甲基苯丙胺对大鼠小胶质细胞的损伤及炎性相关基因表达的影响

Damage of cells and effects of inflammatory related genes induced by methamphetamine in rat microglial cells

目的:观察甲基苯丙胺(methamphetamine,Meth)对原代小胶质细胞的损伤及炎性相关基因表达的影响。方法:原代培养SD胎鼠小胶质细胞,利用MTT和原位末端转移酶标记技术(TUNEL)分别检测Meth引起小胶质细胞活力和凋亡的变化。采用实时荧光定量聚合酶链反应法(real-time PCR)观察Meth对小胶质细胞炎性相关因子mRNA表达的影响。ELISA及试剂盒法检测Meth作用后小胶质细胞白介素(interleukin,IL)-6、肿瘤坏死因子(tumor necrosis factor,TNF)-α、诱导型一氧化氮合酶(inducible nitric oxide synthase,i NOS)的分泌改变。结果:MTT实验显示,Meth降低小胶质细胞活力,呈浓度依赖性,浓度为200μmol/L时与对照组比较,差异有统计学意义(P<0.01)。TUNEL实验结果显示200μmol/L Meth可引起细胞凋亡,与对照组比较,差异有统计学意义(P<0.05)。q-PCR结果显示Meth作用于小胶质细胞24 h可降低IL-24、一氧化氮合酶3(nitric oxide synthase 3,NOS3)表达水平,上调Peli3、Sigma受体1(Sigma receptor 1,Sig1-R)、IL-1β、IL-6、Toll样受体4(Toll like receptor 4,TLR4)的表达水平,差异有统计学意义(P<0.05)。此外,蛋白水平亦发现,Meth可促进IL-6和TNF-α的分泌。结论:Meth可降低小胶质细胞活力,诱导小胶质细胞凋亡,并引起IL-1β、IL-1R、IL-6、TLR4等炎性因子mRNA表达变化,促进IL-6和TNF-α的分泌,进而可能损伤中枢神经系统,产生神经毒性。

Objective：To explore the effects of cell injury and the expression of inflammation related genes in rat microglial cells induced by methamphetamine （Meth）.Methods：Cell viability and apoptosis induced by Meth were detected by MTT and in situ end labeling （TUNEL） after primary culture of SD fetal rat microglial cells.The effect of Meth on the expression of inflammatory cytokines mRNA in the microglial cells was evaluated by real time fluorescence quantitative polymerase chain reaction （real-time PCR）.The secretion of interleukin （IL）-6,tumor necrosis factor （TNF）-α and inducible nitric oxide synthase （i NOS）in microglia cells induced by Meth were detected by ELISA method.Results：MTT showed that Meth reduced microglia viability in a concentration dependent manner （25,50,100,and 200 μmol/L） with statistically significant at a concentration of 200 μmol/L （P 0.05）.TUNEL results showed that200 μmol/L Meth significantly contributed to cell apoptosis compared with the control group （P0.05）.Q-PCR results showed that Meth reduced the expression levels of IL-24 and nitric oxide synthase 3 （NOS3） in microglial cells for 24 hours.However,the expression levels of Peli3,Sigma receptor 1 （sig1）-R,IL-1,IL-6 and Toll like receptor 4 （TLR4） were up-regulated.In addition,protein levels were also confirmed that Meth promoted the secretion of IL-6 and TNF-α.Conclusion：Meth exposure obviously contributes to microglial damage.Moreover,it causes IL-1β,IL-1R,IL-6,TLR4 and other inflammatory factors mRNA expression changes,and promotes the secretion of IL-6 and TNF-α,which may damage the central nervous system,resulting in neurotoxicity.