【作者】 李莉；

【导师】 赵涌；

【作者基本信息】 重庆医科大学 ， 病理学与病理生理学， 2021， 硕士

【摘要】 背景脑缺血再灌注损伤的发生发展是一个复杂的病理生理过程,其中,氧化应激反应与脑缺血再灌注损伤继发性神经细胞死亡密切相关。研究发现机体内源性抗氧化系统的激活对脑缺血再灌注损伤后神经细胞的存活有至关重要的作用。PARK7(又称DJ-1),是通过进化选择的多功能蛋白之一,在抗细胞凋亡,介导细胞信号转导以及抗机体氧化应激等方面发挥至关重要的作用。近年来,人们发现DJ-1依靠其良好的抗氧化作用在保护神经细胞方面发挥重要功能。DJ-1与帕金森疾病的发病机制密切相关,DJ-1在帕金森疾病中能够保护神经元免受氧化应激损伤。最近,DJ-1在缺血性脑卒中的保护作用受到了广泛的研究,但具体的机制仍不清楚。转录因子NF-E2相关因子2(Nrf2)是转录因子家族成员之一,它通过与抗氧化反应元件(ARE)结合作为机体抗氧化防御系统的主调节器。DJ-1作为抗氧化蛋白,在Nrf2活性调控中发挥重要作用。然而,DJ-1是通过上调Nrf2发挥抗氧化保护作用还是通过调节Nrf2的亚细胞定位,促进Nrf2核转移,激活其抗氧化功能,目前尚未有研究报道。目的本研究旨在探讨DJ-1在脑缺血再灌注氧化应激损伤中的作用及其相关调控机制,为进一步防治脑缺血再灌注氧化应激损伤提供理论依据。方法(1)本实验以雄性成年SD大鼠为研究对象,通过建立大鼠大脑中动脉栓塞再灌注(Middle Cerebral Artery Occlusion/Reperfusion,MCAO/R)模型从而对大鼠体内脑缺血再灌注损伤进行模拟。(2)构建大鼠的MCAO/R模型,并于建模前24h左侧脑室注射DJ-1 si RNA,观察干扰DJ-1表达对MCAO/R后各组大鼠神经学功能损伤、脑含水量以及脑组织形态学的影响。(3)采用ELISA实验方法检测超氧化物歧化酶(superoxide dismutase,SOD)和丙二醛(malondialdehyde,MDA)分析干扰DJ-1表达后对MCAO/R后各组大鼠脑组织氧化应激状态的影响。(4)采用Western Blot实验方法分析干扰DJ-1对MCAO/R后各组大鼠脑组织中DJ-1,Nrf2,HO-1以及NQO1的表达水平的影响。(5)构建大鼠的MCAO/R模型,分别于建模前1月对SD大鼠的左侧大脑皮质注射DJ-1过表达腺相关病毒载体(Adeno-Associated Virus,AAV)。(6)采用Western Blot实验方法分析过表达DJ-1后对MCAO/R后各组大鼠脑组织中DJ-1,Nrf2,HO-1以及NQO1的表达水平的影响。(7)采用免疫荧光染色实验观察过表达DJ-1后对MCAO/R后各组Nrf2的表达及入核情况。结果(1)与MCAO/R组相比,DJ-1 si RNA组的神经功能学评分、脑含水量均明显增加;HE和Nissl染色显示脑缺血区神经元损伤进一步加重;SOD含量进一步降低,MDA含量进一步增加。(2)干扰DJ-1以后,其蛋白水平明显降低,同时核内外Nrf2及其下游的HO-1和NQO1也明显降低。(3)过表达DJ-1以后,其蛋白水平明显增加,同时核内外Nrf2及其下游的HO-1和NQO1明显增加。结论DJ-1作为体内重要的神经保护因子,可以减少大鼠脑缺血再灌注氧化应激损伤,并可能是通过激活Nrf2信号通路来实现这一作用的。更多还原

【Abstract】 BackgroudThe occurrence and development of cerebral ischemia-reperfusion injury is a complex pathophysiological process.Among them,oxidative stress is closely related to neuronal death following cerebral ischemia-reperfusion injury.Studies have found that the activation of endogenous antioxidant system plays a crucial role in the survival of nerve cells after cerebral ischemia-reperfusion injury.Dj-1(also known as PARK7,or PARK7)is one of the multifunctional proteins selected through evolution.It plays an important role in anti-apoptosis,mediating cell signal transduction and resisting oxidative stress in the body.Recently,it has been found that Dj-1 plays an important role in protecting nerve cells by relying on its good antioxidant effect.Dj-1 is closely related to the pathogenesis of Parkinson’s disease.Dj-1 can protect neurons from oxidative stress damage in Parkinson’s disease.Recently,the protective effect of DJ-1 in ischemic stroke has been extensively studied,but the specific mechanism remains unclear.Transcription factor NF-E2-related factor 2,a member of the transcription factor family,acts as a master regulator of the body’s antioxidant defense system through binding with antioxidant response elements(ARE).As an antioxidant protein,dj-1 plays an important role in the regulation of Nrf2 activity.However,whether DJ-1 promotes the nuclear transfer of Nrf2 and activates its antioxidant function by upregulation of Nrf2 or by regulating the subcellular localization of Nrf2 has not been reported yet.ObjectiveThe purpose of this study was to explore the role of DJ-1 in cerebral ischemia-reperfusion oxidative stress injury and its related regulatory mechanism,so as to provide theoretical basis for further prevention and treatment of cerebral ischemia-reperfusion oxidative stress injury.Methods(1)In this study,male adult SD rats were selected as research subjects,and Middle Cerebral Artery Occlusion(MCAO)model was established to simulate Cerebral ischemia reperfusion(I/R)injury in rats.(2)DJ-1 si RNA was injected into the left ventricle 24 h before MCAO/R,to observe neurological functional scores cerebral water content and the effect of brain tissue morphology in DJ-1 si RNA group after MCAO.(3)To analyze the effects of interference of DJ-1 expression on oxidative stress level after MCAO/R,superoxide dismutase(SOD)and malondiadehyde(MDA)were measured by ELISA.(4)Western Blot was used to analyze the effect of interference of DJ-1on the expression of DJ-1,Nrf2,HO-1 and NQO1 in brain tissue of rats after MCAO/R.(5)Adeno-Associated Virus(AAV)of Dj-1 overexpression was injected into the left cerebral cortex of SD rats one month before MCAO/R.(6)Western Blot was used to analyze the effect of overexpression of DJ-1 on the expression of DJ-1,Nrf2,HO-1 and NQO1 in brain tissue of rats after MCAO/R.(7)Immunofluorescence staining was used to observe expression and nucleation of Nrf2 after MCAO/R.Results(1)Compared with MCAO group,the neurofunctional scores and brain water content of DJ-1 si RNA group were significantly increased.HE and Nissl staining showed further aggravation of neuronal injury in cerebral ischemia area.SOD content was further reduced and MDA content was further increased.(2)After interference with DJ-1,the protein level of Nrf2 and its downstream HO-1 and NQO1 were also significantly reduced.(3)After overexpression of DJ-1,the protein level of DJ-1,Nrf2 and their downstream HO-1 and NQO1 were significantly increased.ConclusionAs an important neuroprotective factor in vivo,DJ-1 can reduce oxidative stress injury caused by cerebral ischemia reperfusion in rats,which may be realized by activating Nrf2 signaling pathway.更多还原