Leptin promotes wound healing in the skin

Abstract

Introduction: Leptin, a 16 kDa anti-obesity hormone, exhibits various physiological properties. Interestingly, skin wound healing was proven to delay in leptin-deficient ob/ob mice. However, little is known on the mechanisms of this phenomenon. In this study, we attempted to elucidate a role of leptin in wound healing of skin.

Methods: Immunohistochemical analysis was performed to confirm the expression of the leptin receptor (Ob-R) in human and mouse skin. Leptin was topically administered to chemical wounds created in mouse back skin along with sustained-release absorbable hydrogel. The process of wound repair was histologically observed and the area of ulceration was measured over time. The effect of leptin on the proliferation, differentiation and migration of human epidermal keratinocytes was investigated.

Results: Ob-R was expressed in epidermal cells of human and mouse skin. Topical administration of leptin significantly promoted wound healing. Histological analysis showed more blood vessels in the dermal connective tissues in the leptin-treated group. The proliferation, differentiation/function and migration of human epidermal keratinocytes were enhanced by exogenous leptin.

Conclusion: Topically administered leptin was proven to promote wound healing in the skin by accelerating proliferation, differentiation/function and migration of epidermal keratinocytes and enhancing angiogenesis around the wounded area. These results strongly suggest that topical administration of leptin may be useful as a treatment to promote wound healing in the skin.

Fig 1. Immunohistochemical localization of Ob-R in human and mouse skin.

(A) Immunohistochemical staining for Ob-R. Ob-R was expressed in epidermal cells of humen skin (arrows). DAB and hematoxylin staining. (B) Negative control. (C) Immunohistochemical staining for Ob-R. Epidermal cells of mouse skin, particularly prickle/gananular cells, were positive for Ob-R (arrows). DAB and hematoxylin staining. (D) Negative control. (E) Immunohistochemical staining for Ob-R in mouse skin. Ob-R was also expressed in hair follicles (arrow). DAB and hematoxylin staining. (F) Negative control. Scale bars = 20 μm.

Fig 2. Effect of leptin on wound healing in mouse skin.

(A) Chemical wounds created in mouse back skin by applying two pieces of filter paper (12x12mm each) soaked with 20% sodium hypochlorite for 5 minutes. (B and C) Skin wound healing at day 4 after wound creation. No significant difference in wound healing was noted between leptin-treated group and control group. Values are mean ± SE from 12 animals per group. (D and E) Skin wound healing at day 8 after wound creation. Significantly enhanced re-epithelialization of the wound was observed in leptin-treated group compared with control group. Values are mean ± SE from 12 animals per group. **P < 0.01. (F) Histological findings during wound repair at day 8 after wound creation. Spaces between the two arrowheads show ulcerated area without epithelial lining. H-E staining. (a) Leptin-reated group. (b) Control group. Scale bars = 500 μm.

Fig 3. Number of blood vessels in the dermal connective tissue beneath the ulcerated area.

(A) At 4 days after initial wounding, no significant difference in the number of CD31-positive cells between Leptin-treated group and control group. (B) At 8 days, more CD31-positive cells were observed in the dermal connective tissue beneath the ulcerated area of leptin-treated group compared with control group. Values are mean ± SE from 5 animals per group. *P < 0.05. (C) Changes in body weight. (D) Changes in serum levels of blood sugar. (E) Changes in serum levels of AST. (F) Changes in serum levels of ALT. None of these laboratory parameters were significantly affected by leptin application. Values are mean ± SE from 5 animals per group.

Fig 4. Effect of leptin on human epidermal keratinocytes.

(A) Ob-R mRNA expression in human epidermal keratinocytes. (B) Ob-R protein expression in human epidermal keratinocytes. (C) Effect of leptin on the proliferation of human epidermal keratinocytes. Leptin enhanced cell proliferation at a concentration equal to and more than 10 ng/mL. Values are mean ± SE (n = 8). *P < 0.05. **P < 0.01. ***P < 0.001.

Fig 5. Effect of leptin on the expression of mRNA encoding G3PDH and Cytokeratin 13, Cytokeratin 14 and Transglutaminase I in human epidermal keratinocytes.

(A) Gene expression was analyzed by semi-quantitative RT-PCR analysis. (B-D) Effect of leptin on the expression of mRNA encoding Cytokeratin 13 (B), Cytokeratin 14 (C) and Transglutaminase I (D). Gene expression was analyzed by quantitative RT-PCR analysis. Values are mean expression ± SD of each gene (n = 8). ***P < 0.001.

Fig 6. Effect of leptin on the migration of human epidermal keratinocytes.

Leptin enhanced the migration of cells. Values are mean ± SE from two independent experiments performed in triplicate. *P < 0.05