Limits...
Astragaloside IV Downregulates β-Catenin in Rat Keratinocytes to Counter LiCl-Induced Inhibition of Proliferation and Migration.

Li FL, Li X, Wang YF, Xiao XL, Xu R, Chen J, Fan B, Xu WB, Geng L, Li B - Evid Based Complement Alternat Med (2012)

Bottom Line: The effects on cell proliferation were evaluated by the MTS/PMS colorimetric assay, effects on cell migration were determined by a wound-healing scratch experiment, effects on the cell cycle were analyzed by flow cytometry, and effects on protein expression were analyzed by immunoblotting and immunofluorescence.LiCl strongly inhibited cell proliferation and migration, up-regulated β-catenin expression, and down-regulated proliferating cell nuclear antigen (PCNA) expression.AS-IV treatment attenuat the inhibition of proliferation and migration, significantly reducing the enhanced β-catenin expression, and recovering PCNA and β-tubulin expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai 200437, China.

ABSTRACT
Re-epithelialization is a crucial step towards wound healing. The traditional Chinese medicine, Astragalus membranaceus (Fisch) Bge, has been used for hundreds of years for many kinds of ulcerated wounds. Recent research has identified the active compound in this drug as astragaloside IV (AS-IV), but the underlying molecular mechanisms of its therapeutic action on keratinocytes remain poorly understood. In this study, we used an in vitro model of ulcer-like wound processes, lithium chloride (LiCl)-induced cultured mouse keratinocytes, to investigate the effects of AS-IV treatment. The effects on cell proliferation were evaluated by the MTS/PMS colorimetric assay, effects on cell migration were determined by a wound-healing scratch experiment, effects on the cell cycle were analyzed by flow cytometry, and effects on protein expression were analyzed by immunoblotting and immunofluorescence. LiCl strongly inhibited cell proliferation and migration, up-regulated β-catenin expression, and down-regulated proliferating cell nuclear antigen (PCNA) expression. AS-IV treatment attenuat the inhibition of proliferation and migration, significantly reducing the enhanced β-catenin expression, and recovering PCNA and β-tubulin expression. Thus, AS-IV mediates mouse keratinocyte proliferation and migration via regulation of the Wnt signaling pathway. Down-regulating β-catenin to increase keratinocyte migration and proliferation is one mechanism by which AS-IV can promote ulcerated wound healing.

No MeSH data available.


Related in: MedlinePlus

AS-IV attenuates LiCl-induced β-tubulin downregulation. Keratinocytes were treated with LiCl (20 mM) and LiCl (20 mM) + AS-IV (80 μg/mL) for 24 h, and β-tubulin was determined by immunofluorescence (a, merged images of K14/β-tubulin/DAPI were shown) and immunoblotting ((c), GAPDH protein expression was used as the protein loading control). (b) Cell numbers of β-tubulin-positive keratinocytes per 100 total cells are presented. AS-IV significantly attenuated LiCl-induced β-tubulin downregulation. *P < 0.05, **P < 0.01. Scale bar = 100 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3368212&req=5

fig8: AS-IV attenuates LiCl-induced β-tubulin downregulation. Keratinocytes were treated with LiCl (20 mM) and LiCl (20 mM) + AS-IV (80 μg/mL) for 24 h, and β-tubulin was determined by immunofluorescence (a, merged images of K14/β-tubulin/DAPI were shown) and immunoblotting ((c), GAPDH protein expression was used as the protein loading control). (b) Cell numbers of β-tubulin-positive keratinocytes per 100 total cells are presented. AS-IV significantly attenuated LiCl-induced β-tubulin downregulation. *P < 0.05, **P < 0.01. Scale bar = 100 μm.

Mentions: Tubulin protein is a key mediator of cell migration [16]. To determine whether LiCl treatment affected β-tubulin expression, we incubated primary keratinocytes with LiCl alone or with AS-IV. Changes in β-tubulin expression levels were evaluated by immunofluoresence following staining of treated cells with a β-tubulin-specific antibody (Figure 8(a)), and by immunoblotting (Figure 8(c)). β-tubulin was expressed in most of the untreated primary keratinocytes, but LiCl-treatment reduced β-tubulin expression. As shown in Figure 8(b), β-tubulin was expressed in only 15% of LiCl-treated cells but was expressed in 27% of cells treated with LiCl and AS-IV. In the DMSO-treated control cultures, 80% of cells expressed β-tubulin. Together, our findings indicate that LiCl is able to cause downregulation of β-tubulin, which was resolved, at least in part, by treatment with AS-IV. Thus, overexpression of β-catenin during the wound healing process may impair healing by reducing β-tubulin expression, and subsequently inhibiting keratinocyte migration. Since AS-IV can attenuate LiCl-induced downregulation of β-tubulin, it may represent a promising therapeutic agent for improving migration and promoting wound healing.


Astragaloside IV Downregulates β-Catenin in Rat Keratinocytes to Counter LiCl-Induced Inhibition of Proliferation and Migration.

Li FL, Li X, Wang YF, Xiao XL, Xu R, Chen J, Fan B, Xu WB, Geng L, Li B - Evid Based Complement Alternat Med (2012)

AS-IV attenuates LiCl-induced β-tubulin downregulation. Keratinocytes were treated with LiCl (20 mM) and LiCl (20 mM) + AS-IV (80 μg/mL) for 24 h, and β-tubulin was determined by immunofluorescence (a, merged images of K14/β-tubulin/DAPI were shown) and immunoblotting ((c), GAPDH protein expression was used as the protein loading control). (b) Cell numbers of β-tubulin-positive keratinocytes per 100 total cells are presented. AS-IV significantly attenuated LiCl-induced β-tubulin downregulation. *P < 0.05, **P < 0.01. Scale bar = 100 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3368212&req=5

fig8: AS-IV attenuates LiCl-induced β-tubulin downregulation. Keratinocytes were treated with LiCl (20 mM) and LiCl (20 mM) + AS-IV (80 μg/mL) for 24 h, and β-tubulin was determined by immunofluorescence (a, merged images of K14/β-tubulin/DAPI were shown) and immunoblotting ((c), GAPDH protein expression was used as the protein loading control). (b) Cell numbers of β-tubulin-positive keratinocytes per 100 total cells are presented. AS-IV significantly attenuated LiCl-induced β-tubulin downregulation. *P < 0.05, **P < 0.01. Scale bar = 100 μm.
Mentions: Tubulin protein is a key mediator of cell migration [16]. To determine whether LiCl treatment affected β-tubulin expression, we incubated primary keratinocytes with LiCl alone or with AS-IV. Changes in β-tubulin expression levels were evaluated by immunofluoresence following staining of treated cells with a β-tubulin-specific antibody (Figure 8(a)), and by immunoblotting (Figure 8(c)). β-tubulin was expressed in most of the untreated primary keratinocytes, but LiCl-treatment reduced β-tubulin expression. As shown in Figure 8(b), β-tubulin was expressed in only 15% of LiCl-treated cells but was expressed in 27% of cells treated with LiCl and AS-IV. In the DMSO-treated control cultures, 80% of cells expressed β-tubulin. Together, our findings indicate that LiCl is able to cause downregulation of β-tubulin, which was resolved, at least in part, by treatment with AS-IV. Thus, overexpression of β-catenin during the wound healing process may impair healing by reducing β-tubulin expression, and subsequently inhibiting keratinocyte migration. Since AS-IV can attenuate LiCl-induced downregulation of β-tubulin, it may represent a promising therapeutic agent for improving migration and promoting wound healing.

Bottom Line: The effects on cell proliferation were evaluated by the MTS/PMS colorimetric assay, effects on cell migration were determined by a wound-healing scratch experiment, effects on the cell cycle were analyzed by flow cytometry, and effects on protein expression were analyzed by immunoblotting and immunofluorescence.LiCl strongly inhibited cell proliferation and migration, up-regulated β-catenin expression, and down-regulated proliferating cell nuclear antigen (PCNA) expression.AS-IV treatment attenuat the inhibition of proliferation and migration, significantly reducing the enhanced β-catenin expression, and recovering PCNA and β-tubulin expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai 200437, China.

ABSTRACT
Re-epithelialization is a crucial step towards wound healing. The traditional Chinese medicine, Astragalus membranaceus (Fisch) Bge, has been used for hundreds of years for many kinds of ulcerated wounds. Recent research has identified the active compound in this drug as astragaloside IV (AS-IV), but the underlying molecular mechanisms of its therapeutic action on keratinocytes remain poorly understood. In this study, we used an in vitro model of ulcer-like wound processes, lithium chloride (LiCl)-induced cultured mouse keratinocytes, to investigate the effects of AS-IV treatment. The effects on cell proliferation were evaluated by the MTS/PMS colorimetric assay, effects on cell migration were determined by a wound-healing scratch experiment, effects on the cell cycle were analyzed by flow cytometry, and effects on protein expression were analyzed by immunoblotting and immunofluorescence. LiCl strongly inhibited cell proliferation and migration, up-regulated β-catenin expression, and down-regulated proliferating cell nuclear antigen (PCNA) expression. AS-IV treatment attenuat the inhibition of proliferation and migration, significantly reducing the enhanced β-catenin expression, and recovering PCNA and β-tubulin expression. Thus, AS-IV mediates mouse keratinocyte proliferation and migration via regulation of the Wnt signaling pathway. Down-regulating β-catenin to increase keratinocyte migration and proliferation is one mechanism by which AS-IV can promote ulcerated wound healing.

No MeSH data available.


Related in: MedlinePlus