基于线粒体功能失调与Nrf2介导的抗氧化应激探索柚皮素对脑缺血再灌注损伤的神经保护作用

发布时间：2018-03-12 17:40

本文选题：脑缺血再灌注损伤　切入点：线粒体功能失调　出处：《南方医科大学》2017年博士论文　论文类型：学位论文

【摘要】：一、目的为探讨柚皮素对脑缺血再灌注损伤的神经保护作用及其可能机制,本研究以SD大鼠为实验对象,分别采用体外原代皮层神经细胞培养糖氧剥夺/再灌注(oxygen and glucose deprivation/reperfusion,OGD/Rep)模型和大鼠大脑中动脉缺血再灌注(MACO)模型,从体外和动物模型不同角度探索柚皮素干预对脑缺血再灌注损伤的保护作用及其与线粒体功能失调以及核因子E2相关因子(Nrf2)通路调控的抗氧化应激的关系,明确柚皮素对脑缺血再灌注损伤的神经保护作用的分子机理。二、方法体外研究实验:1、用NES-FITC及DAPI对对数生长期的大鼠皮层神经元细胞进行免疫荧光染色鉴定。2、SD大鼠大脑皮层神经细胞建立OGD/Rep模型。3、(1)用不同浓度NAR干预48h后,检测细胞凋亡情况。(2)检测凋亡蛋白Capase-3、bax、bcl-2、CytC蛋白和基因表达水平。(3)电镜观察各组线粒体结构,检测其膜电势和腺苷酸含量变化。(4)检测ROS、MDA和GSH水平及SOD活性。4、(1)siRNA-Nrf2、OENrf2 干扰 Nrf2 表达,行 OGD/Rep 处理,再给予80MmNAR处理48h后,检测细胞凋亡情况。(2)检测ROS、MDA和GSH水平及SOD活性。(3)检测Nrf2蛋白转移情况以及其与ARE结合的情况。(4)检测Nrf2、Keap1、HO-1和NOQ1蛋白和基因表达水平。体内研究实验:1、利用线栓法建立大鼠大脑中动脉缺血再灌注(MCAO)动物模型,给予不用浓度NAR处理,术后进行神经行为学评分、TTC染色、脑含水量测定以及HE染色。2、给予MCAO模型不同浓度NAR处理72h后,(1)电镜扫描观察各组线粒体结构,检测其膜电势和腺苷酸含量变化;(2)检测脑组织内ROS、MDA和GSH水平及SOD活性;(3)观察脑组织神经细胞凋亡情况,检测脑组织Nrf2、Keap1、HO-1、NQO1 蛋白表达情况,检测 Nrf2、Keap1、HO-1、NQO1基因表达情况,同时检测Nrf2的DNA结合活力和Keap1亚硝基化情况。三、结果体外研究结果:1、与正常对照相比,模型组细胞活性低,凋亡率高,差异显著(P0.05),与模型组对比,不同浓度NAR预处理后,细胞活性提高、凋亡率下降,NAR-H组最为显著(P0.05)。2、与正常组比较,模型组Capase-3,bax及CytC表达上调,bcl-2表达下调(P0.05)。与模型组相比,经过不同浓度NAR预处理后,Capase-3,bax及CytC表达下调,bcl-2表达上调。其中NAR-H组最为显著(P0.05)。3、NAR预处理可改善线粒体形态,提高其膜电位及腺苷酸合成能力,提高 SOD、GSH水平,降低 ROS、MDA水平。4、与模型组比较,转染OENrf2组和NAR组细胞活性提高,凋亡率下降,OENrf2组最为显著(P0.05);转染Nrf2-siRNA组则相反(P0.05)。5、与模型组比较,转染OENrf2组和NAR组SOD、GSH水平提高,ROS、MDA水平下降,OENrf2组最为显著(P0.05);转染Nrf2-siRNA组则无明显差异(P0.05)。7、与模型组比较,转染OENrf2组和NAR组细胞质Nrf2蛋白表达增加,而胞浆表达减少,OENrf2组最为显著(P0.05);转染Nrf2-siRNA组差异无统计学意义(P0.05)。8、NAR诱导Nrf2表达,促进Nrf2通路下游蛋白Nrf2、Keap1、HO-1和NOQ1的表达。体内研究结果:1、NAR预处理组脑梗死面积、神经行为学评分和脑水含量均显著优于模型组。2、NAR预处理组线粒体正常结构增加、膜电位增强、腺苷酸合成能力提高,氧化应激水平降低,与模型对照组相比,差异具有统计学意义。3、NAR预处理组细胞凋亡减少,提高Keap1巯基亚硝基化水平、增强Nrf2 DNA活性,激活Nrf2通路下游Nrf2、Keap1、HO-1和NOQ1的表达。四、结论柚皮素对体外细胞培养氧糖剥夺/再灌注模型和大鼠脑缺血再灌注模型中的神经细胞均具有明显的保护作用,该作用与其改善线粒体结构和功能失调以及激活Nrf2介导的抗氧化应激活性相关。
[Abstract]:First, to explore the neuroprotective effect of naringin on cerebral ischemia reperfusion injury and its possible mechanism, in this study, SD rats were used respectively in primary cultured cortical neurons of oxygen and glucose deprivation / reperfusion (oxygen and glucose deprivation/reperfusion, OGD/Rep) model and rat cerebral ischemia / reperfusion (MACO) model in vitro and animal models from different angles to explore the protective effect of naringenin intervention on cerebral ischemia reperfusion injury and mitochondrial dysfunction and nuclear factor E2 related factor (Nrf2) pathway between oxidative stress, the specific molecular mechanism of naringenin on cerebral ischemia reperfusion injury neuroprotection. Two methods in vitro experiments: 1, logarithmic growth period for the NES-FITC and DAPI in rat cortical neurons were identified by immunofluorescence staining of.2 and SD in rat cerebral cortex of God The establishment of OGD/Rep cell model of.3 (1) with different concentrations of NAR after 48h intervention, the apoptosis was detected. (2) detection of apoptosis protein Capase-3, Bax, Bcl-2, the expression level of CytC protein and gene. (3) observed in mitochondria electron microscope, to test the change of membrane potential and adenosine (4) detection. ROS, MDA and GSH level and the activity of SOD.4 (1) siRNA-Nrf2, the expression of OENrf2 interference Nrf2 OGD/Rep treating, then given 80MmNAR after 48h treatment, the apoptosis was detected. (2) detection of ROS, MDA and GSH level and SOD activity. (3) the detection of Nrf2 protein and ARE and its transfer (4). Combined detection of Nrf2, Keap1, HO-1 and NOQ1 protein level and gene expression. In vivo experiment: 1, using the suture method in rats cerebral ischemia reperfusion (MCAO) animal model, give no concentration NAR treatment, postoperative neurobehavioral score, TTC staining, content determination with the brain .2 and HE staining, MCAO 72h model to deal with different concentrations of NAR (1), scanning electron microscope to observe the structure of mitochondria, to test the change of membrane potential and adenylate content; (2) detection in the brain tissue of ROS, MDA and GSH level and SOD activity; (3) to observe the brain tissue apoptosis of neural cells, detection of brain Nrf2, Keap1, HO-1, NQO1 protein expression, Nrf2 detection, Keap1, HO-1, NQO1 gene expression, and detection of Nrf2 DNA binding activity and Keap1 nitrosylation. Three results in vitro results: 1, compared with the normal control, model group cell activity is low, the apoptosis rate is high, significant difference (P0.05), compared with the model group, different concentration of NAR after pretreatment, cell activity increased, apoptosis rate decreased, NAR-H group (P0.05.2), compared with the normal group, Capase-3 model group, expression of Bax and CytC, bcl-2 expression (P0.05). Compared with the model group, after different concentration The degree of NAR after pretreatment, Capase-3, Bax and CytC expression, bcl-2 expression. The NAR-H group (P0.05.3), NAR preconditioning can improve mitochondrial morphology, membrane potential and improve their ability to improve SOD, adenylate synthesis, reduce ROS, GSH level, MDA level of.4, compared with the model group to improve the transfection of OENrf2 group and NAR group cell activity, decreased apoptosis rate, OENrf2 group (P0.05); transfection group Nrf2-siRNA (P0.05) in.5, compared with the model group, OENrf2 transfection group and NAR group SOD, GSH level, ROS, MDA decreased, OENrf2 group (P0.05); there was no obvious difference between the transfected Nrf2-siRNA group (P0.05.7), compared with the model group, OENrf2 transfection group and NAR group of cytoplasmic Nrf2 protein expression increased and cytoplasmic expression decreased, OENrf2 group (P0.05); there was no significant difference in transfection group Nrf2-siRNA (P0.05).8, Nrf2 induced expression of NAR, promote Nrf2 pathway The downstream protein Nrf2, Keap1, HO-1 and NOQ1 expression in vivo. Results: 1, NAR pretreatment group infarct size, neurological behaviors and brain water content were significantly better than the.2 model group, NAR pretreatment group increased mitochondrial membrane potential of normal structure, enhance, improve the level of oxidative stress adenylate synthesis ability, lower than with the model group, the difference was statistically significant.3, NAR pretreatment group to reduce cell apoptosis, improve Keap1 S-nitrosylation level, Nrf2 enhanced DNA activity, activation of the Nrf2 pathway downstream of Nrf2, Keap1, HO-1 and NOQ1 expression. Four, the conclusion of naringenin on cultured cells of oxygen glucose deprivation / reperfusion model rats cerebral ischemia reperfusion model in nerve cells has a significant protective effect, and the effect of improving the structure and function of mitochondria dysfunction and activation of Nrf2 mediated activation of oxidative stress.

【学位授予单位】：南方医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R743

【相似文献】