Zhang Ting, Gong Xiaoli, Hu Guanzheng, Wang Xiaomin
Department of Neurobiology, Capital Medical University, Beijing Institute for Brain Disorders and Key Laboratory for Neurodegenerative Disorder, Ministry of Education, No. 10 Xitoutiao, Youanmenwai, Fengtai District, Beijing, 100069, China.
Department of Physiology, Capital Medical University, No. 10 Xitoutiao, Youanmenwai, Fengtai District, Beijing, 100069, China.
J Neuroinflammation. 2015 Mar 14;12:50. doi: 10.1186/s12974-015-0275-y.
Microglia are key players for the inflammatory responses in the central nervous system. Suppression of microglial activation and the resulting production of proinflammatory molecules are considered a promising strategy to alleviate the progression of neurodegenerative disorders. Triptolide was demonstrated as a potent anti-inflammatory compound both in vitro and in vivo. The present study explored potential signal pathways of triptolide in the lipopolysaccharide (LPS)-induced inflammatory response using primary rat microglial cells.
Microglial cells were pretreated with triptolide and stimulated with LPS. To investigate the anti-inflammatory effect of triptolide, we used Griess reagent and Western blot for NO release and iNOS expression, respectively. Moreover, we applied microglia-conditioned medium to neuronal cells and used the MTS assay to test cell viability. We found that triptolide inhibited LPS-induced NO and iNOS synthesis in microglial cells, which in turn protected neurons. To evaluate the involvement of the EP2 pathway, we used real-time PCR and Western blot to determine EP2 expression. We found that LPS induced a large increase in EP2 expression in microglia, and triptolide almost completely inhibited LPS-induced EP2 expression. Using the selective EP2 agonist butaprost and the EP2 antagonist AH6809, we determined that triptolide inhibited LPS-stimulated NO production in microglia mainly through the EP2 pathway. Additionally, by further treating triptolide-treated microglia with the downstream PKA-specific activator 6-Bnz-cAMP or the Epac-specific activator 8-pCPT-2-O-Me-cAMP, we found that 6-Bnz-cAMP but not 8-pCPT-2-O-Me-cAMP increased NO production in triptolide-LPS treated microglia. These results indicate that the EP2-PKA pathway is very important for triptolide's effects.
Triptolide inhibits LPS-stimulated NO production in microglia via a signaling mechanism involving EP2 and PKA. This finding may help establish the pharmacological function of triptolide in neurodegenerative disorders. Moreover, the observation of inflammatory EP2 signaling in primary microglia provides important evidence that EP2 regulates innate immunity in the central nervous system.
小胶质细胞是中枢神经系统炎症反应的关键参与者。抑制小胶质细胞活化及由此产生的促炎分子被认为是缓解神经退行性疾病进展的一种有前景的策略。雷公藤内酯醇在体外和体内均被证明是一种有效的抗炎化合物。本研究使用原代大鼠小胶质细胞探索了雷公藤内酯醇在脂多糖（LPS）诱导的炎症反应中的潜在信号通路。
小胶质细胞先用雷公藤内酯醇预处理，然后用LPS刺激。为研究雷公藤内酯醇的抗炎作用，我们分别使用格里斯试剂和蛋白质免疫印迹法检测一氧化氮（NO）释放和诱导型一氧化氮合酶（iNOS）表达。此外，我们将小胶质细胞条件培养基应用于神经元细胞，并使用MTS法检测细胞活力。我们发现雷公藤内酯醇抑制小胶质细胞中LPS诱导的NO和iNOS合成，进而保护神经元。为评估EP2信号通路的参与情况，我们使用实时定量聚合酶链反应（qPCR）和蛋白质免疫印迹法测定EP2表达。我们发现LPS诱导小胶质细胞中EP2表达大幅增加，而雷公藤内酯醇几乎完全抑制LPS诱导的EP2表达。使用选择性EP2激动剂布他前列素和EP2拮抗剂AH6809，我们确定雷公藤内酯醇主要通过EP2信号通路抑制LPS刺激的小胶质细胞中NO的产生。此外，通过用下游PKA特异性激活剂6-苄基-cAMP或Epac特异性激活剂8-对氯苯硫基-2'-O-甲基-cAMP进一步处理经雷公藤内酯醇处理的小胶质细胞，我们发现6-苄基-cAMP而非8-对氯苯硫基-2'-O-甲基-cAMP增加了经雷公藤内酯醇-LPS处理的小胶质细胞中NO的产生。这些结果表明EP2-PKA信号通路对雷公藤内酯醇的作用非常重要。
雷公藤内酯醇通过涉及EP2和PKA的信号机制抑制LPS刺激的小胶质细胞中NO的产生。这一发现可能有助于确立雷公藤内酯醇在神经退行性疾病中的药理功能。此外，在原代小胶质细胞中观察到炎症性EP2信号传导提供了重要证据，表明EP2调节中枢神经系统的固有免疫。
