Salvianolic acid B prevents epithelial-to-mesenchymal transition through the TGF-beta1 signal transduction pathway in vivo and in vitro - PubMed (original) (raw)
Salvianolic acid B prevents epithelial-to-mesenchymal transition through the TGF-beta1 signal transduction pathway in vivo and in vitro
Qing-Lan Wang et al. BMC Cell Biol. 2010.
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.
Figures
Figure 1
The chemical structure of Sal B.
Figure 2
Sal B ameliorated renal interstitial fibrosis induced by HgCl2 in model rat. (A) Masson's trichrome staining for assessing renal interstitial fibrosis. HgCl2 induced an increase of collagen in kidney section. Sal B and Vit E treatments reduced collagen deposition. (B) Kidney Hyp content determined by Jamall's method. Hyp content of kidneys was increased significantly in the model group as compared with the normal group. In contrast, Sal B and Vit E treatments significantly decreased kidney Hyp content.
Figure 3
The Effects of Sal B on α-SMA and E-Cadherin expression in kidneys of model rats. (A) Immunofluorescence staining of α-SMA and E-cadherin in kidneys. Increased labeling of α-SMA in the tubule was observed in model rats, while it was noted to be decreased by Sal B treatment. The E-cadherin labeling intensity was decreased in model rats, whereas the intensity was maintained in the Sal B treatment group (B) Western blot analyses for α-SMA and E-cadherin expression in renal tissues. Significantly increased α-SMA expression and decreased E-cadherin expression were observed in rats of the model group. Sal B treatment attenuated HgCl2-induced upregulation of α-SMA expression. Moreover, it significantly inhibited the HgCl2-induced decrease of E-cadherin expression. (C) Graphic presentation of the relative expression of α-SMA and E-cadherin. The values are represented as the density of α-SMA or E-cadherin vs GAPDH (%). **P < 0.01 vs normal; *P < 0.05 vs normal; #P < 0.05 vs Model; # #P < 0.01 vs Model.
Figure 4
The Effect of Sal B on TGF-β1, TβRI, Smad2, p-Smad2 and p-Smad3 expression in kidneys of model rats. Western blot analysis showed significantly increased TGF-β1, TβR-I, p-Smad2 and p-Smad3 expressions in rats of the model group. In contrast, Sal B treatment attenuated HgCl2-induced upregulation of TGF-β1, TβR-I, p-Smad2 and p-Smad3 expressions. The values are represented as the density of TGF-β1 or TβR-I vs. GAPDH (%), or p-Smad2 vs. Smad2 (%), or p-Smad3 vs. Smad3 (%). **P < 0.01 vs. normal; *P < 0.05 vs. normal; ## P < 0.01 vs. model; #P < 0.05 vs. model.
Figure 5
The effect of Sal B on the activity of MMP-2 in kidneys of model rats. The activity of MMP-2 was assessed by gelatin zymography. The results showed the activity of MMP-2 was increased in rats of model group when compared with that of normal group. Sal B significantly down-regulated the activity of MMP-2. **P < 0.01 vs. normal; # P < 0.05 vs. Model.
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.
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.
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.
Figure 9
Sal B blocked α-SMA 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) shows the increased labeling intensity of α-SMA observed after TGF-β1 treatment, while its increase becomes reversed after 1 μM and 10 μM Sal B and SB-431542 treatments. The blue-colored stain is nuclear counterstaining with Hoechst 33258. (B) Western blot analysis for α-SMA. α-SMA expression was increased when the cells were exposed to TGF-β1, whereas treatment with 1 μM and 10 μM Sal B and SB-431542 significantly attenuated the up-regulation of α-SMA by TGF-β1. (C) Graphic presentation of the relative expression of α-SMA. The values are represented as 100% vs. GAPDH. **_P <_0.01 vs. control; # #P < 0.01 vs. TGF-β1.
Figure 10
Sal B down-regulated MMP-2 and MMP-9 activation induced by TGF-β1 in HK-2 cells. 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 Sal B, (3) 2.5 ng/ml TGF-β1 plus 10 μM Sal B, (4) 2.5 ng/ml TGF-β1 plus 10 μM SB-431542 for 24 h. The activation of MMP-2 and MMP-9 derived from cells in the conditioned media were analyzed by gelatin zymographic analysis. The results show that TGF-β1 induced pro-MMP-2, the activation of pro-MMP-2, and MMP-9 secretion. 1 μM Sal B, 10 μM Sal B, and SB-431542 significantly decreased the secretion of these MMPs after being induced by TGF-β1. The values are as shown as folds vs. Control. **P < 0.01 vs. control; # #P < 0.01 vs. TGF-β1.
Figure 11
Sal B attenuated TGF-β1-induced TβR-I and Smad2/3 phosphorylation and up-regulated Smad7 expression in HK-2 cells. 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. The expression of TβR-I, Smad2, Smad3, p-Smad2/3 and Smad7 was detected by Western Blot. There was no significant change between each group in Smad2/3, but the expression of phosphorylated Smad2/3 and TβR-I was significantly increased. Smad7 expression was decreased after incubation with TGF-β1. Treatment with 1 μM and 10 μM Sal B and SB-431542 significantly attenuated the TGF-β1-induced up-regulation of p-Smad2/3 and TβR-I and increased Smad7 expression. The values are represented as the density of p-Smad2/3, TβR-I or Smad7 vs. GAPDH (%). **P < 0.01 vs. control; #P < 0.05 vs. TGF-β1, # #P < 0.01 vs. TGF-β1.
References
- Li MX, Liu BC. Epithelial to mesenchymal transition in the progression of tubulointerstitial fibrosis. Chin Med J (Engl) 2007;120(21):1925–1930. - PubMed
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