Guo Mengdi, Cao Qian, Xia Shengnan, Cao Xiang, Chen Jian, Qian Yi, Bao Xinyu, Xu Yun
Department of Neurology, Nanjing Drum Tower Hospital, Affiliated Hospital of Medical School, Nanjing University, Nanjing 210008, Jiangsu Province, China.
Jiangsu Key Laboratory for Molecular Medicine, Medical School of Nanjing University, Nanjing 210008, Jiangsu Province, China.
J Transl Int Med. 2023 Jul 5;11(2):156-168. doi: 10.2478/jtim-2023-0090. eCollection 2023 Jul.
Overactivated glial cells, especially microglia, are core components in the progression of pathologic neuroinflammation, and the application of anti-inflammatory reagents has been regarded as a potential therapy in the management of infarction/reperfusion (I/R) brain injury. This research aims to clarify the anti-inflammatory efect of a novel lipophilic compound N-(2-[4-tert-butylphenyl]-2-[pyrrolidine-1-yl]ethyl)-7-methyl-4-oxo-4H-chromene-2-carboxamide (named CP-07 in this study) in LPS-stimulated BV2 cell line and primary mouse microglia, and its therapeutic effect on I/R brain injury.
Cell Counting Kit-8 assay was used to determine the maximal nontoxic dose of CP-07. The mRNA levels of representative proinflammatory cytokines were determined by quantitative real-time polymerase chain reaction both and . TTC staining was performed to calculate infarct volumes while behavioral tests were used to assess the neurological deficits at 24 h after middle cerebral artery occlusion (MCAO). Flow cytometry analysis and immunofluorescence staining were performed to calculate the percentage of pro-inflammatory microglia .A selective JAK2/STAT3 pathway inhibitor, AG490 was used to block STAT3 phosphorylation before the CP-07 anti-inflammation tests .
CP-07 could effectively suppress the mRNA levels of IL-6, IL-1β, iNOS and TNF-α induced by lipopolysaccharide (LPS) , and markedly block the evaluation of the fluorescence intensity of Iba-1 in primary mouse microglia. In middle cerebral arteryocclusion models, intraperitoneal injection with 1 mg/kg CP-07 significantly reduced cerebral infarct volumes at 24 h after surgery compared with vehicle treatment group, and promoted the recovery of neurological functions in MCAO mice. Further studies validated that CP-07 administration reduced the percentage of CD86 positive microglia after I/R injury, and the expression level of p-STAT3 was also markedly reduced in both microglial cells and the penumbra tissues. Blocking STAT3 phosphorylation with AG490 could completely eliminate the anti-inflammatory effects of CP-07, at least .
We showed that a newly synthesized compound, CP-07, could effectively reduce the inflammatory responses in LPS-stimulated BV2 cells and primary mouse microglia, and overproduction of cytokines in middle cerebral artery occlusion mouse models by inhibiting STAT3 phosphorylation, leading to a neuroprotective effect on I/R brain injury.
过度活化的胶质细胞，尤其是小胶质细胞，是病理性神经炎症进展的核心组成部分，抗炎试剂的应用已被视为治疗脑梗死/再灌注（I/R）损伤的一种潜在疗法。本研究旨在阐明新型亲脂性化合物N-(2-[4-叔丁基苯基]-2-[吡咯烷-1-基]乙基)-7-甲基-4-氧代-4H-色烯-2-甲酰胺（本研究中命名为CP-07）对脂多糖（LPS）刺激的BV2细胞系和原代小鼠小胶质细胞的抗炎作用及其对I/R脑损伤的治疗效果。
采用细胞计数试剂盒-8法测定CP-07的最大无毒剂量。通过定量实时聚合酶链反应测定代表性促炎细胞因子的mRNA水平。进行TTC染色以计算梗死体积，同时在大脑中动脉闭塞（MCAO）后24小时采用行为学测试评估神经功能缺损。进行流式细胞术分析和免疫荧光染色以计算促炎小胶质细胞的百分比。在CP-07抗炎试验前，使用选择性JAK2/STAT3通路抑制剂AG490阻断STAT3磷酸化。
CP-07能有效抑制脂多糖（LPS）诱导的IL-6、IL-1β、诱导型一氧化氮合酶（iNOS）和肿瘤坏死因子-α（TNF-α）的mRNA水平，并显著阻断原代小鼠小胶质细胞中离子钙接头蛋白1（Iba-1）荧光强度的升高。在大脑中动脉闭塞模型中，与溶剂处理组相比，腹腔注射1mg/kg CP-07在术后24小时显著减小脑梗死体积，并促进MCAO小鼠神经功能的恢复。进一步研究证实，CP-07给药降低了I/R损伤后CD86阳性小胶质细胞的百分比，并且小胶质细胞和半暗带组织中p-STAT3的表达水平也显著降低。用AG490阻断STAT3磷酸化至少可完全消除CP-07的抗炎作用。
我们发现新合成的化合物CP-07可通过抑制STAT3磷酸化有效减轻LPS刺激的BV2细胞和原代小鼠小胶质细胞中的炎症反应，以及大脑中动脉闭塞小鼠模型中细胞因子的过度产生，从而对I/R脑损伤产生神经保护作用。
