Won So Y, Choi Sang-Ho, Jin Byung K
Brain Disease Research Center, Ajou University School of Medicine, Suwon 443-479, South Korea.
J Neuroimmunol. 2009 Sep 29;214(1-2):83-92. doi: 10.1016/j.jneuroim.2009.07.005. Epub 2009 Aug 5.
In the present study, we examine whether prothrombin kringle-2 (pKr-2), a domain of prothrombin distinct from thrombin and a potent microglial activator induces reactive oxygen species (ROS) generation through stimulation of microglial NADPH oxidase activity, and whether this phenomenon contributes to oxidative damage and consequent neurodegeneration. Intracortical injection of pKr-2 caused significant loss of cortical neurons in vivo after seven days, as evident from Nissl staining and immunohistochemical analysis using the neuronal-specific nuclear protein (NeuN) antibody. In parallel, pKr-2-activated microglia and ROS production were observed in rat cortex displaying degeneration of cortical neurons. Reverse transcription-PCR at various time points after pKr-2 administration disclosed early and transient expression of inducible nitric oxide synthase (iNOS) and proinflammatory cytokines, such as interleukin 1beta (IL-1beta). Co-localization of iNOS, IL-1beta, and TNF-alpha within microglia was evident with double-label immunohistochemistry. Additionally, pKr-2 induced upregulation of cytosolic components of NADPH oxidase (p67(phox)), translocation of cytosolic p67(phox) protein to the membrane, and p67(phox) expression in microglia in the cortex in vivo, signifying NADPH oxidase activation. The pKr-2-induced oxidation of proteins and loss of cortical neurons were partially inhibited by DPI, an NADPH oxidase inhibitor, and trolox, an antioxidant. Consistent with our hypothesis, following treatment with pKr-2 in vitro, neurotoxicity was detected exclusively in co-cultures of cortical neurons and microglia, but not in microglia-free neuron-enriched cortical cultures, indicating that microglia are required for pKr-2 neurotoxicity. Our results strongly suggest that pKr-2 as an endogenous compound participates in cortical neuron death through microglial NADPH oxidase-mediated oxidative stress.
在本研究中，我们探究了凝血酶原kringle-2（pKr-2），一种不同于凝血酶的凝血酶原结构域且是一种强效小胶质细胞激活剂，是否通过刺激小胶质细胞NADPH氧化酶活性诱导活性氧（ROS）生成，以及这种现象是否导致氧化损伤及随后的神经退行性变。皮层内注射pKr-2在7天后导致体内皮层神经元显著丢失，这从尼氏染色以及使用神经元特异性核蛋白（NeuN）抗体的免疫组织化学分析中可以明显看出。同时，在显示皮层神经元变性的大鼠皮层中观察到pKr-2激活的小胶质细胞和ROS生成。给予pKr-2后不同时间点的逆转录聚合酶链反应（RT-PCR）揭示了诱导型一氧化氮合酶（iNOS）和促炎细胞因子如白细胞介素1β（IL-1β）的早期和短暂表达。双标记免疫组织化学显示iNOS、IL-1β和肿瘤坏死因子-α（TNF-α）在小胶质细胞内共定位明显。此外，pKr-2在体内诱导皮层小胶质细胞中NADPH氧化酶的胞质成分（p67(phox)）上调、胞质p67(phox)蛋白向膜的转位以及p67(phox)表达，表明NADPH氧化酶被激活。pKr-2诱导的蛋白质氧化和皮层神经元丢失被NADPH氧化酶抑制剂二苯基碘鎓（DPI）和抗氧化剂生育三烯酚（trolox）部分抑制。与我们的假设一致，在体外给予pKr-2处理后，仅在皮层神经元和小胶质细胞的共培养物中检测到神经毒性，而在无微胶质细胞的富含神经元的皮层培养物中未检测到，这表明小胶质细胞是pKr-2神经毒性所必需的。我们的结果强烈表明，pKr-2作为一种内源性化合物通过小胶质细胞NADPH氧化酶介导的氧化应激参与皮层神经元死亡。
