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目的探讨环氧化酶2(COX-2)在化学性缺氧模拟剂氯化钴(CoCl2)诱导永生化人皮肤角质形成细胞(HaCaT)毒性及炎症反应中的作用。方法用CoCl2处理HaCaT细胞,建立化学性缺氧诱导人皮肤角质形成细胞损伤的实验模型。CCK-8比色法检测细胞存活率,ELISA试剂盒检测细胞培养基中白介素-6(IL-6)和白介素-8(IL-8)的水平,Western blot法检测COX-2蛋白的表达。结果 2 000μmol.L-1 CoCl2处理HaCaT细胞0～3 h,可上调COX-2的表达,1 h COX-2表达开始升高,2 h表达达到高峰。用0、500、1 000和2 000μmol.L-1 CoCl2处理HaCaT细胞2 h可剂量依赖性地上调COX-2的表达。2 000μmol.L-1 CoCl2处理HaCaT细胞0～8 h可时间依赖性地降低HaCaT细胞存活率,半数致死时间(LT50)在6 h左右。2 000μmol.L-1 CoCl2处理HaCaT细胞6 h可促进HaCaT细胞释放IL-6和IL-8。选择性COX-2抑制剂(NS-398)可明显对抗2 000μmol.L-1 CoCl2处理引起的细胞毒性及CoCl2诱导的IL-6和IL-8的释放增加。结论 COX-2介导化学性缺氧诱导的HaCaT细胞毒性及炎症损伤作用。
Objective To investigate the role of cyclooxygenase 2 (COX-2) in the induction of immortalized human keratinocytes (HaCaT) and its inflammatory response induced by cobalt chloride. Methods HaCaT cells were treated with CoCl2 to establish an experimental model of chemical hypoxia-induced injury of human keratinocytes. The cell viability was detected by CCK-8 colorimetric assay. The levels of interleukin-6 (IL-6) and interleukin-8 (IL-8) were detected by ELISA kit and the expression of COX-2 protein by Western blot. Results After treated with 2 000 μmol·L-1 CoCl2 for 0-3 h, the expression of COX-2 was up-regulated. The expression of COX-2 began to increase at 1 h and reached a peak at 2 h. HaCaT cells treated with 0, 500, 1 000 and 2 000 μmol·L-1 CoCl2 for 2 h up-regulated the expression of COX-2 in a dose-dependent manner. HaCaT cells treated with 2 000 μmol·L-1 CoCl2 for 0-8 h reduced the survival rate of HaCaT cells in a time-dependent manner, with a median lethal time (LT50) of about 6 h. Treatment of HaCaT cells with 2 000 μmol·L-1 CoCl2 for 6 h promoted the release of IL-6 and IL-8 from HaCaT cells. Selective COX-2 inhibitor (NS-398) significantly antagonized cytotoxicity induced by 2 000 μmol·L-1 CoCl2 and increased CoCl2-induced IL-6 and IL-8 release. Conclusions COX-2 mediates cytotoxicity and inflammatory injury induced by chemical hypoxia in HaCaT cells.