P2X4/NLRP3炎性小体通路介导小胶质细胞白细胞介素-1β的释放

Involvement of P2X4/NLRP3 Inflammasome Pathway in IL-1β Production from Microglia

目的探讨P2X4嘌呤受体和NLRP3炎性小体对炎症反应时小胶质细胞释放白细胞介素-1β(IL-1β)的影响。方法采用脂多糖(LPS)和三磷酸腺苷(ATP)双信号干预建立小胶质细胞激活模型,将处于对数生长期生长分化状态良好的小胶质细胞用于实验,按不同干预方法分为8组:正常对照组、ATP组、LPS组、LPS+ATP组、5-BDBD组、ATP+5-BDBD组、LPS+5-BDBD组和LPS+ATP+5-BDBD组。Real time PCR检测各组NLRP3、P2X4 mRNA表达水平;Western blot技术测定半胱氨酸天冬氨酸蛋白酶-1(caspase-1)蛋白表达水平;酶联免疫吸附法(ELISA)检测上清液中IL-1β释放水平。结果 2 mmol·L^(-1)ATP能够上调小胶质细胞P2X4 mRNA的表达水平,差异有统计学意义(P<0.001);用低浓度(1μg·m L^(-1))LPS初始刺激后,ATP分子能够诱导小胶质细胞中NLRP3 mRNA水平升高(P<0.001)、caspase-1蛋白水平表达上调(P<0.001)、促进IL-1β释放(P<0.01),而P2X4的特异性受体阻断剂5-BDBD抑制上清液中IL-1β水平的上调(P<0.05),差异有统计学意义。结论小胶质细胞中IL-1β的释放有赖于P2X4/NLRP3炎性小体途径。

Aim To investigate the role of P2X4 purinergic receptor and NOD-like receptor 3（NLRP3）-inflammasomein in the release of interleukin1β（IL^（-1）β） from microgliain response to inflammatory injury. Methods Microglia in exponential phase were used for the experiment. There were divided into 8 groups according to different intervention methods： a control group, a ATP group, a LPS group, a LPS＋ATP group, a 5-BDBD group, a ATP＋5-BDBD group, a LPS＋5-BDBD group, a LPS＋ATP＋5-BDBD group. Real-time PCR was used to measure the NLRP3 mRNA and P2 X4 mRNA expression. The protein level of caspase^（-1） was detected by Western blot. The concentration of IL^（-1）β was measured by enzyme-linked immunosorbent assay（ELISA）. Results The results showed that 2 mmol·L^（-1） adenosine triphosphate（ATP）induced P2 X4 mRNA expression（P0.001）. After incubated with LPS（1 μg·m L^（-1）） for about 6 h, then treated with ATP（2 mmol·L^（-1）） for about 3 h, the NLRP3 mRNA expression, caspase^（-1） protein expression and the production of IL^（-1）β（P0.001, P0.001, P0.01, respectively） were induced. Moreover, addition of the P2 X4 purinergic receptor antagonist 5-BDBD reduced the production of IL^（-1）β（P0.05）, and the difference was statistically significant. Conclusion The release of IL^（-1）β is dependent of the P2 X4-NLRP3 inflammasome pathway in microglia. The neuroinflammation mediated by microglia is a causative factor of neurodegenerative disease, such as Parkinson＇s disease（PD）, therefore, the regulation of P2 X4-NLRP3 inflammasome pathway may provide novel treatment protocols for neurodegenerative diseases.