昼夜节律关键脑区褪黑素及其受体在黑质-纹状体多巴胺神经元毁损中的变化及机制研究

Several published articles have documented that melatonin effectively protects against the degeneration of dopaminergic neurons in PD models. However, there are conflicting reports suggesting the negative effects of melatonin recently. For example, melatonin exacerbates 6-OHDA-mediated motor impairments and cannot work as a universal remedy, and we previously reported that the concentrations of melatonin at day and night in both eyes were distinctly increased compared with the vehicle groups in rat model of advanced PD. Similarly, the level of melatonin in the striatum dialysates of same PD model was increased as well. Therefore, we proposed that melatonin might play dual roles in PD progression. In the following study, brain microdialysis combined with HPLC method will be performed to investigate dynamic changes of melatonin and dopamine in the striatum dialysates during the course of 6-OHDA induced PD rats. We also will assess melatonin tissue contents in the retina, suprachiasmatic nucleus and pineal. After applying exogenous melatonin and melatonin antagonists, the distribution and changes of melatonin receptors in above mentioned tissues will detected by RT-PCR, Western blot and immunochemistry during the degeneration of dopaminergic neurons in midbrain. The results will contribute to the understanding of pathogenic mechanisms of PD and to provide new treatment strategies or discovery a new biomarker in different stage of PD.

研究显示褪黑素可保护帕金森病（PD）动物黑质多巴胺细胞，但近有报道，褪黑素可加剧PD的运动症状，且临床褪黑素治疗PD效果欠佳，提示褪黑素很可能在PD进展中发挥双重作用。本组前期研究发现，中晚期PD大鼠纹状体透析液、视网膜组织及PD患者血清褪黑素的昼夜水平均明显高于对照组，但机制未明。拟建立6-OHDA毁损黑质-纹状体多巴胺神经元大鼠，以褪黑素与多巴胺的关系为主线，以昼夜节律系统的关键部位视网膜、视交叉上核、松果体为切入点，在原有研究的基础上，加用外源性褪黑素及其受体拮抗剂、多巴胺受体激动剂和拮抗剂，探讨多巴胺细胞毁损大鼠褪黑素水平升高机制。用神经生化、分子生物学、形态学和影像学等方法，检测上述组织褪黑素及其受体随多巴胺细胞毁损程度的变化，并与血清褪黑素比较，探讨褪黑素和多巴胺的平衡在中脑多巴胺细胞毁损中的作用。旨在从昼夜节律系统了解PD发病机制、寻找潜在PD治疗靶点或干预措施提供理论依据。

帕金森病（PD）主要以中脑黑质多巴胺能神经元丧失，致其投射至纹状体的多巴胺减少。有证据提示，多巴胺与昼夜节律系统相关。本组前期研究发现，PD病人血清或中晚期PD大鼠脑内褪黑素水平升高，提示褪黑素可能在PD进程中发挥双重作用。由于褪黑素可抑制视网膜和纹状体多巴胺的释放，故本课题旨在探讨褪黑素和多巴胺之间的平衡在PD进程中的作用及可能的机制。.用6-OHDA毁损右侧前脑内侧束建立PD大鼠模型，采用脑微透析术和HPLC动态监测不同毁损程度大鼠纹状体内多巴胺和褪黑素的变化，同时比较视网膜、中脑黑质和血清的褪黑素水平。加用褪黑素受体激动剂（AG）和拮抗剂（LU）、以及多巴胺D2受体的激动剂（Quin）和拮抗剂（RA），了解PD大鼠褪黑素与多巴胺之间的关系，SPECT/CT检测不同毁损程度大鼠纹状体内多巴胺转运蛋白（DAT）的变化，Western blot检测大鼠纹状体褪黑素受体MEL-1A/B和DAT的表达量。利用6-OHDA建立的PD细胞及动物模型，探讨镁及镁离子转运体SLC41A1在PD进程的作用。.结果显示，在6-OHDA诱导的PD大鼠，褪黑素水平升高与PD的进程相关，PD大鼠纹状体DAT含量随着病程减少。大鼠脑内单侧注射6-OHDA除了导致毁损侧黑质多巴胺神经元减少外，还可引起毁损脑对侧眼组织多巴胺神经元减少；毁损脑对侧及同侧眼组织褪黑素水平也有不同程度的增加，尤以对侧为著。多巴胺D2受体激动剂可降低PD大鼠血清褪黑素水平，而多巴胺D2受体拮抗剂可增加PD大鼠血清褪黑素水平。褪黑素受体激动剂和拮抗剂可通过调节体内多巴胺与褪黑素的平衡对6-OHDA损伤的神经元细胞起到一定的保护作用。PD大鼠纹状体镁离子转运体SLC41A1表达降低，MgSO4可改善PD大鼠的运动症状，并上调SLC41A1的表达。该研究结果对临床PD的治疗具有一定的指导意义。