腺苷A2A受体诱导巨噬细胞M2极化在慢性低灌注脑白质损伤中的作用及机制

Inflammation play an important role in the white matter lesions induced by chronic cerebral hypoperfusion.It is focus on the modulation of inflammation to explore the treatment of ischemic white matter lesions.Our findings showed that activation of A2A receptor inhibited the inflammation, attenuated white matter lesions induced by chronic cerebral hypoperfusion.It is associated with the inhibition of macrophage activation in the white matter lesions induced by chronic cerebral hypoperfusion.Numerious studies have indicated that a switch of mcrophage from M1 to M2 phenotype promoted the synthesis of anti-inflammatory cytokines and inhibited the inflammation. We have found that there was a switch of mcrophage from M1 to M2 phenotype in white matter lesions induced by chronic cerebral hypoperfusion. Moreover, activation of A2A receptor promoted the macrophage M2 polarization. Therefore we believe that activation of A2A receptor promote macrophage M2 polarization and inhibit the inflammation in the white matter lesions induced by chronic cerebral hypoperfusion. However, the signal pathway remained uncleared. To investigate the signal pathway, time-lapse two-photon spectral confocal imaging and 2DLC-MS/MS was used to exam the role of macrophage M2 polarization in the anti-inflammatory effect of A2A receptor to the white matter lesions induced by chronic cerebral hypoperfusion. Furthermore, we also explore the signal pathway in the macrophage M2 polarization to supply the ideals to investigate the mechanism of white matter lesions induced by chronic cerebral hypoperfusion.

炎性反应在慢性低灌注脑白质损伤中具有重要作用。如何调节炎性反应减轻损伤是探索低灌注脑白质损伤治疗措施的关注点。我们发现腺苷通过A2A受体抑制炎性反应，减轻慢性低灌注脑白质损伤，其机制主要是通过抑制巨噬细胞向脑浸润活化实现的。已证实巨噬细胞M1向M2极化，促进抗炎因子合成，减轻炎性反应。预实验发现低灌注脑白质损伤区存在明显的巨噬细胞M1向M2极化现象，与髓鞘损伤程度负相关。鉴于A2A受体是诱导巨噬细胞M2极化的主要调控因素。因此我们推测A2A受体诱导巨噬细胞M2极化，抑制炎性反应，减轻低灌注脑白质损伤，但其作用机制尚不明确。为此，本项目拟通过临床血和脑脊液标本结合动物实验，采用双光子功能显微成像技术、蛋白质谱分析术等技术，从整体及细胞水平探讨A2A受体诱导巨噬细胞M2极化在慢性低灌注脑白质损伤中的作用，明确A2A受体诱导巨噬细胞M2极化的信号通路。为慢性低灌注脑白质损伤机制及治疗提供新思路。

炎性反应在慢性低灌注脑白质损伤中具有重要作用。如何调节炎性反应减轻损伤是探索低灌注脑白质损伤治疗措施的关注点。腺苷通过A2A受体抑制炎性反应，减轻慢性低灌注脑白质损伤，其机制主要是通过抑制巨噬细胞向脑浸润活化实现的。已证实巨噬细胞M1向M2极化，促进抗炎因子合成，减轻炎性反应。因此推测A2A受体诱导巨噬细胞M2极化，抑制炎性反应，减轻低灌注脑白质损伤，但其作用机制尚不明确。利用C57/BL6小鼠制作慢性低灌注脑白质损伤动物模型，在模型成功后3d，7d，14d，28d采用免疫荧光双标染色、荧光定量PCR检测M1和M2标记物的蛋白及mRNA表达，结果发现慢性低灌注状态下胼胝体区域活化的小胶质细胞/巨噬细胞有M1/M2的转化。随后我们采用腺苷A2A受体基因敲除小鼠制备慢性低灌注脑白质损伤模型，在在模型成功后3d，7d，14d，28d采用免疫荧光双标染色、免疫定量PCR检测M1和M2标记物的蛋白及mRNA表达，结果发现腺苷A2A受体基因敲除诱导慢性低灌注脑白质损伤区域活化的小胶质细胞/巨噬细胞向M1型转化。但其作用机制目前不清。我们采用GFP荧光蛋白小鼠作为供体构建骨髓细胞GFP+嵌合子小鼠，制作慢性低灌注脑白质损伤模型，结果发现慢性低灌注脑白质损伤模型的胼胝体区域有大量外周血巨噬细胞浸润，那么其作用有待进一步研究。我们利用腺苷A2A受体基因敲除小鼠做供体构建骨髓细胞腺苷A2A受体-/-小鼠，构建慢性低灌注脑白质损伤模型。结果发现骨髓来源细胞腺苷A2A受体基因敲除诱导小胶质细胞/巨噬细胞向M1型转化。培养巨噬细胞系RAW264.7，在低糖低氧培养基中培养1h，4h，12h，24h后采用免疫荧光双标染色、免疫定量PCR检测M1和M2标记物的蛋白及mRNA表达，结果发现M1标记物iNOS的表达明显增高，CD16和CD32的表达明显增高，Argine-1的表达下降。CD206的表达无明显差异。构建PPARγ的iRNA慢病毒载体，成功转染巨噬细胞，采用ELISA检测巨噬细胞在低糖低氧培养下炎性因子的表达，结果发现抑制PPARγ的表达显著降低了腺苷A2A受体介导的M2型巨噬细胞的诱导，降低了炎性因子的表达。

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