Discovery of novel functions of brain microglia and their in vivo analysis.

在脑缺血或炎症等病理改变时，小胶质细胞被迅速激活并分泌各种细胞因子，包括TNF-α，对神经元既有伤害作用，也有保护作用。尽管小胶质细胞衍生的TNF-α很重要，但TNF-α的释放是如何调节的仍然未知。近年来研究发现，神经末梢释放的ATP可激活免疫细胞或受损细胞，并通过细胞表面P2受体（至少是g蛋白偶联的P2Y_2和嗜离子性的P2X_7）激活小胶质细胞。在本研究中，我们发现ATP刺激TNF-α的释放，导致mRNA在大鼠培养的脑小胶质细胞中表达。大鼠P2X_7受体选择性拮抗剂Brilliant Blue G可抑制2′和3′- o -（4-苯甲酰苯甲酰）-腺苷5′-三磷酸（BzATP）诱导的TNF-α释放。活化ERK的MEK1抑制剂PD98059和U-0126以及p38 MAP激酶抑制剂SB203580均能抑制atp或bzatp诱导的TNF-α释放，而bzatp刺激的TNF-α mRNA表达被U-0126抑制，而SB203580则不能。这些结果表明，细胞外ATP至少通过P2X_7受体触发大鼠小胶质细胞中TNF-α的释放，并提示TNF-α释放的信号通路涉及ERK和p38 MAP激酶，它们分别在转录和转录后水平上发挥不同的作用。

Upon pathological changes such as brain ischemia or inflammation, microglia are rapidly activated and secrete various cytokines, including TNF-α, which plays not only harmful, but also protective roles for neurons. Despite importance of microglia-derived TNF-α, it remains unknown how the release of TNF-α is regulated. Recently it was revealed that ATP, which is released from nerve terminals, activated immune cells or damaged cells, activates microglia via cell surface P2 receptors, at least G-protein-coupled P2Y_2 and ionotropic P2X_7. In this study, we found that ATP stimulates the release of TNF-α, resulting from mRNA expression in rat cultured brain microglia. The release of TNF-α was maximally elicited by 1 mM ATP and also induced by a P2X_7 selective agonist, 2'-and 3'-O-(4-benzoylbenzoyl)-adenosine 5'-triphosphate (BzATP), and BzATP-induced TNF-α was inhibited by Brilliant Blue G, a selective antagonist of rat P2X_7 receptor. ATP-or BzATP-induced TNF-α release was inhibited by PD98059 and U-0126, inhibitors of MEK1, which activates ERK, and also by SB203580, an inhibitor of p38 MAP kinase, while BzATP-stimulated mRNA expression of TNF-α was inhibited by U-0126, but not by SB203580. These results indicate that extracellular ATP triggers TNF-α release in rat microglia at least via P2X_7 receptor and suggest that signaling to the release of TNF-α involves ERK and p38 MAP kinase, which play distinct roles at the transcriptional and the post-transcriptional levels, respectively.