Zhang Chunyuan, Xing Zeting, Tan Meiyun, Wu Yanwen, Zeng Wei
Department of Anesthesiology, the Affiliated Boai Hospital of Zhongshan, Southern Medical University, Zhongshan, Guangdong 528403, China.
ACS Omega. 2021 Feb 2;6(6):4167-4174. doi: 10.1021/acsomega.0c04799. eCollection 2021 Feb 16.
: Astrocyte-mediated neuroinflammation plays an important role in anesthetic isoflurane-induced cognitive impairment. Roflumilast, a selective inhibitor of phosphodiesterase-4 (PDE-4) used for the treatment of chronic obstructive pulmonary disease (COPD), has displayed a wide range of anti-inflammatory capacity in different types of cells and tissues. In the current study, we aimed to investigate whether roflumilast possesses a protective effect against isoflurane-induced insults in mouse primary astrocytes. : Primary astrocytes were isolated from the cerebral cortices of immature rats. The production of NO was determined using DAF-FM DA staining assay. QRT-PCR and western blot were used to evaluate the expression levels of iNOS, COX-2, and BDNF in the astrocytes treated with different therapies. The gene expressions and concentrations of IL-6 and MCP-1 released by the astrocytes were detected using qRT-PCR and ELISA, respectively. The expression levels of phosphorylated CREB and PGE were determined using western blot and ELISA, respectively. H89 was introduced to evaluate the function of CREB. Recombinant human BDNF and ANA-12 were used to verify the role of BDNF. : The upregulated iNOS, excessive production of NO, IL-6, and MCP-1, and activated COX-2/PGE signaling pathways in the astrocytes induced by isoflurane were significantly reversed by the introduction of roflumilast, in a dose-dependent manner. Subsequently, we found that BDNF could be upregulated by roflumilast, which was verified to be related to the activation of CREB and blocked by H89 (a CREB inhibitor). In addition, the COX-2/PGE signaling pathway activated by isoflurane can be inactivated by recombinant human BDNF. Finally, the regulatory effect of roflumilast against the isoflurane-activated COX-2/PGE signaling pathway was significantly blocked by ANA-12, which is a BDNF inhibitor. : Roflumilast might ameliorate isoflurane-induced inflammation in astrocytes the CREB/BDNF signaling pathway.
星形胶质细胞介导的神经炎症在麻醉药异氟烷诱导的认知功能障碍中起重要作用。罗氟司特是一种用于治疗慢性阻塞性肺疾病（COPD）的磷酸二酯酶-4（PDE-4）选择性抑制剂，在不同类型的细胞和组织中显示出广泛的抗炎能力。在本研究中，我们旨在探讨罗氟司特是否对异氟烷诱导的小鼠原代星形胶质细胞损伤具有保护作用。
原代星形胶质细胞从新生大鼠的大脑皮层中分离出来。使用DAF-FM DA染色法测定一氧化氮（NO）的产生。实时定量聚合酶链反应（QRT-PCR）和蛋白质免疫印迹法用于评估不同处理的星形胶质细胞中诱导型一氧化氮合酶（iNOS）、环氧化酶-2（COX-2）和脑源性神经营养因子（BDNF）的表达水平。分别使用qRT-PCR和酶联免疫吸附测定（ELISA）检测星形胶质细胞释放的白细胞介素-6（IL-6）和单核细胞趋化蛋白-1（MCP-1）的基因表达和浓度。分别使用蛋白质免疫印迹法和ELISA测定磷酸化环磷腺苷反应元件结合蛋白（CREB）和前列腺素E（PGE）的表达水平。引入H89以评估CREB的功能。使用重组人BDNF和ANA-12验证BDNF的作用。
罗氟司特的引入以剂量依赖性方式显著逆转了异氟烷诱导的星形胶质细胞中iNOS上调、NO、IL-6和MCP-1的过量产生以及COX-2/PGE信号通路的激活。随后，我们发现罗氟司特可上调BDNF，这被证实与CREB的激活有关，并被H89（一种CREB抑制剂）阻断。此外，重组人BDNF可使异氟烷激活的COX-2/PGE信号通路失活。最后，ANA-12（一种BDNF抑制剂）显著阻断了罗氟司特对异氟烷激活的COX-2/PGE信号通路的调节作用。
罗氟司特可能通过CREB/BDNF信号通路改善异氟烷诱导的星形胶质细胞炎症。
