852 Short-term ozone exposure stimulates both IGF1R and EGFR to promote diabetic wound healing

Abstract

Ozonated oil, containing the potent disinfectant and oxygen donor ozone (O3), improves wound oxygenation and closure after topical application. However, the mechanism of O3 action on skin cells during wound healing is largely unknown. We hypothesized that IGF1R/EGFR crosstalk and activation of downstream signaling pathways are required for the observed acceleration in wound healing. In a diet-induced obese diabetic mouse model, topical application of O3 oil visibly promoted healing with a 2-3-fold reduction in the epidermal gap and 1.7-fold increase in granulation tissue compared with vehicle-treated mice at 8 days after initiation (when wounds in nondiabetic mice had closed). Immunohistochemical staining showed a 2-2.5 fold increase in BrdU labeling index (p<.01) and a 1.9-2.3 fold increase in keratinocyte (KC) migration (p<.05) at the wound edge at 4-8 days post wounding. Brief exposure of monolayer cultured normal KCs to O3 (20 uM, 2 h) daily increased cell proliferation in high glucose (HG; p<.001) within 24h and normal glucose (NG: p<.01 NG vs. HG+O3) by 72h in WST assays. Exposure to O3 also enhanced cell migration (p<.001, HG vs. HG+O3 by 24h and p<.05 NG vs. NG+O3 by 60h in mouse KCs and p<.01 HG vs. HG+O3 by 6h and p<.05 NG vs. NG+O3 by 18h in human KCs) in scratch assays. Under NG condition, brief O3 exposure increased EGF-stimulated p-ERK by 1.7-fold, p-AKT at Thr 308 and p-PI3K by 2.1-fold and p-AKT at Ser 473 by 2.9-fold, and increased IGF-1 stimulated p-ERK by 1.5-fold. Interestingly, stimulation with EGF or IGF-1 reversed HG-induced inhibition on PI3K/Akt and ERK activation in the presence of O3, suggesting that brief O3 exposure ameliorate skin cell function under HG condition. The increase in KC proliferation and migration caused by O3 exposure was completely inhibited by disrupting EGFR and IGF1R simultaneously. These findings suggest that topical exposure to O3 requires activation of both EGFR and IGF1R to accelerate wound healing.