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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

LiCl induces S phase arrest in keratinocytes. (a) Keratinocytes were exposed to various concentrations of LiCl for 24 h. (b) Keratinocytes were exposed to 20 mM LiCl for 24 h, 48 h, and 72 h. (c) Histogram showing the average amounts of cells in various cell cycle stages from three experiments following a 24 h exposure to different LiCl concentrations. (d) Histogram showing the average distribution of cells in various cell cycle stages from three experiments with LiCl ± AS-IV, and LiCl + EGF treatments.
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fig5: LiCl induces S phase arrest in keratinocytes. (a) Keratinocytes were exposed to various concentrations of LiCl for 24 h. (b) Keratinocytes were exposed to 20 mM LiCl for 24 h, 48 h, and 72 h. (c) Histogram showing the average amounts of cells in various cell cycle stages from three experiments following a 24 h exposure to different LiCl concentrations. (d) Histogram showing the average distribution of cells in various cell cycle stages from three experiments with LiCl ± AS-IV, and LiCl + EGF treatments.

Mentions: LiCl-induced inhibition of growth may result from cell cycle effects. Thus, the cell cycle distribution of LiCl-treated and control cells was determined by measuring the DNA content of Krishan-stained cells using fluorescence-activated cell sorting. As shown in Figure 5, LiCl-treated cells exhibited a higher proportion of cells in the S phase than control cells. Histograms were generated to visualize the percent of cell cycle distributions produced by different LiCl concentrations (Figure 5(b)) and different exposure times (Figure 5(c)). Interestingly, when LiCl-treated cells were exposed to AS-IV, there was remarkable attenuation of induced cell cycle arrest in the S phase. Specifically, the percentage of S phase cells in LiCl-treated keratinocytes cultures went from 48.81 ± 7.43 to 35.28 ± 2.14 upon AS-IV exposure (P < 0.05).


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)

LiCl induces S phase arrest in keratinocytes. (a) Keratinocytes were exposed to various concentrations of LiCl for 24 h. (b) Keratinocytes were exposed to 20 mM LiCl for 24 h, 48 h, and 72 h. (c) Histogram showing the average amounts of cells in various cell cycle stages from three experiments following a 24 h exposure to different LiCl concentrations. (d) Histogram showing the average distribution of cells in various cell cycle stages from three experiments with LiCl ± AS-IV, and LiCl + EGF treatments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig5: LiCl induces S phase arrest in keratinocytes. (a) Keratinocytes were exposed to various concentrations of LiCl for 24 h. (b) Keratinocytes were exposed to 20 mM LiCl for 24 h, 48 h, and 72 h. (c) Histogram showing the average amounts of cells in various cell cycle stages from three experiments following a 24 h exposure to different LiCl concentrations. (d) Histogram showing the average distribution of cells in various cell cycle stages from three experiments with LiCl ± AS-IV, and LiCl + EGF treatments.
Mentions: LiCl-induced inhibition of growth may result from cell cycle effects. Thus, the cell cycle distribution of LiCl-treated and control cells was determined by measuring the DNA content of Krishan-stained cells using fluorescence-activated cell sorting. As shown in Figure 5, LiCl-treated cells exhibited a higher proportion of cells in the S phase than control cells. Histograms were generated to visualize the percent of cell cycle distributions produced by different LiCl concentrations (Figure 5(b)) and different exposure times (Figure 5(c)). Interestingly, when LiCl-treated cells were exposed to AS-IV, there was remarkable attenuation of induced cell cycle arrest in the S phase. Specifically, the percentage of S phase cells in LiCl-treated keratinocytes cultures went from 48.81 ± 7.43 to 35.28 ± 2.14 upon AS-IV exposure (P < 0.05).

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