**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**

**Figure 2.** Effect of leptin on the wound healing of the mouse skin. (A) Histological findings of wound repair of skin at day 8 after initial wounding in leptin-treated group. (B) Histological findings of wound repair of skin at day 8 after initial wounding in control group. Spaces between arrow heads show ulcerative area without epithelium. Wound heal is significantly enhanced in leptin-treated group. (C) Skin wound healing at day 4 after wound creation. No significant difference in wound healing was noted between leptin-treated and control group. (D) Skin wound healing at day 8 after wound creation. Significantly enhanced re-epithelialization was observed in leptin-treated group. \*\*P < 0.01. H-E

staining. Bars: 500 μm.

28 Wound Healing - New insights into Ancient Challenges

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 leptintreated 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.

**Figure 4.** Number of vascular endothelial cells in the dermal connective tissue beneath the ulcerated area. (A) At day 4, after initial wounding, no significant difference in the number of CD31-positive cells between leptin-treated group and control group. (B) At day 8, after initial wounding, more vascular endothelial cells distributed in the connective tissue beneath the ulcer in leptin-treated group compared with control group. \*P < 0.05.

#### **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 (*Ob-R*) (data not shown). To elucidate the effect of leptin on the proliferation of human epidermal keratinocytes, the cells were cultured in the absence or presence of various concentrations of leptin. The results indicated that the proliferation of human keratinocytes was significantly enhanced by leptin at a concentration equal to and more than 10 ng/mL

**Figure 5.** 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. \*P < 0.05, \*\*P < 0.01, \*\*\*P < 0.001.

(**Figure 5**). These findings showed the modest stimulatory effect of leptin on the proliferation of human epidermal keratinocytes.

### **4.3. Effect of leptin on the differentiation/function of human epidermal keratinocytes**

**Figure 4.** Number of vascular endothelial cells in the dermal connective tissue beneath the ulcerated area. (A) At day 4, after initial wounding, no significant difference in the number of CD31-positive cells between leptin-treated group and control group. (B) At day 8, after initial wounding, more vascular endothelial cells distributed in the connective tissue

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 (*Ob-R*) (data not shown). To elucidate the effect of leptin on the proliferation of human epidermal keratinocytes, the cells were cultured in the absence or presence of various concentrations of leptin. The results indicated that the proliferation of human keratinocytes was significantly enhanced by leptin at a concentration equal to and more than 10 ng/mL

**Figure 5.** 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. \*P < 0.05, \*\*P < 0.01, \*\*\*P < 0.001.

beneath the ulcer in leptin-treated group compared with control group. \*P < 0.05.

30 Wound Healing - New insights into Ancient Challenges

**4.2. Effect of leptin on the proliferation 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 keratinocyterelated genes, that is, *Cytokeratin 13*, *Cytokeratin 14,* and *Transglutaminase I*. Accordingly, this analysis detected an elevation in expression levels of these gene expressions in the presence of 100 ng/mL leptin (**Figure 6**). These findings showed that leptin has a stimulatory effect on the differentiation/function of human epidermal keratinocytes.

**Figure 6.** Effect of leptin on the expression of mRNA encoding *Cytokeratin 13*, *Cytokeratin 14,* and *Transglutaminase I* in human epidermal keratinocytes analyzed by quantitative RT-PCR analysis. (A) *Cytokeratin 13*, (B) *Cytokeratin 14*, (C) *Transglutaminase I*. Leptin exerted stimulatory effect on the gene expression of *Cytokeratin 13*, *Cytokeratin 14,* and *Transglutaminase I* at the concentration of 100 ng/mL. \*\*\*P < 0.001.

### **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 [32]. 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.

**Figure 7.** Effect of leptin on the migration of human epidermal keratinocytes. Leptin accelerated the migration of human epidermal keratinocytes, significantly. \*P < 0.05.
