米诺环素下调环氧化酶-2抑制砷诱导小胶质细胞活化的实验研究

目的 探讨米诺环素对砷诱导的小胶质细胞活化和环氧化酶-2（COX-2）表达的影响及可能机制。方法 本实验设置4组:对照组、10μmol/L NaAsO2组、10μmol/L米诺环素组和10μmol/L NaAsO2+10μmol/L米诺环素组，各组BV-2小胶质细胞贴壁生长至对数期后，进行24h染毒处理，然后从形态学观察，ELISA法检测干扰素-γ（INF-γ）、白细胞介素-1β（IL-1β）、白细胞介素-6（IL-6）和肿瘤坏死因子-α（TNF-α）的分泌，Realtime-PCR分析COX-2 mRNA表达，Western blotting分析COX-2蛋白表达四个方面展开研究。结果 形态学观察发现，与对照组相比，各染毒处理组BV-2小胶质细胞均受到不同程度的激活，而与NaAsO2组相比，NaAsO2+米诺环素组细胞激活状态受到一定抑制。ELISA法检测结果显示:各组INF-γ（F=20.165）、IL-1β（F=10.688）、IL-6（F=11.488）和TNF-α（F=11.641）分泌差异均具有统计学意义（P均<0.001）；与NaAsO2组相比，NaAsO2+米诺环素组细胞培养液上清IL-1β、IL-6和TNF-α分泌减少，INF-γ分泌增高（P均<0.05），米诺环素对砷染毒处理的炎性细胞因子分泌有逆向改变作用。Realtime-PCR检测结果显示:各组COX-2 mRNA表达差异具有统计学意义（F=266.427，P<0.001）；与NaAsO2组相比，米诺环素下调砷染毒处理的COX-2 mRNA表达（P<0.001）。Western blotting光带经分析后，各组COX-2蛋白表达差异具有统计学意义（F=74.785，P<0.001）；与NaAsO2组相比，米诺环素减弱砷染毒处理的COX-2蛋白表达（P<0.001）。结论 下调COX-2表达可能是米诺环素抑制砷诱导小胶质细胞活化机制的重要环节。

Inhibition of minocycline in arsenic-induced microglia activation by down-regulating COX-2

Objective To explore the effect of minocycline on arsenic-induced microglia activation and cyclooxygenase-2(COX-2) expression as well as the possible mechanism. Methods In this study, four groups were set up: control group, 10 μmol/L NaAsO2 group, 10 μmol/L minocycline group and 10 μmol/L NaAsO2 + 10μmol/L minocycline group. The BV-2 microglia in each group were treated for 24 h after adherent growth to logarithmic phase, and then the study was carried out from four aspects including morphological observation, ELISA detection of interferon-γ(INF-γ), interleukin-1β(IL-1β), interleukin-6(IL-6) and tumor necrosis factor-α(TNF-α) secretion, Realtime-PCR analysis of COX-2 mRNA expression and Western blotting analysis of COX-2 protein expression. Results Morphological observation showed that compared with the control group, BV-2 microglia in each treatment group were activated in different degrees. While compared with the NaAsO2 group, the activation status of BV-2 microglia in the NaAsO2 + minocycline group was inhibited to some extent. The results of ELISA test indicated that the levels of INF-γ(F=20.165), IL-1β(F=10.688), IL-6(F=11.488) and TNF-α(F=11.641) in each group were statistically significant(P<0.001). Compared with the NaAsO2 group, the level of IL-1β, IL-6, and TNF-α reduced, while the secretion of INF-γ was increased in cell culture supernatant of the NaAsO2 + minocycline group(all P<0.05). Minocycline had effects on the secretion of inflammatory cytokines treated by arsenic. The results of Realtime-PCR test showed that the difference of COX-2 mRNA expression in each group was statistically significant(F=266.427, P<0.001). Compared with NaAsO2 group, minocycline reduced the expression of COX-2 mRNA in cells treated with arsenic(P<0.001). After the Western blotting analysis, the difference of COX-2 protein expression in each group was statistically significant(F=74.785, P<0.001). Compared with NaAsO2 group, minocycline reduced the expression of COX-2 protein in cells treated with arsenic(P<0.001). Conclusion Down-regulation of COX-2 expression may play an important role in the inhibition of arsenic-induced microglia activation by minocycline.