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 inhibition of keratinocyte migration. (a) Mouse primary keratinocytes with LiCl-induced activation of β-catenin were used in the scratch assay. LiCl inhibited keratinocyte migration compared to untreated cells. Inhibition was prominent at 48 h and was sustained through 72 h (**P < 0.01). EGF (positive control) stimulated migration was significant after 72 h, as evidenced by the nearly closed wound. AS-IV attenuated the LiCl-induced keratinocyte migration inhibition effect, as evidenced by the difference in wound closure after 72 h (*P < 0.05, compared with LiCl-treated group). Straight lines demarcate the initial wound area, and dotted lines indicate the migrating front of cells. (b) β-catenin was upregulated at the scratch wound edge. Insets show enlarged images of the dotted rectangle. The white circle indicates the keratinocytes starting to migrate from the nonnuclear β-catenin-positive cell. Scale bar = 100 μm. (c) Histograms showing the average coverage of scratch wounds widths as percentages of the baseline wound width at time of scratch (0) and 24 h, 48 h, and 72 h after LiCl, LiCl + AS-IV, and LiCl + EGF treatments. *P < 0.05, **P < 0.01. Scale bar = 100 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3368212&req=5

fig7: AS-IV attenuates LiCl-induced inhibition of keratinocyte migration. (a) Mouse primary keratinocytes with LiCl-induced activation of β-catenin were used in the scratch assay. LiCl inhibited keratinocyte migration compared to untreated cells. Inhibition was prominent at 48 h and was sustained through 72 h (**P < 0.01). EGF (positive control) stimulated migration was significant after 72 h, as evidenced by the nearly closed wound. AS-IV attenuated the LiCl-induced keratinocyte migration inhibition effect, as evidenced by the difference in wound closure after 72 h (*P < 0.05, compared with LiCl-treated group). Straight lines demarcate the initial wound area, and dotted lines indicate the migrating front of cells. (b) β-catenin was upregulated at the scratch wound edge. Insets show enlarged images of the dotted rectangle. The white circle indicates the keratinocytes starting to migrate from the nonnuclear β-catenin-positive cell. Scale bar = 100 μm. (c) Histograms showing the average coverage of scratch wounds widths as percentages of the baseline wound width at time of scratch (0) and 24 h, 48 h, and 72 h after LiCl, LiCl + AS-IV, and LiCl + EGF treatments. *P < 0.05, **P < 0.01. Scale bar = 100 μm.

Mentions: To test if LiCl-induced overexpression of β-catenin can affect keratinocyte migration during wound healing, we used the in vitro wound scratch assay. Keratinocytes were incubated with LiCl, LiCl and EGF (positive control), and/or AS-IV for 72 h. Keratinocyte migration into the scratch area was observed and the distance was quantified. As shown in Figure 7, LiCl treatment inhibited keratinocyte migration by almost 80%, whereas LiCl and AS-IV promoted migration by 60%, presumably via downregulation of activated β-catenin.


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 inhibition of keratinocyte migration. (a) Mouse primary keratinocytes with LiCl-induced activation of β-catenin were used in the scratch assay. LiCl inhibited keratinocyte migration compared to untreated cells. Inhibition was prominent at 48 h and was sustained through 72 h (**P < 0.01). EGF (positive control) stimulated migration was significant after 72 h, as evidenced by the nearly closed wound. AS-IV attenuated the LiCl-induced keratinocyte migration inhibition effect, as evidenced by the difference in wound closure after 72 h (*P < 0.05, compared with LiCl-treated group). Straight lines demarcate the initial wound area, and dotted lines indicate the migrating front of cells. (b) β-catenin was upregulated at the scratch wound edge. Insets show enlarged images of the dotted rectangle. The white circle indicates the keratinocytes starting to migrate from the nonnuclear β-catenin-positive cell. Scale bar = 100 μm. (c) Histograms showing the average coverage of scratch wounds widths as percentages of the baseline wound width at time of scratch (0) and 24 h, 48 h, and 72 h after LiCl, LiCl + AS-IV, and LiCl + EGF treatments. *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

fig7: AS-IV attenuates LiCl-induced inhibition of keratinocyte migration. (a) Mouse primary keratinocytes with LiCl-induced activation of β-catenin were used in the scratch assay. LiCl inhibited keratinocyte migration compared to untreated cells. Inhibition was prominent at 48 h and was sustained through 72 h (**P < 0.01). EGF (positive control) stimulated migration was significant after 72 h, as evidenced by the nearly closed wound. AS-IV attenuated the LiCl-induced keratinocyte migration inhibition effect, as evidenced by the difference in wound closure after 72 h (*P < 0.05, compared with LiCl-treated group). Straight lines demarcate the initial wound area, and dotted lines indicate the migrating front of cells. (b) β-catenin was upregulated at the scratch wound edge. Insets show enlarged images of the dotted rectangle. The white circle indicates the keratinocytes starting to migrate from the nonnuclear β-catenin-positive cell. Scale bar = 100 μm. (c) Histograms showing the average coverage of scratch wounds widths as percentages of the baseline wound width at time of scratch (0) and 24 h, 48 h, and 72 h after LiCl, LiCl + AS-IV, and LiCl + EGF treatments. *P < 0.05, **P < 0.01. Scale bar = 100 μm.
Mentions: To test if LiCl-induced overexpression of β-catenin can affect keratinocyte migration during wound healing, we used the in vitro wound scratch assay. Keratinocytes were incubated with LiCl, LiCl and EGF (positive control), and/or AS-IV for 72 h. Keratinocyte migration into the scratch area was observed and the distance was quantified. As shown in Figure 7, LiCl treatment inhibited keratinocyte migration by almost 80%, whereas LiCl and AS-IV promoted migration by 60%, presumably via downregulation of activated β-catenin.

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