Role of autophagy during cutaneous wound healing in mice

Abstract

Objective To observe the dynamic changes in autophagy during cutaneous wound healing and investigate its possible contributions to wound repair. Methods Two skin wound of 6 mm in size were inflicted on the back of GFP-LC3 transgenic mice. In 0, 3, 7 and 14 d after skin injury, the skin samples were harvested to prepare paraffin-embedded sections, and GFP immunohistochemical (IHC) staining with anit-LC3 antibody was performed for the dynamic changes of autophagy during wound healing. Double-labeling immunofluorescence assay was utilized for identifying cell types of autophgy in granulation tissues by staining both GFP and macrophage marker F4/80 or myofibroblasts marker α-SMA. The mice were divided into skin defect group and injured mice with 3-methyladenine (3-MA, 10 mg/kg, intra-peritoneal injection for 10 continuous days) treatment group. The dynamic changes of the wound were observed, and the skin tissues were collected in 0, 7 and 10 d after injury to evaluate the effect of autophagy on wound healing after fixation and staining. qRT-PCR was employed to detect the expression levels of α-SMA, TGF-β1, PAI-1, type Ⅰ and Ⅲ collagen in the wound samples. Results The autophagic activity was enhanced and then declined in the process of wound healing, and its strongest activity was observed in proliferative phase (7 d). In the granulation tissues, the activity was stronger in the keratinocytes than the interstitial cells, and the autophagic cells were mainly α-SMA-positive myofibroblasts. There were no significant differences in wound area and width, and epidermis thickness in 7 and 10 d after skin injury between the 3-MA treatment and untreated mice, but the former group had significantly increased proliferation activity of epidermal basal cells (P < 0.05) at 7 d after injury. However, no significant differences were seen in the mRNA levels of α-SMA, TGF-β1, PAI-1 and collagen molecules between the 2 groups of mice. Conclusion There exists stronger autophagic activity in the proliferative phase of cutaneous wound healing, but inhibiting autophagy shows little impact on the process.

Keywords

• skin
• wound healing
• autophagy
• myofibroblasts
• keratinocyte
• gfp-lc3 transgenic mice