Nrf2-ARE信号通路调控脑出血灶周小胶质细胞的机制研究

The morbidity,mortality and disability of intracerebral hemorrhage are still high and the mechanism of its perihematoma damage involves multiple levels and multiple links. Microglia (MG) abnormal activation ,to which great importance is more attached ,can magnify inflammatory reaction and aggravate neurotoxicity.Nrf2-ARE pathway is an important regulation pathway to anti-peroxidation and anti-inflammatory damage.??Microglia (MG),when activated by the Nrf2, will have a nerve protective effect, and when regulated by the NF-κ B, will result in a neurotoxic effect. ??The aim of this study is to explore the mechanism of intracerebral hemorrhage perihematoma damage on the basis of activation of Nrf2-ARE signaling pathway and regulation of MG, inhibition of the NF-κB disintegration through HO-1, and reduction of the possibility of NF-κB transfer into the nucleus to control microglia activation. In this study, two models are adopted: the rat intracerebral hemorrhage model and neuron/microglia co-cultured inflammatory damage model. Flow cytometry, laser confocal microscope, electron microscope and molecular biology techniques will be used to find out the relevant evidence that Nrf2-ARE signaling pathway in intracerebral hemorrhage inhibits the functions of NF-κ B from both in vivo and vitro. The aim of this step is to clarify the mechanism of how to regulate MG neural protective effect and how to suppress the neural toxicity.The ultimate aim of this study is to offer valuable experimental data on treatment of intracerebral hemorrhage by clarification of nerve protective molecular mechanism in MG regulated by Nrf2-ARE pathway in signaling transduction pathway.

脑出血的发病率、致死率和致残率仍很高，灶周损伤机制涉及多个水平、多个环节。灶周小胶质细胞（MG）异常活化可放大炎性反应、加重神经毒性，且愈受重视。Nrf2-ARE通路是抗氧化、抗炎性损伤的重要调节通路，Nrf2激活MG促神经保护作用，NF-κB调控MG神经毒性作用。拟以激活Nrf2-ARE信号通路、MG活化调控为视角，沿着HO-1抑制NF-κB解离、减少NF-κB核内转移调控MG活化的思路，朝着阐述脑出血灶周损伤机制的目标展开工作。采用大鼠脑出血模型和体外神经元/小胶质细胞共培养炎性损伤模型，利用流式细胞仪、激光共聚焦显微镜、电镜和分子生物学等技术，从动物在体、离体两个层面，寻找脑出血后Nrf2-ARE信号通路抑制NF-κB作用的相关证据，为调控MG神经保护、抑制神经毒性作用明确机制。在信号转导通路水平阐明Nrf2-ARE通路调控MG中神经保护的分子机制，为临床脑出血的治疗提供实验依据。

本研究采用激光共聚焦、免疫荧光、western blot、RT-PCR、分子生物学、流式细胞仪等手段，通过体内和体外实验观察了大鼠脑出血后Nrf2-ARE 信号通路与灶周小胶质细胞活化间的关系及其对脑出血灶周神经保护作用，结果发现：（1）Nrf2-ARE信号通路在脑出血急性期后具有抗炎性反应，具有神经保护作用；（2）RA抑制Nrf2解离及转入核,抑制Nrf2-ARE信号通路的抗炎性作用,SFN激活Nrf2-ARE信号通路可提高HO-1抗氧化酶表达,减轻脑出血后灶周炎性反应；（3）大鼠ICH后继发性脑损伤中小胶质细胞发挥着重要的作用， Nrf2-ARE通路通过小胶质细胞发挥神经保护作用；（4）脑出血灶周小胶质细胞HO-1抑制NF-κB信号通路的机制并具有神经保护作用；（5）miR-181C是通过其他NF-κB相关的信号通路抑制NF-κB活化和下调NF-κB下游基因的表达，参与神经保护作用。.上述结果证实了我们的假说—脑出血后激活Nrf2-ARE通路，通过HO-1抑制NF-κB信号产生的神经毒性作用，增强诱导小胶质细胞在脑出血灶周的神经保护作用。HO-1为脑出血继发性脑损伤的治疗提供一个新靶点。