Department of Ophthalmology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, Guangdong, China; Department of Ophthalmology, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou 510120, Guangdong, China.
Department of General Practice, Nanfang Hospital, Southern Medical University, Guangzhou 510515, Guangdong, China.
Int Immunopharmacol. 2023 Nov;124(Pt B):110952. doi: 10.1016/j.intimp.2023.110952. Epub 2023 Sep 24.
The abnormal polarisation of microglial cells (MGs) following retinal ischemia/reperfusion (RIR) initiates neuroinflammation and progressive death of retinal ganglion cells (RGCs), causing increasingly severe and irreversible visual dysfunction. Roflumilast (Roflu) is a promising candidate for treating neuroinflammatory diseases. This study aimed to explore whether Roflu displayed a cytoprotective effect against RIR-induced neuroinflammation and to characterise the underlying signalling pathway.
The effects and mechanism of Roflu against RIR injury were investigated in C57BL/6J mice and the BV2 cell line. We used quantitative real-time PCR and enzyme-linked immunosorbent assay to examine the levels of inflammatory factors. Furthermore, haematoxylin and eosin and immunofluorescence (IF) stainings were used to assess the morphology of the retina and the states of MGs and RGCs. Reactive oxygen species (ROS) levels were examined using a ROS assay kit, while whole-genome sequencing analysis was conducted to identify altered pathways and molecules. Western blotting and IF staining were used to quantify the proteins associated with the nuclear factor erythroid 2-related factor 2 (Nrf2)/stimulator of interferon gene (STING)/nuclear factor kappa beta (NF-κB) pathway.
MG polarisation includes the pro-inflammatory and neurotoxic M1 phenotype as well as the anti-inflammatory and neuroprotective M2 phenotype. Roflu significantly attenuated MG activation and contributed to a shift in the MG phenotype from M1 to M2. Moreover, Roflu decreased ROS release and increased heme oxygenase 1 and NAD(P)H quinone oxidoreductase 1 expression. In vitro and in vivo experiments validated that Roflu exerted its neuroprotective effects primarily by upregulating the Nrf2/STING/NF-κB pathway. However, these effects were abrogated when the Nrf2 expression was inhibited by pharmacological or genetic manipulation.
Roflu suppressed RIR-induced neuroinflammation by driving the shift of MG polarisation from M1 to M2 phenotype, which was mediated by the upregulation of the Nrf2/STING/NK-κB pathway.
视网膜缺血/再灌注（RIR）后小胶质细胞（MGs）的异常极化引发神经炎症和视网膜神经节细胞（RGCs）的进行性死亡，导致视力功能越来越严重和不可逆转的损伤。罗氟司特（Roflu）是一种有前途的治疗神经炎症性疾病的候选药物。本研究旨在探讨 Roflu 是否对 RIR 诱导的神经炎症具有细胞保护作用，并探讨其潜在的信号通路。
在 C57BL/6J 小鼠和 BV2 细胞系中研究了 Roflu 对 RIR 损伤的作用及其机制。我们使用定量实时 PCR 和酶联免疫吸附试验来检测炎症因子的水平。此外，使用苏木精和伊红以及免疫荧光（IF）染色来评估视网膜的形态以及 MGs 和 RGCs 的状态。使用 ROS 测定试剂盒检测活性氧（ROS）水平，同时进行全基因组测序分析以鉴定改变的途径和分子。使用 Western blot 和 IF 染色来定量与核因子红细胞 2 相关因子 2（Nrf2）/干扰素基因刺激物（STING）/核因子 kappa beta（NF-κB）通路相关的蛋白。
MG 极化包括促炎和神经毒性 M1 表型以及抗炎和神经保护 M2 表型。Roflu 显著减弱了 MG 的激活，并促进了 MG 表型从 M1 向 M2 的转变。此外，Roflu 减少了 ROS 的释放并增加了血红素加氧酶 1 和 NAD（P）H 醌氧化还原酶 1 的表达。体外和体内实验验证了 Roflu 主要通过上调 Nrf2/STING/NF-κB 通路发挥其神经保护作用。然而，当通过药理学或遗传操作抑制 Nrf2 表达时，这些作用被阻断。
Roflu 通过驱动 MG 极化从 M1 表型向 M2 表型转变来抑制 RIR 诱导的神经炎症，这是通过上调 Nrf2/STING/NK-κB 通路介导的。
