D3R调控伏隔核小胶质细胞活化影响抑郁样行为的作用及机制

Major depressive disorder (MDD) is the leading cause of worldwide disability and the most prevalent mood disorder. A robust body of data suggested nucleus accumbens (NAc) inflammation was likely involved in depressive-like behavior. Neverthless the mechanism and source of increased NAc inflammation and behavior disturbance in depression are not yet well understood. Our previous results not only found D3R was distributed in microglial cells in the NAc of normal mice, but also revealed that D3R deficiency resulted in microglial activation in the NAc and the depressive-like behavior trigged by D3R deficiency was alleviated by inhibition of microglial activation. In the present study, we will first employ RNA interference (RNAi) to knock down D3R protein levels in the NAc, and then use intracranial injection, behavior testing and RNA overexpression technique in order to study the role of D3R in the NAc mediated the depressive-like behavior; also, we will apply cell transfection, intracranial injection, molecular biology and immunology methods to explore whether or how D3R regulates microglial activation in in vivo and in vitro experiments, and the aim is to identify the role and relevant signaling pathway by which D3R regulates microglial activation and then affects depression-like behavior. This research will reveal the effects and mechanism of D3R in regulating microglia in the NAc and affecting depressive-like behavior, and provide potential molecular and cellular targets for the treatment of MDD.

伏隔核炎症反应在抑郁症发病过程中具有重要作用，但抑郁症中伏隔核炎症反应的发生机制尚不清楚。我们研究观察到，伏隔核小胶质细胞有D3R的表达，D3R缺失引起的抑郁样行为与伏隔核小胶质细胞活化有关。本项目拟构建D3R过表达、表达抑制载体，利用核团注射技术，观察在小鼠伏隔核中上调/下调D3R表达对小鼠抑郁样行为的作用；采用细胞转染、免疫荧光、免疫印迹、流式细胞、ELISA等检测技术，观察在细胞及动物水平上D3R表达上调/下调对小胶质细胞活化的影响，明确D3R调控伏隔核小胶质细胞活化进而参与抑郁样行为的发生；同时，在细胞及动物实验中下调D3R表达，通过抑制剂干预、siRNA等方法抑制D3R下游相关信号通路分子，确定D3R调控小胶质细胞活化的关键信号通路。以期揭示D3R通过调控伏隔核小胶质细胞活化介导伏隔核炎症反应参与抑郁样行为的作用及分子机制，为抑郁症的防治和抗抑郁药物的研发提供新方向和新靶点。

伏隔核炎症反应在抑郁症发病过程中发挥重要作用，但抑郁症中伏隔核炎症反应的发生机制尚不清楚。我们前期研究观察到，伏隔核小胶质细胞有D3R的表达，D3R缺失引起的抑郁样行为与伏隔核小胶质细胞活化有关。本项目通过构建D3R过表达、表达抑制载体，利用核团注射技术观察到，WT小鼠伏隔核下调D3R可以引起小鼠抑郁样行为的发生，D3R敲除小鼠伏隔核回补D3R后能够纠正D3R缺失诱导的小鼠抑郁样行为。在伏隔核下调D3R表达的小鼠模型中，利用流式细胞技术观察到，伏隔核中CD11b标记的小胶质细胞表达增加、促炎型标记物（CD11b+CD86+）表达增加；ELISA法检测发现，伏隔核D3R下调后促炎性细胞因子（TNF-α、IL-1β和IL-6）表达水平明显增加。在D3R敲除小鼠原代小胶质细胞以及BV-2小胶质细胞系通过siRNA方法抑制D3R表达的体外细胞培养中，通过免疫荧光、免疫印迹、流式细胞和ELISA等技术观察到，D3R表达抑制加重了脂多糖引起的CD11b标记的小胶质细胞表达增多、细胞胞体变大以及细胞分支增加等变化；且发现D3R表达抑制后更加促进脂多糖引起的促炎型（CD11b+CD86+）小胶质细胞表达的增多，以及细胞上清中脂多糖引起的促炎性细胞因子（（TNF-α、IL-1β和IL-6）分泌的增加。最后，在BV-2小胶质细胞系下调D3R表达的体外模型中，利用免疫印记法观察到，Akt信号通路的蛋白表达明显上升；在此基础上，给予Akt细胞通路抑制剂MK2206，通过流式细胞法观察到，抑制Akt信号通路可以改善D3R抑制后加重脂多糖诱导促炎性小胶质细胞表达的增加。研究结果表明，D3R通过调控Akt信号通路抑制伏隔核小胶质细胞向促炎型转变进而发挥抗抑郁作用。这将为抑郁症的防治和抗抑郁药物的研发提供新方向和新靶点。