老年脑缺血时IL-4受体调节巨噬/小胶质细胞极化的作用及机制

Neuroinflammation plays an important role in the ischemic brain injury, microglia/macrophage are the main inflammatoy cells in the ischemic brain. Microglia/macrophage can be activated into classical activated (M1) or alternatively activated （M2）cells. M2 macrophages are considered to initiate inflammatory responses, in contrast M2 is characterized by neuro-protection. More evidence indicated that microglia/macrophage polarization can be modulated through different signaling pathway, IL-4/JAK/STAT6 signaling skews macrophage function toward the M2 phenotype. Our pilot data show that IL-1β/IL-6 is highly up-regulated and much more CD68+ cells were found in peri-infarct region in aged ischemic mice brain than that of young mice. Our data also suggest that an increased inflammatory response to acute ischemic injury in the aged mice leads to more severe brain damage and functional deficits. More evidence indicated that IL-4 receptor expression is reduced in the aging brain. Since IL-4 induces macrophage polarization towards M2, we hypothesize that deficiency of IL-4/JAK/STAT6 signaling pathway inhibits activated microglia/macrophage shifting towards an M2 phenotype, therefore induces more M1 phenotype. We will use middle cerebral artery oclution model in aged mice to show that the dynamic of microglial/macrophage polarization in aged ischemic brain. IL-4/JAK/STAT6 will be assessed to see wehther they are involved in the microglia/ macrophage polarization in the aged ischemic brain. Finaly to investigete wehther serere brain injury in aged ischemia is associated with macrglial/macrophage polarization.

神经炎性反应在缺血后脑损伤中起重要作用，巨噬/小胶质细胞是主要的炎性细胞，近来发现其激活后可产生经典激活型（M1）和替代激活型（M2），其中M1与炎性反应有关，而M2则具有神经保护作用。研究也发现上述极化受不同信号通路调控，IL-4/JAK/STAT6信号可促进M2的极化。我们的前期研究发现，与青年鼠相比老年鼠脑缺血后CD68+（M1标志）细胞、IL-1/IL-6等炎性因子增加，脑损伤也较为严重。已有报道老年鼠中IL-4R的表达缺陷，我们推测，老年鼠中IL-4/JAK/STAT6信号通路的缺陷抑制了M2的极化，从而导致M1细胞相应增多，其产生的炎性反应增加了脑组织的损伤。本项目将在老年鼠大脑中动脉栓塞模型中观察缺血后损伤局部M1、M2细胞的分布和数量，在此基础上，探讨老年鼠中IL-4/JAK/STAT6信号改变与巨噬/小胶质细胞M1极化和脑损伤间的关联，从而阐明老年脑缺血神经损伤的机制。

研究背景：研究表明老年患者脑缺血后其神经功能的损伤更严重，神经功能恢复更慢。炎性反应在脑卒中的发病机制中起重要作用。但其在老龄缺血性卒中时的作用及其机制仍不甚清楚。IL-4能诱导巨噬细胞向M2型极化，参与脑缺血后的炎性反应。本研究主要探讨IL-4信号在老龄脑缺血性中的作用及其可能机制。方法：选制备阻断远端大脑中动脉的小鼠脑缺血模型(MCAO)。检测脑梗死/脑萎缩的体积、用墙角试验和移除黏附物能力测试检测脑缺血前后的动物行为学。免疫荧光染色检测脑缺血周边区的CD68+阳性细胞、M1和M2细胞表型及神经元凋亡。ELISA检测缺血脑组织中的白介素-6（IL-6）,白介素-1β（IL-1β）和血管内皮生长因子(VEGF)水平。蛋白免疫印迹法检测紧密连接相关蛋白Claudin-5和ZO-1。伊文思蓝渗出法检测血脑屏障的渗透性。明胶酶谱法 检测MMP-9的表达。同时还检测促血管因子的表达，及CD31染色检测血管新生，Ki67/CD31共染色检测血管内皮增殖。通过骨髓来源的巨噬细胞和HUVEC共培养，观察糖氧剥夺后对IL-4和MI/M2表型表达的影响。这些结果提示老年小鼠脑缺血急性期的神经元凋亡严重，更明显的血脑屏障破坏及严重的脑损伤与急性期的炎性因子IL-1β和IL-6水平相关联。同时脑缺血急性期局部的炎性反应严重，恢复期炎症消退差。通过骨髓来源的巨噬细胞和HUVEC共培养，用糖氧剥夺模拟体内缺血，研究IL-4调节炎症的信号通路。进一步研究证明老龄小鼠缺氧后IL-4水平增高有限，促进STAT6的磷酸化可增加细胞M2表型的ARG和CD206的表达。我们的研究初步提示老年小鼠脑缺血后有更严重的炎性反应和血脑屏障破坏，神经功能恢复差。脑缺血后IL-4 信号通路受损可能与老龄脑严重的神经功能损伤有关。提示调节IL-4信号水平可能成为治疗老年脑卒中的新靶点。