炎症性痛觉睡眠障碍及机制研究

Patients with inflammation or inflammatory diseases always experience pain and/or hyperalgesia accompanied by sleep disorders. However, the mechanism of inflammatory pain-induced sleep disorders is unclear and there's no effective treatment for these sleep disorders in clinic. Studies show that dopamine D2 receptor on nucleus accumbens (NAc) neurons plays an important role in central pain transmission and regulation. While, our previous work has proved that dopamine in NAc is an essential neurotransmitter in sleep-wake regulation and the D2 receptor activation plays a key role in the maintenance of wakefulness. Dopamine D2 receptor belongs to G protein coupled receptor. It is regulated by G protein coupled receptor kinase 2 (GRK2), which is widely expressed in the nerve system. GRK2 belongs to a family of GRKs. It can regulate homologous desensitization of a wide array of G protein-coupled receptors (GPCR). Such GPCR desensitization by GRK2 protects cells against overstimulation. Conversely, a reduced level of GRK2 can enhance cell signaling. GRK2 not only regulates cellular signaling at the level of GPCRs, but also can directly interact with intracellular signaling molecules. In our previous work we have identified nociceptor G protein-coupled-receptor kinase 2 (GRK2) as a novel regulator of inflammatory hyperalgesia. These results suggest that the mechanism of inflammatory pain-induced sleep disorder is probably due to the GRK2 regulating D2 receptor in NAc. We propose that with inflammatory pain, the level of GRK2 in NAc neurons decreases, resulting in the reduced D2 receptor desensitization, the D2 receptor becomes more sensitive to agonist, leading to sleep disorders such as insomnia. To test this hypothesis, in the present study we will first evaluate the GRK2 level in NAc after different inflammation models. At the same time, we will analyze the time course of the sleep disorders after inflammation by recording the EEG, comparing it with the changes of GRK2. To further prove the relationship between the changes of GRK2 and sleep disorders after inflammation, we will construct lentiviral gene vector with GRK2 siRNA, inject it into NAc to produce continuously reduced expression of GRK2 in NAc neurons. The EEG of the mice is then recorded. To investigate whether the GRK2 regulate sleep via D2 receptor, we will also use D2 receptor gene knockout mice to test if GRK2 siRNA injection will produce sleep disorder in these D2 receptor knockout mice.

炎症性痛觉的病人伴有明显的睡眠障碍，但机制未明，故缺乏有效的防治手段。伏隔核多巴胺D2受体在中枢痛觉传导中有重要作用。本课题组研究发现伏隔核的多巴胺是调节睡眠-觉醒的重要递质，D2受体的激活对觉醒维持起到关键作用。D2受体属G蛋白偶联受体，受G蛋白偶联受体激酶2(GRK2)的调节。我们研究证明神经元胞体GRK2蛋白水平持续下调是慢性炎症性痛觉发生的关键。这些结果提示炎症性痛觉睡眠障碍发生的机制可能与伏隔核GRK2调节D2受体有关。我们假设在炎症性痛觉时伏隔核神经元胞内GRK2蛋白水平降低，使伏隔核中的D2受体去敏化被抑制，受体对激动剂的反应性增加，导致失眠等睡眠障碍的发生。本研究将利用D2受体基因敲除小鼠、慢病毒基因载体抑制特定脑区伏隔核GRK2蛋白表达等手段，研究炎症性痛觉时伏隔核GRK2水平的改变对睡眠-觉醒调节的影响，并进一步探索GRK2和D2受体在炎症性痛觉睡眠障碍中的作用及机制。

本课题我们首先研究了小鼠炎症痛模型和痛觉敏化模型中，睡眠障碍的表现形式。结果发现，2%角叉菜胶引起的进行性炎症，不影响睡眠觉醒的总时间，但可导致明显的睡眠片段化。模型小鼠小于16秒的觉醒片段（brief awakening）数量明显增加。中枢H1受体阻断剂Pyrilamine能明显减少这种短暂的觉醒片段，但不影响小鼠痛阈。提示中枢组胺对持续性炎性痛引起的睡眠障碍有改善作用。.痛觉敏化模型小鼠，在敏化期再次受到炎性介质EPI的刺激后，EPI引起的痛觉过敏时程明显延长。但小鼠的睡眠-觉醒时间和睡眠结构参数等均未有明显改变。提示痛觉敏化可能是改变了外周初级感觉神经元可塑性，而对中枢调控睡眠的神经元没有影响。.利用神经病理性疼痛模型发现小鼠坐骨神经结扎（PSL）导致的神经病理性疼痛可引起显著的睡眠障碍。普瑞巴林和钩吻碱甲均有镇痛作用。且两者白天给药可明显延长神经病理性疼痛模型小鼠的NREM睡眠时间，减少觉醒。而晚上给药则对小鼠睡眠觉醒没有影响。组化结果发现，PSL能增加模型小鼠前扣带回皮层(ACC)脑区神经元c-fos的表达。普瑞巴林和钩吻碱甲可以减少这种c-fos表达的增加，提示ACC可能是调节疼痛引起的睡眠障碍的重要脑区。.神经痛小鼠的痛阈值在一天中有规律的波动，这种波动与睡眠觉醒相关，提示了光照对疼痛和睡眠的影响。我们探索了不同的光照对疼痛和睡眠的影响，其中光照对疼痛的影响还在进一步深入研究中。而光照对睡眠的影响的研究发现，与传统理论不同，不仅白光和蓝光可以影响睡眠觉醒，传统上认为无法被小鼠视觉系统感知的红光也会影响小鼠的睡眠觉醒。该结果不仅丰富了睡眠觉醒调节和节律调节的理论，也为指导夜间动物行为学实验，避免不合理光照影响动物的睡眠觉醒，并获得稳定可靠的实验结果提供了依据。