Cho S, Kim Y, Cruz M O, Park E M, Chu C K, Song G Y, Joh T H
Department of Neurology and Neuroscience, Weill Medical College of Cornell University at Burke Medical Research Institute, 785 Mamaroneck Ave., White Plains, NY 10605, USA.
Glia. 2001 Mar 15;33(4):324-33. doi: 10.1002/1098-1136(20010315)33:4<324::aid-glia1031>3.0.co;2-m.
Excessive proinflammatory cytokine and NO production by activated microglia play a role in neurodegenerative disorders. To investigate whether the neuroprotectant N-acetyl-O-methyldopamine (NAMDA) downregulates genes associated with microglial activation, we measured gene expression of TNF-alpha, IL-1beta, inducible nitric oxide synthase (NOS2), and an associated cofactor synthesis gene, GTP cyclohydrolase I (GTPCH) in LPS-stimulated microglia cells in the presence or absence of NAMDA. The temporal pattern of cytokine gene expression showed that LPS (0.2 microg/ml) increased TNF-alpha and IL-1beta gene expression at 1 and 3 h, which was repressed by cotreatment of NAMDA. Similarly, LPS also induced GTPCH and NOS2 gene expression at 3 and 6 h, and cotreatment of NAMDA repressed the induction with parallel reduction of nitrite, an oxidative metabolite of nitric oxide. Since transcription factor NF-kappaB is involved in regulating expression of these genes, the effects of NAMDA on NF-kappaB nuclear translocation and DNA binding in immunostimulated microglia were investigated. We found that neither LPS-induced NF-kappaB translocation nor DNA binding activity was affected by cotreatment with NAMDA in BV-2 microglia. On the other hand, NAMDA increased intracellular cAMP levels and potentiated LPS-induced phosphorylated cAMP-responsive element binding protein (pCREB) expression. Treatment with adenosine 3'5'-cyclic monophosphothioate, a specific inhibitor of cAMP-dependent protein kinase (PKA), reversed not only NAMDA-induced pCREB upregulation but also NAMDA-induced repression of TNF-alpha and IL-1beta gene transcription. The data demonstrate that NAMDA represses LPS-induced proinflammatory cytokines gene expression via a cAMP-dependent protein kinase pathway. Thus, repressing proinflammatory cytokines and NOS2 gene expression in activated microglia by NAMDA may provide new therapeutic strategies for ischemic cerebral disease as well as other neurodegenerative diseases.
活化的小胶质细胞产生过多的促炎细胞因子和一氧化氮（NO）在神经退行性疾病中起作用。为了研究神经保护剂N-乙酰-O-甲基多巴胺（NAMDA）是否下调与小胶质细胞活化相关的基因，我们在存在或不存在NAMDA的情况下，测量了脂多糖（LPS）刺激的小胶质细胞中肿瘤坏死因子-α（TNF-α）、白细胞介素-1β（IL-1β）、诱导型一氧化氮合酶（NOS2）以及相关辅助因子合成基因鸟苷三磷酸环化水解酶I（GTPCH）的基因表达。细胞因子基因表达的时间模式表明，LPS（0.2微克/毫升）在1小时和3小时增加TNF-α和IL-1β基因表达，而NAMDA共处理可抑制这种增加。同样，LPS在3小时和6小时也诱导GTPCH和NOS2基因表达，NAMDA共处理可抑制这种诱导，并同时减少一氧化氮的氧化代谢产物亚硝酸盐。由于转录因子核因子κB（NF-κB）参与调节这些基因的表达，因此研究了NAMDA对免疫刺激的小胶质细胞中NF-κB核转位和DNA结合的影响。我们发现，在BV-2小胶质细胞中，NAMDA共处理既不影响LPS诱导的NF-κB转位，也不影响其DNA结合活性。另一方面，NAMDA增加细胞内cAMP水平，并增强LPS诱导的磷酸化cAMP反应元件结合蛋白（pCREB）表达。用环磷腺苷酸3',5'-环硫代磷酸酯（一种cAMP依赖性蛋白激酶（PKA）的特异性抑制剂）处理，不仅逆转了NAMDA诱导的pCREB上调，还逆转了NAMDA诱导的TNF-α和IL-1β基因转录抑制。数据表明，NAMDA通过cAMP依赖性蛋白激酶途径抑制LPS诱导的促炎细胞因子基因表达。因此，NAMDA抑制活化小胶质细胞中的促炎细胞因子和NOS2基因表达可能为缺血性脑病以及其他神经退行性疾病提供新的治疗策略。
