中华皮肤科杂志 ›› 2011, Vol. 44 ›› Issue (3): 195-198.

• 论著 • 上一篇    下一篇

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

杨春涛1,凌宏忠2,曾凡钦3,张辉4,杨战利5,傅路6,莫利球7,韩艳芳8,冯鉴强9   

  1. 1. 中山大学中山医学院
    2. 广州中山医科大学附属第一医院皮肤科
    3. 广州市中山大学第二医院皮肤科
    4. 重庆，第三军医大学附属西南医院全军肝胆外科研究所
    5. 广州，中山大学中山医学院生理学教研室
    6. 广州，中山大学中山医学院生理教研室
    7. 中山大学附属第一医院黄埔院区麻醉科
    8. 广州 中山大学附属第二医院皮肤科
    9. 中山大学
  • 收稿日期:2010-10-22 修回日期:2010-11-23 出版日期:2011-03-15 发布日期:2011-03-10
  • 通讯作者: 冯鉴强 E-mail:fengjq-sums@163.com
  • 基金资助:

    广东省科技计划项目

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

  • Received:2010-10-22 Revised:2010-11-23 Online:2011-03-15 Published:2011-03-10

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摘要：

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2 mmol/L CoCl2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 ~ 4 h，可促进NF-κB亚基P65的磷酸化，在0.5 h时P65亚基开始磷酸化，1.5 h时P65亚基的磷酸化水平达到高峰，约为对照组的6.6倍，而4 h基本恢复到正常水平。CoCl2处理HaCaT细胞0 ~ 6 h，可时间依赖性地降低细胞活力，2、4和6 h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6 h，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-κB亚基P65介导CoCl2诱导的HaCaT细胞毒性及炎症反应。

关键词: 核因子-kappa B

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