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Astragalus and Paeoniae Radix Rubra extract (APE) inhibits hepatic stellate cell activation by modulating transforming growth factor-β/Smad pathway.

Huang W, Li L, Tian X, Yan J, Yang X, Wang X, Liao G, Qiu G - Mol Med Rep (2014)

Bottom Line: The results demonstrated that APE (5‑80 µg/ml) significantly inhibited fetal bovine serum‑induced cell proliferation in a dose‑dependent manner.The results further indicated that APE treatment not only reduced PAI‑1 expression, but also increased uPA expression in a dose‑dependent manner.In conclusion, APE exerted inhibitory effects on cell proliferation, invasion and activation of HSCs, and the mechanisms underlying these effects may involve the TGF‑β1/Smad pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Scientific Research, Xi'an Medical College, Xi'an, Shaanxi 710061, P.R. China.

ABSTRACT
Previous studies have shown that Astragalus and Paeoniae Radix Rubra extract (APE) is capable of protecting against liver fibrosis in rats. The hypothesis of the present study was that APE exerts its anti‑fibrotic effect by mediating the transforming growth factor β (TGF‑β)/Smad signaling pathway. In order to investigate this hypothesis, a series of assays were designed to detect the effects of APE on cell proliferation, cell invasion and the activation of hepatic stellate cells (HSCs). In addition, the effects of APE on the TGF‑β/Smad signaling pathway were explored, with the aim of elucidating the underlying mechanisms. HSCs were initially isolated from normal rat liver. A number of assays were then employed in order to evaluate the effects of APE on the function of these cells. Cell proliferation was investigated using an MTT assay and cell invasion was observed with the use of transwell invasion chambers. Collagen synthesis was measured with a 3H‑proline incorporation assay and expression of α‑smooth muscle actin was used to determine the extent of HSC activation. Protein expression induced by TGF‑β1 in HSCs was investigated by western blot and immunofluorescence analyses. Plasminogen activator inhibitor type1 (PAI‑1) and urokinase‑type plasminogen activator (uPA) transcriptional activity was measured using reverse transcription polymerase chain reaction. The results demonstrated that APE (5‑80 µg/ml) significantly inhibited fetal bovine serum‑induced cell proliferation in a dose‑dependent manner. Cell invasion and activation of HSCs induced by TGF‑β1 were disrupted by treatment with APE in a dose‑dependent manner. TGF‑β1 was observed to increase the phosphorylation of Smad2/3, while APE administered at higher doses produced inhibitory effects on Smad2/3 phosphorylation. In addition, administration of APE abrogated the TGF‑β1‑induced reduction in Smad‑7 expression in a dose‑dependent manner. The results further indicated that APE treatment not only reduced PAI‑1 expression, but also increased uPA expression in a dose‑dependent manner. In conclusion, APE exerted inhibitory effects on cell proliferation, invasion and activation of HSCs, and the mechanisms underlying these effects may involve the TGF‑β1/Smad pathway.

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Effects of APE on TGF-β1-induced invasion and activation of HSCs. (A and B) Number of cells resulting from stimulation of HSCs by TGF-β1 treatment exhibited a significant, dose-dependent suppression when treated with APE (10, 20, 40 or 80 μg/ml) (magnification, ×100). (C) TGF-β1-stimulated HSC collagen synthesis was significantly and dose-dependently decreased following treatment with APE (10, 20, 40 or 80 μg/ml). (D) Expression of α-SAM was significantly decreased following treatment with APE (10, 20, 40 or 80 μg/ml) compared with that in the control. ##P<0.05 compared with control group and **P<0.05 compared with group treated with TGF-β1 alone. APE, Astragalus and Paeoniae Radix Rubra extract; TGF-β1, transforming growth factor-β1; α-SAM, α-sterile alpha motif; HSC, hepatic stellate cells.
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f1-mmr-11-04-2569: Effects of APE on TGF-β1-induced invasion and activation of HSCs. (A and B) Number of cells resulting from stimulation of HSCs by TGF-β1 treatment exhibited a significant, dose-dependent suppression when treated with APE (10, 20, 40 or 80 μg/ml) (magnification, ×100). (C) TGF-β1-stimulated HSC collagen synthesis was significantly and dose-dependently decreased following treatment with APE (10, 20, 40 or 80 μg/ml). (D) Expression of α-SAM was significantly decreased following treatment with APE (10, 20, 40 or 80 μg/ml) compared with that in the control. ##P<0.05 compared with control group and **P<0.05 compared with group treated with TGF-β1 alone. APE, Astragalus and Paeoniae Radix Rubra extract; TGF-β1, transforming growth factor-β1; α-SAM, α-sterile alpha motif; HSC, hepatic stellate cells.

Mentions: The invasion capability of HSCs was investigated using a Transwell invasion assay. As shown in Fig. 1A, HSCs moved to the lower compartment of the chambers across the Matrigel-coated polycarbonate membrane when stimulated by treatment with TGF-β1. Treatment with APE (10, 20, 40, 80 μg/ml) significantly reduced the number of TGF-β1-stimulated cells that invaded across the polycarbonate membrane. In accordance with previous results, this effect occurred in a dose-dependent manner (23). Thus, TGF-β1-induced HSC invasion into the bottom chamber and across the collagen IV/collagen I-coated polycarbonate membrane was inhibited by APE (Fig. 1A and B).


Astragalus and Paeoniae Radix Rubra extract (APE) inhibits hepatic stellate cell activation by modulating transforming growth factor-β/Smad pathway.

Huang W, Li L, Tian X, Yan J, Yang X, Wang X, Liao G, Qiu G - Mol Med Rep (2014)

Effects of APE on TGF-β1-induced invasion and activation of HSCs. (A and B) Number of cells resulting from stimulation of HSCs by TGF-β1 treatment exhibited a significant, dose-dependent suppression when treated with APE (10, 20, 40 or 80 μg/ml) (magnification, ×100). (C) TGF-β1-stimulated HSC collagen synthesis was significantly and dose-dependently decreased following treatment with APE (10, 20, 40 or 80 μg/ml). (D) Expression of α-SAM was significantly decreased following treatment with APE (10, 20, 40 or 80 μg/ml) compared with that in the control. ##P<0.05 compared with control group and **P<0.05 compared with group treated with TGF-β1 alone. APE, Astragalus and Paeoniae Radix Rubra extract; TGF-β1, transforming growth factor-β1; α-SAM, α-sterile alpha motif; HSC, hepatic stellate cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4337737&req=5

f1-mmr-11-04-2569: Effects of APE on TGF-β1-induced invasion and activation of HSCs. (A and B) Number of cells resulting from stimulation of HSCs by TGF-β1 treatment exhibited a significant, dose-dependent suppression when treated with APE (10, 20, 40 or 80 μg/ml) (magnification, ×100). (C) TGF-β1-stimulated HSC collagen synthesis was significantly and dose-dependently decreased following treatment with APE (10, 20, 40 or 80 μg/ml). (D) Expression of α-SAM was significantly decreased following treatment with APE (10, 20, 40 or 80 μg/ml) compared with that in the control. ##P<0.05 compared with control group and **P<0.05 compared with group treated with TGF-β1 alone. APE, Astragalus and Paeoniae Radix Rubra extract; TGF-β1, transforming growth factor-β1; α-SAM, α-sterile alpha motif; HSC, hepatic stellate cells.
Mentions: The invasion capability of HSCs was investigated using a Transwell invasion assay. As shown in Fig. 1A, HSCs moved to the lower compartment of the chambers across the Matrigel-coated polycarbonate membrane when stimulated by treatment with TGF-β1. Treatment with APE (10, 20, 40, 80 μg/ml) significantly reduced the number of TGF-β1-stimulated cells that invaded across the polycarbonate membrane. In accordance with previous results, this effect occurred in a dose-dependent manner (23). Thus, TGF-β1-induced HSC invasion into the bottom chamber and across the collagen IV/collagen I-coated polycarbonate membrane was inhibited by APE (Fig. 1A and B).

Bottom Line: The results demonstrated that APE (5‑80 µg/ml) significantly inhibited fetal bovine serum‑induced cell proliferation in a dose‑dependent manner.The results further indicated that APE treatment not only reduced PAI‑1 expression, but also increased uPA expression in a dose‑dependent manner.In conclusion, APE exerted inhibitory effects on cell proliferation, invasion and activation of HSCs, and the mechanisms underlying these effects may involve the TGF‑β1/Smad pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Scientific Research, Xi'an Medical College, Xi'an, Shaanxi 710061, P.R. China.

ABSTRACT
Previous studies have shown that Astragalus and Paeoniae Radix Rubra extract (APE) is capable of protecting against liver fibrosis in rats. The hypothesis of the present study was that APE exerts its anti‑fibrotic effect by mediating the transforming growth factor β (TGF‑β)/Smad signaling pathway. In order to investigate this hypothesis, a series of assays were designed to detect the effects of APE on cell proliferation, cell invasion and the activation of hepatic stellate cells (HSCs). In addition, the effects of APE on the TGF‑β/Smad signaling pathway were explored, with the aim of elucidating the underlying mechanisms. HSCs were initially isolated from normal rat liver. A number of assays were then employed in order to evaluate the effects of APE on the function of these cells. Cell proliferation was investigated using an MTT assay and cell invasion was observed with the use of transwell invasion chambers. Collagen synthesis was measured with a 3H‑proline incorporation assay and expression of α‑smooth muscle actin was used to determine the extent of HSC activation. Protein expression induced by TGF‑β1 in HSCs was investigated by western blot and immunofluorescence analyses. Plasminogen activator inhibitor type1 (PAI‑1) and urokinase‑type plasminogen activator (uPA) transcriptional activity was measured using reverse transcription polymerase chain reaction. The results demonstrated that APE (5‑80 µg/ml) significantly inhibited fetal bovine serum‑induced cell proliferation in a dose‑dependent manner. Cell invasion and activation of HSCs induced by TGF‑β1 were disrupted by treatment with APE in a dose‑dependent manner. TGF‑β1 was observed to increase the phosphorylation of Smad2/3, while APE administered at higher doses produced inhibitory effects on Smad2/3 phosphorylation. In addition, administration of APE abrogated the TGF‑β1‑induced reduction in Smad‑7 expression in a dose‑dependent manner. The results further indicated that APE treatment not only reduced PAI‑1 expression, but also increased uPA expression in a dose‑dependent manner. In conclusion, APE exerted inhibitory effects on cell proliferation, invasion and activation of HSCs, and the mechanisms underlying these effects may involve the TGF‑β1/Smad pathway.

Show MeSH
Related in: MedlinePlus