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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 affects keratinocyte morphology but not cell viability. Treatment with 20 mM and 40 mM LiCl caused cell size to enlarge (a, red arrow). (b) Average cell size from three experiments. (c) LiCl treatment had no effect on keratinocyte viability, as evidenced by the cell viability assay.
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fig4: LiCl affects keratinocyte morphology but not cell viability. Treatment with 20 mM and 40 mM LiCl caused cell size to enlarge (a, red arrow). (b) Average cell size from three experiments. (c) LiCl treatment had no effect on keratinocyte viability, as evidenced by the cell viability assay.

Mentions: To investigate the normal function of the Wnt signaling pathway in keratinocytes, we used LiCl to activate the Wnt/β-catenin signaling pathway. The effect of LiCl on keratinocyte proliferation was examined by the MTS/PMS assay and measurement of BrdU incorporation into DNA during DNA synthesis. As shown in Figure 2(a), keratinocytes were treated with different concentrations (0~40.0 mM/L) of LiCl for 24, 48, and 72 h. Controls were subconfluent and synchronous keratinocytes were treated with DMSO solvent alone for the indicated times. Compared to the control group, LiCl-treated keratinocytes exhibited consistent growth retardation. The half maximal (50%) inhibitory concentration (IC50) values (Figure 2(a), dotted line) of growth achieved after LiCl-treatment for 24, 48, and 72 h were 25.85 ± 5.03, 16.16 ± 0.95, and 12.35 ± 1.38 mM, respectively. Lower concentrations of LiCl (<5 mM) did not inhibit keratinocyte growth. Higher concentrations of LiCl (>5 mM) produced significant and dose-dependent inhibition of keratinocyte proliferation (Figure 2(a), circle). The BrdU incorporation assay also revealed a concentration-dependent decrease in BrdU-positive cells in response to different dosages of LiCl. As shown in Figure 3(a), the amount of BrdU-positive cells following LiCl-treatment of 10 mM (57.91 ± 6.57%), 20 mM (23.48 ± 3.19%), and 40 mM (9.67 ± 2.45%) was significantly less than those in the DMSO-treatment control group (71.81 ± 6.79%; all P < 0.01). Cell viability was unaffected by LiCl-treatment at all doses (Figure 4(c)). These data unambiguously confirm that LiCl is able to exert potent growth inhibitory effects on keratinocytes in a concentration- and time-dependent manner. These results also indicate that LiCl-treatment inhibits keratinocyte proliferation without affecting their viability.


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 affects keratinocyte morphology but not cell viability. Treatment with 20 mM and 40 mM LiCl caused cell size to enlarge (a, red arrow). (b) Average cell size from three experiments. (c) LiCl treatment had no effect on keratinocyte viability, as evidenced by the cell viability assay.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3368212&req=5

fig4: LiCl affects keratinocyte morphology but not cell viability. Treatment with 20 mM and 40 mM LiCl caused cell size to enlarge (a, red arrow). (b) Average cell size from three experiments. (c) LiCl treatment had no effect on keratinocyte viability, as evidenced by the cell viability assay.
Mentions: To investigate the normal function of the Wnt signaling pathway in keratinocytes, we used LiCl to activate the Wnt/β-catenin signaling pathway. The effect of LiCl on keratinocyte proliferation was examined by the MTS/PMS assay and measurement of BrdU incorporation into DNA during DNA synthesis. As shown in Figure 2(a), keratinocytes were treated with different concentrations (0~40.0 mM/L) of LiCl for 24, 48, and 72 h. Controls were subconfluent and synchronous keratinocytes were treated with DMSO solvent alone for the indicated times. Compared to the control group, LiCl-treated keratinocytes exhibited consistent growth retardation. The half maximal (50%) inhibitory concentration (IC50) values (Figure 2(a), dotted line) of growth achieved after LiCl-treatment for 24, 48, and 72 h were 25.85 ± 5.03, 16.16 ± 0.95, and 12.35 ± 1.38 mM, respectively. Lower concentrations of LiCl (<5 mM) did not inhibit keratinocyte growth. Higher concentrations of LiCl (>5 mM) produced significant and dose-dependent inhibition of keratinocyte proliferation (Figure 2(a), circle). The BrdU incorporation assay also revealed a concentration-dependent decrease in BrdU-positive cells in response to different dosages of LiCl. As shown in Figure 3(a), the amount of BrdU-positive cells following LiCl-treatment of 10 mM (57.91 ± 6.57%), 20 mM (23.48 ± 3.19%), and 40 mM (9.67 ± 2.45%) was significantly less than those in the DMSO-treatment control group (71.81 ± 6.79%; all P < 0.01). Cell viability was unaffected by LiCl-treatment at all doses (Figure 4(c)). These data unambiguously confirm that LiCl is able to exert potent growth inhibitory effects on keratinocytes in a concentration- and time-dependent manner. These results also indicate that LiCl-treatment inhibits keratinocyte proliferation without affecting their viability.

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