Abstract:

Objective To explore whether the phosphorylation of NF-κB P65 subunit is involved in the cytotoxicity to and inflammation in an immortal human keratinocyte cell line HaCaT during cobalt chloride （CoCl2）-induced chemical hypoxia. Methods HaCaT cells were treated with CoCl2 of 2 mmol/L to set up a chemical hypoxia-induced cell model of injury. Then, RNA interference was used to down-regulate the expression of P65 in CoCl2-induced HaCaT cells. After additional culture, cell viability was tested by cell counting kit-8 （CCK-8）, the levels of interleukin 6 （IL-6） and interleukin 8 （IL-8） were detected by ELISA kits, phosphorylated and total P65 protein was measured by Western blot. Results The exposure of HaCaT cells to 2 mmol/L CoCl2 for 0 to 4 hours enhanced the phosphorylation of P65, which began at 0.5 hour, peaked at 1.5 hours, and restored to the normal level at 4 hours, and the level of P65 phosphorylation was about 6.6 times that in the untreated control group. The CoCl2 of 2 mmol/L decreased the cell viability of HaCaT cells in a time dependent manner, and a significant difference was observed in the viability of HaCaT cells between CoCl2-treated and untreated HaCaT cells at 2, 4, and 6 hours （P < 0.05, 0.01, 0.01）. The release of IL-6 and IL-8 from HaCaT cells was also promoted by CoCl2 treatment. The knockdown of P65 expression with siRNA markedly suppressed the CoCl2-induced cytotoxicity to and increase in the release of IL-6 and IL-8 from HaCaT cells, despite of an increment in cell viability by about 11%. Conclusion The phosphorylated P65 subunit mediates CoCl2-induced cytotoxicity and inflammatory injury to HaCaT cells.

Key words: NF-kappa B