Nr4a2在抑郁大鼠海马工作记忆损害中的作用机制研究

Cognitive symptoms, one of the most important symptoms of depression, affect the recovery and prognosis of the depressive patients. Related to the mechanism of cognitive dysfunction of depression disorders, it shows that the transcription factor Nr4a2 directly participates in the regulation of memory formation, storage and transformation, as well as the function of the hippocampus working memory circuit; Nr4a2 gene knockout mice (Nr4a2+/-) exhibit symptoms of working memory impairment and depression-like behavior, and learning and overexpression of Nr4a2 in hippocampus can reverse the symptoms mentioned above, suggesting that down-regulated expression of Nr4a2 may be involved in the cognitive function of depression disorders especially in the regulation of impaired working memory in hippocampus. This study intends to adopt male Sprague-Dawley rats to establish the chronic unpredictable mild stress animal model of depression, using the method of RNA interference (RNAi), gene overexpression and brain stereotaxic technique to target at directional regulation of Nr4a2 expression in specific hippocampus, combined with the intervention of different mechanism of antidepressants (Fluoxetine, Agomelatine and Vortioxetine), and together with animal behavior test, immunohistochemistry, RT-PCR and DNA microarray technology, et al techniques, to research the role of Nr4a2 played in the mechanism of impaired working memory in hippocampus and to explore the effective intervention for cognitive dysfunction of depression disorders. The research may provide scientific basis for further study the mechanism of cognitive dysfunction in depression, and may be beneficial to choose the effective intervention drug in future.

认知症状是抑郁症核心症状之一，影响患者康复和预后。有关抑郁症认知功能损害的发生机制，研究发现转录因子Nr4a2直接参与调控记忆的形成、存储与转化过程，影响海马工作记忆回路功能；Nr4a2+/-小鼠表现出工作记忆受损和抑郁样行为，而学习与海马过表达Nr4a2能逆转上述现象，提示Nr4a2表达失调可能涉及抑郁症认知功能尤其是海马工作记忆损害的调控过程。本研究拟采用雄性Sprague-Dawley大鼠构建慢性不可预知温和应激抑郁动物模型，运用Nr4a2 RNA干扰(RNAi)或过表达以及脑立体定位技术定向调控海马Nr4a2表达，结合不同机制抗抑郁药(氟西汀、阿戈美拉汀和伏硫西汀)干预，采用动物行为学、免疫组织化学、RT-PCR和高通量基因表达谱分析等技术，研究Nr4a2在抑郁大鼠海马工作记忆损害中的作用机制。本研究将为进一步阐明抑郁症认知功能损害发生机制及探索干预药物优化选择提供科学依据。

转录因子Nr4a2（又称Nurr1）直接参与调控记忆的形成、存储与转化过程，Nr4a2还经由Bdnf、Leptin和Fndc5/Irisin参与了能量调节相关过程，Nr4a2+/-小鼠表现出工作记忆受损和抑郁样行为，提示Nr4a2表达失调可能涉及抑郁症(Major depressive disorder, MDD)认知功能损害的调控过程。本课题项目开展了如下研究：1.探索Nr4a2在健康C57BL/6小鼠脑内的表达。结果发现，Nr4a2蛋白主要在前额叶、内嗅皮层、黑质、下丘脑后部、僵核和内嗅皮层表达比较多，尤其是内嗅皮层和黑质、僵核部位表达丰度最高。而海马仅下托有少量表达，海马其他部位及其他脑区均无表达；2. 优化并证实了成功构建CUMS抑郁小鼠模型的方法。采用雄性C57BL/6小鼠，分别进行4周、6周和8周的慢性不可预知温和应激（CUMS）刺激，结合强迫游泳实验、悬尾实验、蔗糖偏好实验及旷场实验，发现CUMS刺激8周为最佳，抑郁小鼠表现出明显的快感缺失和绝望状态，且焦虑行为不显著，提示经过优化刺激模式后进行8周的CUMS刺激，可成功构建CUMS抑郁小鼠模型；3. 探索基线CUMS抑郁模型小鼠Nr4a2及其相关信号蛋白（Otx2, Gsk3Β和Bdnf）、能量代谢相关信号蛋白（Leptin、Pgc1α和Fndc5/Irisin）的表达情况。结果表明，与对照小鼠相比，基线CUMS抑郁模型小鼠的Nr4a2及相关蛋白无显著变化，而海马、前额叶、肝脏的Leptin和Fndc5/Irisin蛋白表达水平显著升高，提示Nr4a2及其相关信号通路可能与抑郁症的病理机制发生并不相关，但能量相关蛋白可能参与了抑郁症的病理过程；4. 探索了不同机制抗抑郁药物艾司西酞普兰（ESC）和伏硫西汀（VOT）干预对CUMS抑郁小鼠的行为学、体重的影响，及其对Nr4a2及相关蛋白、能量代谢相关蛋白表达的影响。结果显示，ESC和VOT干预对小鼠的抑郁及认知功能发生显著变化，但Nr4a2及相关蛋白的表达并未发生显著改变，而随着药物剂量增加，Fndc5/Irisin呈下调趋势，而Pgc1α呈下调趋势，目前仍在扩大样本量确认。综上，本研究证实Nr4a2及其相关信号蛋白并未直接参与调控抑郁症认知功能改变的过程，但本研究结果为能量相关蛋白可能参与抑郁症的病理生理过程提供了直接的实验证据。