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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

β-catenin is differentially regulated by LiCl and AS-IV. Stabilization of β-catenin in keratinocytes was followed by immunofluorescence staining with a β-catenin-specific antibody (a) and Western blot analysis (b). (a) DMSO (negative control) and EGF (positive control) treated keratinocytes exhibited normal β-catenin membrane expression. The LiCl-treated group showed stabilized β-catenin, which was downregulated following AS-IV treatment. (b) GAPDH protein expression was used as the protein loading control. (c) Western blot analysis revealed that PCNA expression, which is related to cell proliferation, was downregulated 50% by LiCl compared to the control cells, and upregulated to 80% by AS-IV. GAPDH expression was used as a protein loading control. Asterisks indicate statistically significant differences between control and LiCl-treated cells. The scale bar in the first panel of (a) represents 100 μm, and is applicable to both sections.
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fig6: β-catenin is differentially regulated by LiCl and AS-IV. Stabilization of β-catenin in keratinocytes was followed by immunofluorescence staining with a β-catenin-specific antibody (a) and Western blot analysis (b). (a) DMSO (negative control) and EGF (positive control) treated keratinocytes exhibited normal β-catenin membrane expression. The LiCl-treated group showed stabilized β-catenin, which was downregulated following AS-IV treatment. (b) GAPDH protein expression was used as the protein loading control. (c) Western blot analysis revealed that PCNA expression, which is related to cell proliferation, was downregulated 50% by LiCl compared to the control cells, and upregulated to 80% by AS-IV. GAPDH expression was used as a protein loading control. Asterisks indicate statistically significant differences between control and LiCl-treated cells. The scale bar in the first panel of (a) represents 100 μm, and is applicable to both sections.

Mentions: We investigated the effect of AS-IV on β-catenin expression in primary keratinocytes using two standard assays, namely immunofluorescence staining and Western blot analysis. As expected, keratinocytes incubated with EGF showed normal β-catenin membrane expression, but those treated with LiCl exhibited prominent nuclear β-catenin staining. LiCl-induced nuclear β-catenin was attenuated by AS-IV exposure, as evidenced by remarkably less β-catenin nuclear staining (Figure 6(a)). Similarly, Western blot analysis indicated that LiCl-treated keratinocytes expressed over 5-fold more β-catenin than the control cells. In addition, AS-IV exposure of LiCl-treated cells led to nearly 3-fold less β-catenin that that in the control group (Figure 6(b)) and additionally, PCNA, which indicating the cell proliferation, was downregulated 50% by LiCl compared with DMSO control, and upregulated to 80% by AS-IV (Figure 6(c)).


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)

β-catenin is differentially regulated by LiCl and AS-IV. Stabilization of β-catenin in keratinocytes was followed by immunofluorescence staining with a β-catenin-specific antibody (a) and Western blot analysis (b). (a) DMSO (negative control) and EGF (positive control) treated keratinocytes exhibited normal β-catenin membrane expression. The LiCl-treated group showed stabilized β-catenin, which was downregulated following AS-IV treatment. (b) GAPDH protein expression was used as the protein loading control. (c) Western blot analysis revealed that PCNA expression, which is related to cell proliferation, was downregulated 50% by LiCl compared to the control cells, and upregulated to 80% by AS-IV. GAPDH expression was used as a protein loading control. Asterisks indicate statistically significant differences between control and LiCl-treated cells. The scale bar in the first panel of (a) represents 100 μm, and is applicable to both sections.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig6: β-catenin is differentially regulated by LiCl and AS-IV. Stabilization of β-catenin in keratinocytes was followed by immunofluorescence staining with a β-catenin-specific antibody (a) and Western blot analysis (b). (a) DMSO (negative control) and EGF (positive control) treated keratinocytes exhibited normal β-catenin membrane expression. The LiCl-treated group showed stabilized β-catenin, which was downregulated following AS-IV treatment. (b) GAPDH protein expression was used as the protein loading control. (c) Western blot analysis revealed that PCNA expression, which is related to cell proliferation, was downregulated 50% by LiCl compared to the control cells, and upregulated to 80% by AS-IV. GAPDH expression was used as a protein loading control. Asterisks indicate statistically significant differences between control and LiCl-treated cells. The scale bar in the first panel of (a) represents 100 μm, and is applicable to both sections.
Mentions: We investigated the effect of AS-IV on β-catenin expression in primary keratinocytes using two standard assays, namely immunofluorescence staining and Western blot analysis. As expected, keratinocytes incubated with EGF showed normal β-catenin membrane expression, but those treated with LiCl exhibited prominent nuclear β-catenin staining. LiCl-induced nuclear β-catenin was attenuated by AS-IV exposure, as evidenced by remarkably less β-catenin nuclear staining (Figure 6(a)). Similarly, Western blot analysis indicated that LiCl-treated keratinocytes expressed over 5-fold more β-catenin than the control cells. In addition, AS-IV exposure of LiCl-treated cells led to nearly 3-fold less β-catenin that that in the control group (Figure 6(b)) and additionally, PCNA, which indicating the cell proliferation, was downregulated 50% by LiCl compared with DMSO control, and upregulated to 80% by AS-IV (Figure 6(c)).

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