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TMF and glycitin act synergistically on keratinocytes and fibroblasts to promote wound healing and anti-scarring activity

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ABSTRACT

Keratinocyte-fibroblast interactions are critical for skin repair after injury. During the proliferative phase of wound healing, proliferation, migration and differentiation of these cells are the major mechanisms leading to tissue remodeling. We have previously reported that glycitin, a major soy isoflavone, stimulates dermal fibroblast proliferation; and the phytochemical, 4′,6,7-trimethoxyisoflavone (TMF), induces migration of HaCaT keratinocyte cells. We therefore investigated whether these compounds display synergistic effects on skin cells during wound healing in vitro and in vivo. Co-treatment with TMF and glycitin synergistically promotes the proliferation and migration of both keratinocytes and dermal fibroblasts, with a 1:1 ratio of these compounds showing the greatest efficacy in our co-culture system. This keratinocyte-fibroblast interaction occurred via the secretion of TGF-β, and the induction of differentiation and proliferation was confirmed in both indirect and direct co-culture assays. In an excisional and burn wound animal model, mice treated with a 1:1 ratio of TMF and glycitin showed faster wound closure, regeneration and scar reduction than even the positive control drug. These data indicate that two isoflavones, TMF and glycitin, act synergistically to promote wound healing and anti-scarring and could potentially be developed together as a bioactive therapeutic for wound treatment.

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Glycitin and TMF act synergistically to promote the proliferation and migration of HaCaT keratinocytes and human dermal fibroblasts in culture. (a) Proliferation of dermal fibroblasts treated with either glycitin and TMF, mixed in varying ratios or conditioned media from HaCaT keratinocytes treated with same conditions for 24 h, as measured by MTT assay. (b) Scratch wound healing assay with dermal fibroblasts treated as described in a for 24 h. Migration distance was measured using the ImageJ program. (c) Proliferation of HaCaT keratinocytes treated with either glycitin and TMF, mixed in varying ratios or conditioned media from dermal fibroblasts treated with the same conditions for 24 h, as measured by MTT assay. (d) Scratch wound healing assay with HaCaT keratinocytes treated as described in c for 24 h. Migration distance was measured using the ImageJ program. *P<0.05 as compared with control, **P<0.001 as compared with control, G: glycitin, T: TMF. TMF, 4′,6,7-trimethoxyisoflavone.
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fig2: Glycitin and TMF act synergistically to promote the proliferation and migration of HaCaT keratinocytes and human dermal fibroblasts in culture. (a) Proliferation of dermal fibroblasts treated with either glycitin and TMF, mixed in varying ratios or conditioned media from HaCaT keratinocytes treated with same conditions for 24 h, as measured by MTT assay. (b) Scratch wound healing assay with dermal fibroblasts treated as described in a for 24 h. Migration distance was measured using the ImageJ program. (c) Proliferation of HaCaT keratinocytes treated with either glycitin and TMF, mixed in varying ratios or conditioned media from dermal fibroblasts treated with the same conditions for 24 h, as measured by MTT assay. (d) Scratch wound healing assay with HaCaT keratinocytes treated as described in c for 24 h. Migration distance was measured using the ImageJ program. *P<0.05 as compared with control, **P<0.001 as compared with control, G: glycitin, T: TMF. TMF, 4′,6,7-trimethoxyisoflavone.

Mentions: To determine the effect of treatment with mixture of glycitin and TMF on keratinocytes and fibroblast cells, we mixed these compounds at various ratios: G:T=1:1, G:T=1:2 and G:T=2:1 (G: glycitin, T: TMF). We then measured the proliferation and migration of human dermal fibroblast cells that were either treated with the different combinations of the two compounds or incubated with conditioned media from HaCaT keratinocyte cells treated with the same stimuli. We observed increased proliferation of fibroblasts treated with G:T=1:1, 1:2 and 2:1but did not see the same effect in cells treated with other combinations of glycitin and TMF or the HaCaT cell-conditioned media (Figure 2a). Fibroblast migration was increased in all G:T treated groups, whereas treatment with conditioned media did not increase migration. We note that the migratory distance of fibroblasts treated with conditioned media actually decreased as compared with the untreated control group (Figure 2b). This suggests that treatment with glycitin and TMF induces HaCaT cell to produce factors that inhibit fibroblast migration.


TMF and glycitin act synergistically on keratinocytes and fibroblasts to promote wound healing and anti-scarring activity
Glycitin and TMF act synergistically to promote the proliferation and migration of HaCaT keratinocytes and human dermal fibroblasts in culture. (a) Proliferation of dermal fibroblasts treated with either glycitin and TMF, mixed in varying ratios or conditioned media from HaCaT keratinocytes treated with same conditions for 24 h, as measured by MTT assay. (b) Scratch wound healing assay with dermal fibroblasts treated as described in a for 24 h. Migration distance was measured using the ImageJ program. (c) Proliferation of HaCaT keratinocytes treated with either glycitin and TMF, mixed in varying ratios or conditioned media from dermal fibroblasts treated with the same conditions for 24 h, as measured by MTT assay. (d) Scratch wound healing assay with HaCaT keratinocytes treated as described in c for 24 h. Migration distance was measured using the ImageJ program. *P<0.05 as compared with control, **P<0.001 as compared with control, G: glycitin, T: TMF. TMF, 4′,6,7-trimethoxyisoflavone.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig2: Glycitin and TMF act synergistically to promote the proliferation and migration of HaCaT keratinocytes and human dermal fibroblasts in culture. (a) Proliferation of dermal fibroblasts treated with either glycitin and TMF, mixed in varying ratios or conditioned media from HaCaT keratinocytes treated with same conditions for 24 h, as measured by MTT assay. (b) Scratch wound healing assay with dermal fibroblasts treated as described in a for 24 h. Migration distance was measured using the ImageJ program. (c) Proliferation of HaCaT keratinocytes treated with either glycitin and TMF, mixed in varying ratios or conditioned media from dermal fibroblasts treated with the same conditions for 24 h, as measured by MTT assay. (d) Scratch wound healing assay with HaCaT keratinocytes treated as described in c for 24 h. Migration distance was measured using the ImageJ program. *P<0.05 as compared with control, **P<0.001 as compared with control, G: glycitin, T: TMF. TMF, 4′,6,7-trimethoxyisoflavone.
Mentions: To determine the effect of treatment with mixture of glycitin and TMF on keratinocytes and fibroblast cells, we mixed these compounds at various ratios: G:T=1:1, G:T=1:2 and G:T=2:1 (G: glycitin, T: TMF). We then measured the proliferation and migration of human dermal fibroblast cells that were either treated with the different combinations of the two compounds or incubated with conditioned media from HaCaT keratinocyte cells treated with the same stimuli. We observed increased proliferation of fibroblasts treated with G:T=1:1, 1:2 and 2:1but did not see the same effect in cells treated with other combinations of glycitin and TMF or the HaCaT cell-conditioned media (Figure 2a). Fibroblast migration was increased in all G:T treated groups, whereas treatment with conditioned media did not increase migration. We note that the migratory distance of fibroblasts treated with conditioned media actually decreased as compared with the untreated control group (Figure 2b). This suggests that treatment with glycitin and TMF induces HaCaT cell to produce factors that inhibit fibroblast migration.

View Article: PubMed Central - PubMed

ABSTRACT

Keratinocyte-fibroblast interactions are critical for skin repair after injury. During the proliferative phase of wound healing, proliferation, migration and differentiation of these cells are the major mechanisms leading to tissue remodeling. We have previously reported that glycitin, a major soy isoflavone, stimulates dermal fibroblast proliferation; and the phytochemical, 4&prime;,6,7-trimethoxyisoflavone (TMF), induces migration of HaCaT keratinocyte cells. We therefore investigated whether these compounds display synergistic effects on skin cells during wound healing in vitro and in vivo. Co-treatment with TMF and glycitin synergistically promotes the proliferation and migration of both keratinocytes and dermal fibroblasts, with a 1:1 ratio of these compounds showing the greatest efficacy in our co-culture system. This keratinocyte-fibroblast interaction occurred via the secretion of TGF-&beta;, and the induction of differentiation and proliferation was confirmed in both indirect and direct co-culture assays. In an excisional and burn wound animal model, mice treated with a 1:1 ratio of TMF and glycitin showed faster wound closure, regeneration and scar reduction than even the positive control drug. These data indicate that two isoflavones, TMF and glycitin, act synergistically to promote wound healing and anti-scarring and could potentially be developed together as a bioactive therapeutic for wound treatment.

No MeSH data available.


Related in: MedlinePlus