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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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A 1:1 mixture of glycitin and TMF reduces scarring an in vivo burn wound model. (a) Burn wound closure after treatment for 10 days. (b) Wound sites were treated every day for 8 weeks; hair was then removed, and the scars were photographed using a digital camera or (c) the skin testing machine. (d) Scar size was measured 8 weeks after wounding using the Image J program. (e–h) Skin tissues were isolated, fixed and stained with both hematoxylin/eosin and Masson's trichrome. Epidermal thickness and collagen content were measured using the Image J program. (i) Western blot analysis of skin tissue isolated from wound sites 4 weeks and (j) 8 weeks after wounding. *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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fig6: A 1:1 mixture of glycitin and TMF reduces scarring an in vivo burn wound model. (a) Burn wound closure after treatment for 10 days. (b) Wound sites were treated every day for 8 weeks; hair was then removed, and the scars were photographed using a digital camera or (c) the skin testing machine. (d) Scar size was measured 8 weeks after wounding using the Image J program. (e–h) Skin tissues were isolated, fixed and stained with both hematoxylin/eosin and Masson's trichrome. Epidermal thickness and collagen content were measured using the Image J program. (i) Western blot analysis of skin tissue isolated from wound sites 4 weeks and (j) 8 weeks after wounding. *P<0.05 as compared with control, **P<0.001 as compared with control, G: glycitin, T: TMF. TMF, 4′,6,7-trimethoxyisoflavone.

Mentions: We next assessed the effectiveness of glycitin/TMF treatment to prevent scarring using a murine burn wound model. Burns were made by searing the dorsal skin of C57BL/6 mice with a hot iron. These wounds were then treated using the same conditions as described for the excision wound model, except in this case, burns were treated for 8 weeks. As predicted, we observed that burns treated with the glycitin/TMF mixture showed faster healing than the other groups (Figure 6a). After 8 weeks, we checked hair growth in the scar area, since this can indicate remodeling of skin, and found that the glycitin/TMF-treated group showed the most hair growth (Figure 6b). We then removed the hair over the burn site and measured the size of the scars; we found that scars treated with glycitin/TMF were significantly reduced, as compared with those treated with the DMSO control (Figure 6c and d). Specifically, whereas the scars from the positive controls and DMSO-treated groups showed thickened epidermis and excessive collagen deposition, both epidermal thickness and collagen deposition were reduced in the TMF and glycitin/TMF co-treated groups (Figure 6e and h). The glycitin/TMF co-treated group, in particular, showed improved regeneration of appendages and a fully formed panniculus (Figure 6e and f). Because glycitin has no effect on re-epithelialization and contributes to the synthesis of collagen, we excluded the group treated with glycitin only in the burn wound experiment.


TMF and glycitin act synergistically on keratinocytes and fibroblasts to promote wound healing and anti-scarring activity
A 1:1 mixture of glycitin and TMF reduces scarring an in vivo burn wound model. (a) Burn wound closure after treatment for 10 days. (b) Wound sites were treated every day for 8 weeks; hair was then removed, and the scars were photographed using a digital camera or (c) the skin testing machine. (d) Scar size was measured 8 weeks after wounding using the Image J program. (e–h) Skin tissues were isolated, fixed and stained with both hematoxylin/eosin and Masson's trichrome. Epidermal thickness and collagen content were measured using the Image J program. (i) Western blot analysis of skin tissue isolated from wound sites 4 weeks and (j) 8 weeks after wounding. *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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Show All Figures
getmorefigures.php?uid=PMC5382558&req=5

fig6: A 1:1 mixture of glycitin and TMF reduces scarring an in vivo burn wound model. (a) Burn wound closure after treatment for 10 days. (b) Wound sites were treated every day for 8 weeks; hair was then removed, and the scars were photographed using a digital camera or (c) the skin testing machine. (d) Scar size was measured 8 weeks after wounding using the Image J program. (e–h) Skin tissues were isolated, fixed and stained with both hematoxylin/eosin and Masson's trichrome. Epidermal thickness and collagen content were measured using the Image J program. (i) Western blot analysis of skin tissue isolated from wound sites 4 weeks and (j) 8 weeks after wounding. *P<0.05 as compared with control, **P<0.001 as compared with control, G: glycitin, T: TMF. TMF, 4′,6,7-trimethoxyisoflavone.
Mentions: We next assessed the effectiveness of glycitin/TMF treatment to prevent scarring using a murine burn wound model. Burns were made by searing the dorsal skin of C57BL/6 mice with a hot iron. These wounds were then treated using the same conditions as described for the excision wound model, except in this case, burns were treated for 8 weeks. As predicted, we observed that burns treated with the glycitin/TMF mixture showed faster healing than the other groups (Figure 6a). After 8 weeks, we checked hair growth in the scar area, since this can indicate remodeling of skin, and found that the glycitin/TMF-treated group showed the most hair growth (Figure 6b). We then removed the hair over the burn site and measured the size of the scars; we found that scars treated with glycitin/TMF were significantly reduced, as compared with those treated with the DMSO control (Figure 6c and d). Specifically, whereas the scars from the positive controls and DMSO-treated groups showed thickened epidermis and excessive collagen deposition, both epidermal thickness and collagen deposition were reduced in the TMF and glycitin/TMF co-treated groups (Figure 6e and h). The glycitin/TMF co-treated group, in particular, showed improved regeneration of appendages and a fully formed panniculus (Figure 6e and f). Because glycitin has no effect on re-epithelialization and contributes to the synthesis of collagen, we excluded the group treated with glycitin only in the burn wound experiment.

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