Zhu Xiao-Yu, Guo Sheng-Ya, Xia Bo, Li Chun-Qi, Wang Lei, Wang Yong-Hui
State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing, Jiangsu Province 210009, China; Hunter Biotechnology, Inc, F1A, building 5, No. 88 Jiangling Road, Binjiang, Zone, Hangzhou City, Zhejiang Province 310051, China.
Hunter Biotechnology, Inc, F1A, building 5, No. 88 Jiangling Road, Binjiang, Zone, Hangzhou City, Zhejiang Province 310051, China.
J Pharmacol Toxicol Methods. 2019 Jul-Aug;98:106585. doi: 10.1016/j.vascn.2019.106585. Epub 2019 May 18.
RAR-related orphan receptor-γt (RORγt) directs differentiation of proinflammatory T helper 17 cells and is a potential therapeutic target for chronic autoimmune and inflammatory diseases including multiple sclerosis. In this study, zebrafish at days post fertilization treated with ethidium bromide (EB) at a concentration of 75 μM for 72 h were determined as the optimum conditions for the demyelination model development. Zebrafish motility was recorded automatically using a video-track motion detector and quantitative myelin assay was measured by FluoroMyelin staining. A well-known remyelination agent thyroxine (T4) was tested to confirm whether EB-induced motility and myelin damage could be rescued. Two RORγt lead inhibitors GSK805 and SR1001 were assessed for their therapeutic effects on remyelination, axon regeneration, motor neuron promotion and anti-inflammation. T4 significantly improved EB-induced motility dysfunction and myelin damage and promoted myelin basic protein (MBP) regeneration in the demyelinated zebrafish. GSK805 and SR1001 enhanced remyelination in a dose-dependent manner and promoted MBP regeneration. Both GSK805 and SR1001 markedly recovered EB-induced axon and motor neuron damage, and exhibited significantly inhibitory effects of neutrophil infiltration and macrophage recruitment. These results indicate that EB treatment can induce zebrafish demyelination; and the zebrafish demyelination model in combination with quantitative motility and myelin assays is a predictive, reproducible and relatively high throughput screening for rapidly in vivo identification of remyelination compounds and RORγt inhibitors.
维甲酸相关孤儿受体γt（RORγt）指导促炎性辅助性T细胞17的分化，是包括多发性硬化症在内的慢性自身免疫性和炎性疾病的潜在治疗靶点。在本研究中，将受精后用浓度为75μM的溴化乙锭（EB）处理72小时的斑马鱼确定为脱髓鞘模型建立的最佳条件。使用视频跟踪运动检测器自动记录斑马鱼的运动，并通过荧光髓磷脂染色测量定量髓磷脂测定。测试了一种著名的髓鞘再生剂甲状腺素（T4），以确认是否可以挽救EB诱导的运动和髓磷脂损伤。评估了两种RORγt先导抑制剂GSK805和SR1001对髓鞘再生、轴突再生、运动神经元促进和抗炎的治疗效果。T4显著改善了EB诱导的运动功能障碍和髓磷脂损伤，并促进了脱髓鞘斑马鱼中髓鞘碱性蛋白（MBP）的再生。GSK805和SR1001以剂量依赖性方式增强髓鞘再生并促进MBP再生。GSK805和SR1001均显著恢复了EB诱导的轴突和运动神经元损伤，并对中性粒细胞浸润和巨噬细胞募集表现出显著的抑制作用。这些结果表明，EB处理可诱导斑马鱼脱髓鞘；并且结合定量运动和髓磷脂测定的斑马鱼脱髓鞘模型是一种用于在体内快速鉴定髓鞘再生化合物和RORγt抑制剂的预测性、可重复且相对高通量的筛选方法。
