Hypoxia of skin is an important physiopathological process in many diseases, such as pressure ulcer, diabet-ic ulcer, and varicose ulcer. Although cellular injury and inflammation have been involved in hypoxia-induced dermatic injury, the underlying mechanisms remain largely unknown. This study was conducted to investigate the effects of cobalt chloride (CoCl₂), a hypoxia-mimicking agent, on human skin keratinocytes (HaCaT cells) and to explore the possible molecular mechanisms. Exposure of HaCaT cells to CoCl₂ reduced cell viability and caused overproduction of reactive oxygen species (ROS) and oversecretion of interleukin-6 (IL-6) and interleukin-8 (IL-8). Importantly, CoCl₂ exposure elicited overexpression of cyclooxygenase-2 (COX-2) and phosphorylation of nuclear factor-kappa B (NF-ĸB) p65 subunit. Inhibition of COX-2 by NS-398, a selective inhibitor of COX-2, significantly repressed the cytotoxicity, as well as secretion of IL-6 and IL-8 induced by CoCl₂. Inhibition of NF-ĸB by PDTC (a selective inhibitor of NF-ĸB) or genetic silencing of p65 by RNAi (Si-p65), attenuated not only the cytotoxicity and secretion of IL-6 and IL-8, but also overexpression of COX-2 in CoCl₂-treated HaCaT cells. Neutralizing anti-IL-6 or anti-IL-8 antibody statistically alleviated CoCl₂-induced cytotoxicity in HaCaT cells. N-acetyl-L-cysteine (NAC), a well characterized ROS scavenger, obviously suppressed CoCl₂-induced cytotoxicity in HaCaT cells, as well as secretion of IL-6 and IL-8. Additionally, NAC also repressed overexpression of COX-2 and phosphorylation of NF-ĸB p65 subunit induced by CoCl₂ in HaCaT cells. In conclusion, our results demonstrated that oxidative stress mediates chemical hypoxia-induced injury and inflammatory response through activation of NF-ĸB?COX-2 pathway in HaCaT cells.

Keywords: chemical hypoxia, cyclooxygenase-2, inflammation, keratinocytes, nuclear factor-kappa B, oxidative stress