星形胶质细胞Nrf2/ARE-Prx2通路介导氧化应激损伤参与卒中后抑郁机制的研究

About 1/3 stroke patients suffer from post stroke depression (PSD). This prevalence is so higher than the incidence of major depression disorder, which means that stroke patients are more vulnerable to depression than healthy individuals. PSD impaired the outcome of stroke patients effectively. Unfortunately, the pathophysiology of PSD remains elusive. Accumulating evidence including the most recent advance indicates that Nrf2/ARE-Prx2 dependent persistent oxidative damage results in stress-induced vulnerability to depression. However whether this mechanism contributes to PSD is still unclear. Previous studies from us were focused on the involvement of Nrf2/ARE signaling pathway in the antioxidation induced by neuroprotective agents and the anti-inflammation effect induced by anti-depressants. Our pilot study showed that the expression of Nrf2, Trx1, Prx2 was reduced and the contents of MDA, 4-HNE, protein carbonylation, 8-OHdG and ROS was elevated in PSD mice, as compared with that in control mice. These results indicate that Nrf2/ARE dependent persistent oxidative stress induced by cerebral ischemia/reperfusion plays a crucial role in the vulnerability to PSD but need more detail data. Moreover, astrocyte is the main regulator of oxidative/redox hemostasis in brain via Nrf2/ARE pathway, so whether astrocyte driven Nrf2/ARE-Prx2 pathway contributes to the pathogenesis of PSD also should be explored in the present study. We will establish the PSD model and introduce Nrf2 knockout mice, then use Nrf2 agonist t-BHQ, opotogenetic method, neurobehavioral test, molecular biology method and other techniques to clarify whether astrocytic Nrf2/ARE dependent oxidative stress participates the pathogenesis of PSD. This study will provide more experimental evidences for understanding the pathophysiology of PSD and reveal a novel target for the prevention and treatment of PSD.

卒中后抑郁（PSD）严重影响患者预后且缺乏有效防治手段，其形成与卒中后氧化应激关联密切但具体机制不清。脑内氧化/抗氧化微环境主要受星形胶质细胞调控，Nrf2/ARE是其抗氧化功能主要执行者。研究提示Nrf2/ARE通过Prx2介导氧化应激、参与抑郁形成。申请者预实验结果发现：PSD模型脑内Nrf2、Trx1、Prx2表达下调，氧化应激产物MDA、4-HNE、羰基化蛋白质及8-OHdG表达增加。据此我们推测Nrf2/ARE-Prx2通路介导卒中后持续性氧化应激、引起PSD样行为，但需更多证据且是否星形胶质细胞通过这一信号通路参与PSD形成仍不明确。为此课题组拟引入Nrf2基因敲除鼠，构建PSD模型，应用药理学调控、光遗传学技术、在体微透析、分子生物学及神经行为学等技术阐明星形胶质细胞Nrf2/ARE-Prx2通路介导的氧化应激在PSD发病机制中的地位；为临床PSD防治提供新的干预思路及靶点。

卒中后抑郁发病率高，目前尚无有效治疗措施且其具体发病机制并不明确。核因子E2相关因子（Nrf2）是氧化应激、炎症反应的重要调节者，在卒中后抑郁发病中的作用尚不明确。本课题拟首先明确重复经颅磁刺激治疗抑郁症是否和Nrf2所介导的抗炎作用相关，其次进一步阐明Nrf2抗炎、抗氧化作用的上游机制，最后，在卒中后抑郁模型上，进一步明确Nrf2-Prx2信号通路及其所调节的氧化应激、线粒体功能是否参与卒中后抑郁、焦虑样行为产生，Nrf2特异性激动剂Tempol能够改善卒中后抑郁样行为；最后明确是否星形胶质细胞Nrf2介导了炎症反应以及卒中后抑郁样行为。按照预期研究计划，本课题首先在前期研究的基础上进一步明确了Nrf2所调节的抗炎症反应与经颅磁刺激抗抑郁作用之间的关联，而Nrf2-siRNA不仅逆转了rTMS的抗炎作用，也逆转了rTMS对Nrf2的上调作用；同时，进一步阐明了GSK-3β磷酸化促进Nrf2核转位，从而发挥抗氧化、抗炎作用的调节作用；最后，在前期研究基础上，首先创建了较为稳定的卒中后抑郁模型，利用该模型，明确了Nrf2-Prx2信号通路下调导致线粒体功能障碍、氧化应激损伤，最终使动物出现抑郁、焦虑样行为；而Tempol补充性治疗能够外源性上调Nrf2，激活其抗氧化作用并有效改善卒中后抑郁样行为；最后，利用特异性下调星形胶质细胞内Nrf2的腺病毒，明确了星形胶质细胞Nrf2调节炎症反应的关键作用；光遗传学技术激活星形胶质细胞能够激活Nrf2核转位，改善动物的抑郁样行为。通过上述研究，本课题进一步研究了Nrf2-Prx2信号通路调节炎症反应、氧化应激反应的具体上游机制，明确了该信号通路在卒中后抑郁中的发病机制，为卒中后抑郁发病机制阐明提供了理论依据；最后也为未来rTMS、外源性补充Tempol治疗卒中后抑郁提供了理论支撑。