Leptin, a 16 kDa circulating anti-obesity hormone, has many physiological properties such as body weight homeostasis, lipid metabolism, hematopoiesis, thermogenesis, ovarian function, bone formation, and angiogenesis. Interestingly, a certain study showed that skin wound healing delayed in leptin deficient ob/ob mice. However, little has been known about the physiological role of leptin in skin wound healing. In this chapter, we introduce whether local and single-dose administration of leptin exerted a promotive influence on the skin wound healing. Immunohistochemical analysis revealed that leptin receptor was expressed in mouse epidermal cells. In addition, topical administration of leptin promoted the healing of chemical burn wounds created on the back skin of mice without any side effects. Then, the mechanisms of the promotive effect of leptin on the wound healing of the skin were demonstrated immunohistochemical and biological analysis; namely, leptin stimulated angiogenesis in the connective tissue beneath the wounded area and the cell proliferation, differentiation/function, and migration of human epidermal keratinocytes. These findings revealed the possible and promising usefulness of leptin as a new wound-healing promoting agent.
- wound healing
- new promoting agent
- local administration
Leptin, the product of
The surface of the body is covered by skin to communicate with the external environment and to protect deeper tissues and organs by separating them from the external environment such as chemical, mechanical, and thermal stresses, infections, and dehydration [21, 22]. Normal dermal wound repair processes, such as inflammation, angiogenesis, contraction, deposition of extracellular matrix, granulation tissue formation, epithelialization, and remodeling, require various cellular and molecular signals . In this biological process, skin fibroblasts interact with surrounding cells such as keratinocytes, inflammatory cells, and endothelial cells [21, 24]. Fibroblasts produce extracellular matrix, glycoproteins, adhesive molecules, and various cytokines [25, 26]. The lack of these signals may result in poor healing of wounds such as diabetic ulcers [27, 28].
A certain study showed that skin wound healing delayed in leptin deficient
2. Localization of leptin receptor in mouse skin
An immunohistochemical analysis of mouse skin using anti-leptin receptor antibody revealed that leptin receptor was expressed in prickle and granular cells of epidermis (Figure 1). These findings showed that epidermal cells are target of leptin.
3. Effect of leptin on the wound healing of the skin
To elucidate the effect of leptin on the wound healing of the skin, mouse skin chemical burn model was used. Eighteen 6-week-old male ICR mice were fed a normal diet and maintained under a 12-h light/12-h dark cycle. Chemical wounds were created on the back skin by applying two pieces (12 × 12 mm) of filter paper soaked with 20% sodium hypochlorite for 5 min. Wound formation was verified next day, and the wounds were covered with 15 g (12 × 12 × 1 mm) of MedGel (Med GEL Corp., Tokyo, Japan) containing 10 μL of 100 ng/mL leptin (R&D Systems, Minneapolis, USA) or phosphate-buffered saline (PBS) as a control. This hydrogel-contained leptin or PBS was attached to the chemical burn site and dressed. The size of the ulcer was measured on day 4 and 8 after burn formation, and the skin tissue around the wound was obtained for histological analysis. In consequence, at day 4, slightly enhanced re-epithelialization was observed in leptin-treated group, but no significant difference was noted between leptin-treated and control group. In contrast, at day 8, significantly enhanced re-epithelialization was observed in leptin-treated group (Figure 2). These experiments showed that the wound area decreased much faster in the leptin-treated group compared with the control group. These findings demonstrated that single and local administration of leptin using bioabsorbable hydrogel promoted the wound healing of skin.
Meanwhile, body weight (BW), and levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT) or blood sugar (BS) were not affected through experiment period, showing that topically administered leptin had no systemic adverse effects (Figure 3). These findings certify that topically administered leptin is capable of promoting wound healing of the skin without any systemic adverse effects in this period. However, unfortunately, we could not elucidate whether local and single administration of leptin could avoid or not the influence of its side effect over a long period. This issue should be elucidated in the future investigation.
4. Mechanism of the promotive effect of leptin on the wound healing of the skin
4.1. Effect of leptin for angiogenesis on the wound healing of the skin
To elucidate the mechanism of the promotive effect of leptin on the wound healing of the skin, first, the influence of leptin on the angiogenesis in the connective tissues beneath the wound in the skin was revealed by histological analysis. The localization of blood vessels was analyzed by immunohistochemistry by using anti-CD31 antibody. Then, at day 4, after initial wounding, no significant difference on the number of CD31-positive cells was detected between leptin-treated and control group. However, at day 8, after initial wounding, the number of CD31-positive cells significantly increased in leptin-treated group (Figure 4). These findings demonstrated that leptin stimulates angiogenesis in the connective tissue beneath the ulcer, and promotes wound healing in the skin by accelerating the supply of nutritions, oxygen, and even some bioactive substances.
4.2. Effect of leptin on the proliferation of human epidermal keratinocytes
To reveal another possible mechanism underlying the promotive effect of leptin on the skin wound healing, cell biological analyses were performed using human epidermal keratinocytes on the premise that the cells were proven to express the mRNA and protein of leptin receptor (
4.3. Effect of leptin on the differentiation/function of human epidermal keratinocytes
Next, the effect of leptin on the differentiation/function of human keratinocytes was demonstrated using quantitative RT-PCR analysis of the expression of mRNA encoding keratinocyte-related genes, that is,
4.4. Effect of leptin on the migration of human epidermal keratinocytes
Moreover, to elucidate the effect of leptin on cell migration around the skin wounded area, scratch assay using human epidermal keratinocytes was performed. The assay was performed using CytoSelect Wound Healing Assay kit (Cell Biolabs Inc., San Diego, USA) according to the manufacturer’s instructions. After preparation, the cells were treated with or without 100 ng/mL of leptin. Images of wound healing were captured using a phase-contrast microscope at 0, 3, 6, 9, 12, 18, and 24 h after the preparation. The area of open wound field was calculated by using ImageJ software . Consequently, the significant effect was not observed during initial 12 h. However, the area without cells decreased significantly in leptin-treated group compared with control group from 18 to 24 h (Figure 7). This assay revealed that leptin significantly accelerated the migration of human epidermal keratinocytes.
Leptin is capable of promoting wound healing of skin by influencing epidermal keratinocytes proliferation, differentiation/function and migration, and angiogenesis in the connective tissue beneath the wounded area. Moreover, we showed that single dose and topically administration of leptin could promote wound healing in the skin without any side effects by using an adequate drug delivery system . In addition to these findings, our previous study demonstrated that local administration of leptin could promote wound healing in the oral mucosa by enhancing epithelial cell migration and angiogenesis in the connective tissue beneath the wound . Taken together, leptin is proven to play physiological roles in wounded area not only as a systemic hormone but also as a local growth factor. Importantly, these findings presented in this chapter declared the possible and promising usefulness of leptin as a new wound-healing promoting agent.
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