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

在脑缺血或炎症等病理变化时，小胶质细胞迅速活化并分泌包括TNF-α在内的多种细胞因子，对神经元既有损伤作用，又有保护作用。尽管小胶质细胞源性TNF-α的重要性，但TNF-α的释放是如何调节的仍不清楚。近年来研究发现，ATP通过细胞表面P2受体，至少是G蛋白偶联的P2Y_2和亲离子的P2X_7，激活小胶质细胞，ATP可从神经末梢、激活的免疫细胞或损伤的细胞中释放。在本研究中，我们发现ATP刺激培养的大鼠脑小胶质细胞释放TNF-α，导致mRNA的表达。1 mM ATP能最大程度地诱导TNF-α的释放，P2X_7受体选择性激动剂2 ′-和3 ′-O-（4-苯甲酰基苯甲酰基）-腺苷5 ′-三磷酸（BzATP）也能诱导TNF-α的释放，大鼠P2X_7受体选择性拮抗剂Brilliant Blue G可抑制BzATP诱导的TNF-α释放。ATP或BzATP诱导的TNF-α释放被激活ERK的MEK 1抑制剂PD 98059和U-0126以及p38 MAP激酶抑制剂SB 203580抑制，而BzATP刺激的TNF-α mRNA表达被U-0126抑制，但不被SB 203580抑制。这些结果表明，细胞外ATP至少通过P2 X7受体触发大鼠小胶质细胞TNF-α的释放，并提示ERK和p38 MAP激酶参与了TNF-α释放的信号转导，它们分别在转录和转录后水平发挥不同的作用。

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.