neuritin抑制神经炎症改善糖尿病中枢神经病变的作用机制研究

Our being studied NSFC project showed that reversing the down-regulated neuritin expression in brain improved central neuropathy of diabetic rats. But the protective mechanism of neuritin on diabetic central neuropathy is still unclear. Our recent studies showed that neuritin suppressed TRAF6/NF-κB signaling pathway to inhibit lipopolysaccharide-induced microglial inflammation. So we speculate: neuritin regulates TRAF6/NF-κB signaling pathway to improve diabetic central neuropathy. In the project, we will construct lentiviral expression vectors of neuritin and mice with brain-specific knockout neuritin, and analyze the impact of overexpression and knockout of neuritin in brain on diabetic central neuropathy and neuroinflammation in vivo; the technologies, such as Western blotting, real-time PCR and EMSA, are used to investigate the effect of neuritin on microglia activation through TRAF6/NF-κB signaling pathway in microglia-neuron coculture. This project will provide experimental basis that neuritin regulates signaling pathway of microglial inflammation to inhibit neuroinflammation and to improve diabetic central neuropathy.

我们前期研究证明恢复糖尿病大鼠脑组织中neuritin水平可改善中枢神经病变。但其作用机制尚未阐明。新近实验证实，neuritin可抑制TRAF6/NF-κB信号通路拮抗脂多糖诱导的小胶质细胞炎症反应。推测neuritin可能通过调控该信号通路改善糖尿病中枢神经病变。本项目拟构建脑组织特异性高表达neuritin的转基因大鼠和特异性敲除neuritin小鼠，在体分析脑组织高表达和敲除neuritin对糖尿病中枢神经病变和炎症反应的影响；体外使用neuritin刺激小胶质细胞和小胶质细胞与神经细胞共培养，采用Western blotting、real-time PCR、EMSA等技术分析neuritin通过TRAF6/NF-κB信号通路对小胶质细胞活化的影响。本项目将为neuritin调控小胶质细胞炎症信号通路以抑制神经炎症反应来改善糖尿病中枢神经病变提供实验依据。

糖尿病中枢神经病变的发病率和致残率高，临床疗效并不理想，发病机制有待阐明。已结题青年项目（编号81100597）证明恢复糖尿病大鼠脑组织中神经营养因子neuritin水平可改善中枢神经病变。本项目采用CMV-Loxp-Stop-Loxp系统构建高表达neuritin小鼠，与海马特异性CaMKII-Cre小鼠杂交获转基因小鼠，再与db/m小鼠繁殖获得高表达neuritin的2型糖尿病（db/db）小鼠；采用Loxp-Stop-Loxp系统构建敲除neuritin小鼠，与CaMKII-Cre小鼠杂交获敲除小鼠，再与db/m小鼠杂交获得海马特异性敲除neuritin的db/db小鼠，观察海马高表达或敲除neuritin对糖尿病中枢神经病变和炎症反应的影响；体外使用siRNA干扰neuritin或重组neuritin，观察是否通过TRAF6/NF-κB信号通路影响小胶质细胞的炎症反应及神经细胞的轴突数量和长度。mRNA和蛋白表达检测证实成功获海马特异性高表达neuritin和敲除neuritin的db/db小鼠。与基础饲料喂养的db/m小鼠比较，高表达neuritin和NF-κB抑制剂治疗4周均显著改善了高脂饲料喂养的db/db小鼠的躯体感觉诱发电位和神经传导速度，降低血糖，改善海马超微病理结构，下调血清、海马中促炎症因子水平和上调血清、海马中抗炎症因子水平，但敲除neuritin有加剧db/db小鼠的上述神经病变和炎症反应的趋势。除证实已发现的神经细胞表达neuritin外，首次证明neuritin也表达于小鼠小胶质细胞和小胶质细胞株BV2细胞。与db/m小鼠比较，高表达neuritin和NF-κB抑制剂均明显抑制db/db小鼠海马小胶质细胞的活化，抑制海马（特别是小胶质细胞）中TRAF6/NF-κB信号通路的激活。但敲除neuritin有进一步激活db/db小鼠海马小胶质细胞及其炎症信号通路的趋势。离体实验证实，neuritin通过neuralized抑制脂多糖诱导BV2细胞的炎症反应，并增加了脂多糖刺激的神经细胞轴突数量和长度；neuralized通过neuritin的p75受体调控脂多糖刺激BV2细胞中TRAF6/NF-κB通路的激活。本项目将为neuritin调控小胶质细胞TRAF6/NF-κB炎症信号通路以抑制神经炎症反应来改善糖尿病中枢神经病变提供实验依据。