磷酸化NF—κB亚基P65介导化学性缺氧诱导的HaCaT细胞炎症损伤

Phosphorylation of NF-κB P65 subunit mediates chemical hypoxia-induced inflammatory injury in HaCaT cells

目的探讨核因子-κB（NF—κB）亚基P65磷酸化是否参与化学性缺氧模拟剂氯化钴（CoCl2）诱导的永生化人皮肤角质形成细胞（HaCaT）毒性作用及炎症反应。方法用2mmol／LCoCl2处理HaCaT细胞，建立化学性缺氧诱导HaCaT细胞损伤的体外模型。RNA干扰法下调NF—κB亚基P65的表达。细胞计数试剂盒-8比色法检测细胞存活率；ELISA法检测培养基中IL-6和IL-8的含量；Western印迹法检测总量P65及磷酸化P65的蛋白表达。结果CoCl2处理HaCaT细胞0～4h，可促进NF—κB亚基P65的磷酸化，在0．5h时P65亚基开始磷酸化，1．5h时P65亚基的磷酸化水平达到高峰，约为对照组的6．6倍，而4h基本恢复到正常水平。CoCl2处理HaCaT细胞0～6h，可时间依赖性地降低细胞活力，2．4和6h的细胞存活率与对照组比较，P值分别〈0．05、〈0．01及〈0．01。CoCl2处理6h，还引起IL-6和IL-8的释放显著增加。RNA干扰法下调P65的表达后，CoCl2处理引起的HaCaT细胞毒性作用被明显减弱，即使细胞存活率升高了11％左右，下调P65表达还明显抑制了CoCl2处理引起的IL-6和IL-8释放增多。结论磷酸化NF—KB亚基P65介导CoCl2诱导的HaCaT细胞毒性及炎症反应。

Objective To explore whether the phosphorylation of NF-κB P65 subunit is involved in the eytotoxicity 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 COC12 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 COC12 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 CoClrtreated 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 CoCl：-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 CoC12-induced cytotoxicity and inflammatory injury to HaCaT cells.