane permeability.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Effects of Sal B on cell viability and toxicology in HK-2 cells. (A) Cell viability was determined by the alamarBlue assay. The results were expressed as a percentage in the reduction of alamarBlue. 1 μM and 10 μM of Sal B had no significant effect (P > 0.05) on cell viability during each incubation time period, whereas 100 μM of Sal B decreased the viability of HK-2 cells by 11% (P < 0.05) and 20% (P < 0.05), when compared with control cells after 12 h and 24 h of incubation, respectively. (B) Cell toxicology was determined by high content screening (HCS) assay. HK-2 cells were plated in a 96-well plate at a density of 4,000 cells/well. Cells were incubated with 1-100 μM of Sal B for 24 h and then stained with 1 × MPCT1 Fluor Dye. Cell images from the various fluorescent dye stains were taken by HCS (×200). There were no obvious changes in nuclear morphology, membrane permeability, or lysosomal mass/pH after incubation with 1 μM and 10 μM of Sal B when compared with control cells. However, 100 μM of Sal B presented as an obvious cytotoxicity to cells. As shown by the indicator dye, there are fewer nuclei and an increase in membrane permeability.
Mentions: Cell viability was determined by the alamarBlue assay. The results were expressed as the percentage of reduction in alamarBlue. 1 μM and 10 μM of Sal B had no significant effect (P > 0.05) on cell viability during each incubation period, whereas 100 μM of Sal B decreased the viability of HK-2 cells by 11% (P < 0.05) and 20% (P < 0.05) when compared to control cells after 12 h and 24 h of incubation, respectively (Figure 6A).

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