Tubacin抑制脂多糖诱导活化的小胶质细胞炎症介质释放的实验研究

Experimental study on Tubacin inhibiting release of inflammatory mediators in lipopolysaccharide activated microglial cells

目的探讨组蛋白去乙酰化酶6（HDAC6）选择性抑制剂Tubacin对脂多糖（LPS）诱导活化的小胶质细胞炎症介质释放的影响及其作用机制。方法将BV一2小胶质细胞分为4组：正常对照组（常规培养）、LPS组（给予100ng／mLLPS处理以诱导活化）、Tubacin组（给予1μmol／LTubacin处理、、实验组（给予100ng／mLLPs＋1μmol／LTubacin处理、采用ELISA法检测细胞上清液中白介素-6（IL-6）、肿瘤坏死因子-α（TNF-α含量；采用Griess法和Westernblotting检测细胞内一氧化氮（NO）释放量及诱导型一氧化氮合酶（iNOS）蛋白表达；采用化学荧光法和黄嘌呤氧化酶法检测细胞内活性氧（ROS）含量及细胞上清液中超氧化物歧化酶（SOD）活性。结果与正常对照组比较，LPS组BV-2小胶质细胞IL-6、TNF-α含量和NO释放量明显增加，iNOS蛋白表达显著上调，ROS含量明显增加，SOD活性显著降低，差异均有统计学意义（P〈0．05）。与LPS组比较，实验组BV-2小胶质细胞IL-6、TNF-α含量和NO释放量显著降低，iNOS蛋白表达明显下调，ROS含量显著降低，SOD活性明显升高，差异均有统计学意义（P〈0．05）。结论Tubacin能显著抑制LPS诱导活化的小胶质细胞炎症介质的释放．其作用可能是通过降低活化小胶质细胞的氧化应激水平而实现的。

Objective To investigate the inhibitory effect ofTubacin, a selective inhibition of histone deacetylase 6 （HDAC6）, on the release of inflammatory mediators in lipopolysaecharide （LPS） activated microglias and its underlying mechanism. Methods BV-2 microglias were divided into control group （conventional culture）, LPS group （100 ng/mL LPS）, Tubacin treatment group （1 μmol/L Tubacin） and experimental group （LPS 100 ng/mL＋Tubacin 1 μmol/L）. The levels ofinterleukin -6 （IL-6） and tumor necrosis factor-or （TNF-α） were detected by enzyme linked immunosorbent assay （ELISA）. The production of nitric oxide （NO） was assayed by Griess reagent and the expression of inducible nitric oxide synthase （iNOS） was measured by Western blotting. The oxidative stress levels of BV-2 cells were determined by reactive oxygen species （ROS） and superoxide dismutase （SOD） assays. Results As compared with those in the control group, the productions of IL-6, TNF-α and NO were notably increased, the iNOS protein expression was significantly up-regulated, the ROS level was apparently elevated and the SOD activity was significantly decreased in the LPS group （P〈0.05）. As compared with those in the LPS group, the productions of IL-6, TNF-α and NO were notably decreased, the iNOS protein expression was significantly down-regulated, the ROS level was apparently lessened and the SOD activity was significantly increased in the experiment group （/9〈0.05）. Conclusion Tubacin curbs the release of inflammatory mediators in activated microglial cells induced by LPS, whose effect may be achieved through decreasing oxidative stress levels in LPS activated microglial cells.