皮层扩散性抑制预处理在大鼠缺血再灌注损伤中的神经保护作用机制研究

发布时间：2018-04-25 10:47

本文选题：皮层扩散性抑制预处理 + 缺血耐受　；参考：《吉林大学》2016年博士论文

【摘要】：背景:皮层扩散性抑制(Cortical spreading depression,CSD),基于其与梗死周围去极化波(peri-infarct depolarization,PID)的相似性,已经成为研究缺血半暗带向梗死中心区演变机制的良好实验模型。很多研究都已证实,预先诱导CSD波可增加脑组织对抗缺血性损伤的耐受性,但是其潜在的病理生理机制仍不清楚。近年来,很多研究都已证明,自噬在脑缺血性损伤及脑对缺血耐受方面起重要作用。自噬在缺血/再灌注损伤中的作用成为脑保护研究领域的热点,其中AMPK-m TOR-自噬通路研究较为广泛。然而,其明确的作用在各种研究中结果不尽相同,且机制尚不明确,仍需要进一步探讨。以往关于CSD预处理机制方面的研究多集中在能量代谢、一氧化氮、神经生长因子、外生基因表达等方面,尚缺乏CSD预处理和自噬在缺血耐受方面相关性的研究。本实验应用大鼠大脑中动脉阻塞模型(Middle cerebral artery occlusion,MCAO),探讨CSD预处理的病理生理机制,进一步明确自噬在缺血再灌注损伤中神经保护作用机制及AMPK-m TOR-自噬通路在缺血耐受中的作用,为缺血性脑血管病的治疗和预防提供新靶点。目的:通过研究CSD预处理,进一步探讨缺血半暗带向梗死中心区演变的机制。同时也首次将CSD预处理与自噬及其相关通路、自噬在缺血耐受中的作用联系起来,并进一步明确自噬在缺血再灌注损伤中的作用及机制。方法:(1)皮层给1 mol/L氯化钾溶液湿敷2小时诱导皮层扩散性抑制波(CSD),通过脑电图监测及电生理记录直流场电位(direct current potential,DC)变化的方法证实CSD波的发生及其特征,建立CSD预处理的实验模型,对照组用1 mol/L的氯化钠溶液湿敷2小时。(2)CSD预处理与AMPK-m TOR-自噬相关通路研究。112只雄性Wistar大鼠随机分组:盐水对照组;预处理组(0、3、6、12、24小时)组;3-MA(i.c.v.)+CSD组;溶剂对照(i.c.v.)+CSD组;Compound C(i.p)+CSD组;溶剂对照(i.p)+CSD组。盐水对照组皮层给予1 mol/L氯化钠溶液湿敷2小时,12小时后取材;预处理组皮层给予1 mol/L氯化钾溶液湿敷2小时,分别在0、3、6、12、24小时后取材;各给药组在CSD预处理术前30 min分别给予自噬抑制剂3-methyladenine(3-MA,200 nmol,i.c.v.)和AMPK抑制剂Compound C(CC,20 mg/kg,i.p)。Western blotting检测各组皮层自噬标记物LC3-II,Beclin-1,P62水平,AMPK-m TOR通路蛋白p-AMPK(Thr172)/AMPK,p-P70S6K(Thr389)、ULK1水平;免疫荧光检测LC3-II阳性细胞数及分布,透射电镜检测自噬小体水平。(3)CSD预处理在缺血再灌注损伤中的神经保护作用。84只雄性Wistar大鼠随机分组:盐水对照组,CSD预处理(0、3、6、12、24小时)组,3-MA组,溶剂对照组。盐水对照组:盐水预处理后12小时,行MCAO缺血2小时,再灌注12小时;CSD预处理组:分别在CSD预处理后0,3,6,12,24小时后分别行MCAO缺血2小时,再灌注12小时;3-MA组:CSD预处理前30分钟给予自噬抑制剂3-MA(200 nmol,i.c.v.),预处理后12小时后行MCAO缺血2小时,再灌注12小时;溶剂对照组:CSD预处理前30分钟给予相同体积的溶剂(i.c.v.),预处理后12小时后后行MCAO缺血2小时,再灌注12小时。应用TTC染色法计算各组梗死体积,Longa法评估神经功能缺损,并进行相关统计学分析。(4)自噬在缺血再灌注损伤中神经保护作用的机制初步探讨。30只雄性Wistar大鼠随机分5组:假手术组(Sham),MCAO组,CSD+MCAO组,3-MA+CSD+MCAO组,溶剂+CSD+MCAO组。通过reverse transcriptase-PCR、Western blotting检测MCAO缺血再灌注损伤后半暗带区皮层凋亡相关蛋白Bcl-2、Caspase12,内质网应激相关蛋白GRP78、XBP1,缝隙连接蛋白Connexin 36的m RNA水平及蛋白水平;TUNEL及免疫荧光双标染色显示凋亡细胞的比例及细胞类型。结果:(1)皮层给予1 mol/L氯化钾溶液可引起诱导侧皮层脑电图波幅的持续性下降,由117.25±3.51μV下降到78.36±3.46μV(P0.01),而频率和波形没有明显改变,脑电活动被抑制;电生理学改变为直流场电位的负翻转,证明CSD预处理模型建立成功,而对照组没有相应脑电图及电生理改变。(2)CSD预处理对自噬及AMPK-m TOR通路的影响:Western blotting示,CSD可增加大鼠预处理侧皮层LC3-II,Beclin-1水平及p-AMPK(Thr172)/AMPK比率,减少P62和p-P70S6K(Thr389)水平,这种改变在预处理后12小时达到高峰;应用AMPK抑制剂Compound C(20 mg/kg)可下调皮层LC3-II,p-AMPK(Thr172)/AMPK比率,上调P62和p-P70S6K(Thr389)水平。免疫荧光显示,LC3-II阳性细胞数在预处理组明显高于对照组(P0.01),且主要分布于神经元细胞中(87.2%),神经胶质细胞中较少(2.89%);应用3-MA(P0.01)及CC(P0.05)后,LC3-II阳性细胞数减少。透射电镜显示预处理组皮层自噬小体的数量明显高于对照组(P0.05)。(3)CSD预处理可显著降低大鼠MCAO后的脑梗死体积,改善功能学评分,且这种保护作用在预处理后12小时达到高峰(P0.05);应用自噬的抑制剂3-MA(200 nmol,i.c.v.)可减少CSD预处理对梗死体积及神经功能评分的影响(P0.05)。(4)CSD预处理可减少缺血再灌注损伤的半暗带区皮层内凋亡相关标记物Caspase12蛋白质水平和m RNA水平,增加抗凋亡标记物Bcl-2蛋白和m RNA水平,TUNEL染色显示CSD预处理明显减少半暗带区神经元细胞凋亡的比例(P0.01);CSD可降低内质网应激标记物GRP78的蛋白质水平和m RNA水平,减少XBP-1蛋白水平,增加其m RNA水平。应用自噬抑制剂3-MA后可逆转上述改变。CSD预处理后神经元表面主要缝隙连接蛋白Connexin 36的蛋白质和m RNA水平无明显改变(P0.05)。结论:(1)皮层持续给予1 mol/L氯化钾溶液2小时可引起预处理侧皮层脑电活动的抑制,脑电图波幅降低,诱导负性直流DC波的产生,成功建立CSD预处理模型;而给予1 mol/L氯化钠溶液未引起上述改变。(2)CSD可通过AMPK-m TOR通路诱导自噬激活,且激活的自噬主要分布在神经元细胞中,胶质细胞中少见。(3)CSD预处理具有明确的神经保护作用,减少梗死体积,改善神经功能评分,且在预处理后12小时达高峰。(4)CSD预处理诱导的自噬可减少大鼠缺血再灌注损伤半暗带区皮层的神经元凋亡,抑制内质网应激的过度激活,从而产生神经保护作用,而对神经元表面主要缝隙连接蛋白Connexin 36没有影响。
[Abstract]:Background: Cortical spreading depression (CSD), based on its similarity to peri-infarct depolarization (PID) around infarct, has become a good experimental model to study the evolution mechanism of ischemic penumbra to infarct center. Many studies have confirmed that pre induced CSD wave can increase brain tissue antagonism. The tolerance of ischemic injury, but its potential pathophysiological mechanism is still unclear. In recent years, many studies have shown that autophagy plays an important role in ischemic brain damage and cerebral ischemia tolerance. The role of autophagy in ischemia / reperfusion injury has become a hot spot in the field of brain protection, in which the autophagy pathway of AMPK-m TOR- is studied However, its clear role is not the same in various studies, and the mechanism is still unclear. The previous studies on the mechanism of CSD preconditioning are mainly focused on energy metabolism, nitric oxide, nerve growth factor, exogenic gene expression, and so on, and lack of CSD preconditioning and autophagy in ischemic tolerance. In this study, the Middle cerebral artery occlusion (MCAO) model of rat middle cerebral artery (MCAO) was used to explore the pathophysiological mechanism of CSD preconditioning, and to further clarify the mechanism of the neuroprotective effect of autophagy in ischemic reperfusion injury and the role of the autophagy pathway of AMPK-m TOR- in ischemic tolerance. Objective: to provide new targets for the treatment and prevention of tubular diseases. Objective: to further explore the mechanism of the evolution of the ischemic penumbra to the center of the infarct through the study of CSD preconditioning. At the same time, the role of CSD preconditioning and autophagy and its related pathway, the role of autophagy in the ischemic tolerance, and the further study of autophagy in the ischemic reperfusion injury Methods: (1) (1) cortical diffusion inhibition wave (CSD) was induced by wet compress of 1 mol/L potassium chloride solution for 2 hours. The occurrence and characteristics of CSD wave were confirmed by electroencephalogram monitoring and electrophysiological recording DC field potential (direct current potential, DC), and the experimental model of CSD pretreatment was established, and 1 mol/L chlorination was used in the control group. Sodium solution wet compress for 2 hours. (2) CSD preconditioning and AMPK-m TOR- autophagy pathway related pathway study of.112 male Wistar rats randomly divided into saline control group, pretreated group (0,3,6,12,24 hour) group; 3-MA (i.c.v.) +CSD group; solvent control (i.c.v.) +CSD group; Compound restraint group; solvent control group. Saline control group was given 1 chlorine chloride. The sodium chloride solution was applied for 2 hours and 12 hours later. The pretreated group was treated with 1 mol/L potassium chloride solution for 2 hours and after 0,3,6,12,24 hours, respectively. The drug groups were given a autophagic inhibitor 3-methyladenine (3-MA, 200 nmol, i.c.v.) and Compound C of AMPK inhibitors before CSD pretreatment (CC, 20, 20, respectively). Ting detection of autophagic markers LC3-II, Beclin-1, P62 level, p-AMPK (Thr172) /AMPK, p-P70S6K (Thr389), ULK1 level, immunofluorescent detection of positive cell number and distribution, transmission electron microscopy to detect the level of autophagic corpuscle. (3) the neuroprotective effect of preconditioning in ischemia reperfusion injury R rats were randomly divided into two groups: saline control group, CSD preconditioning (0,3,6,12,24 hour) group, 3-MA group, solvent control group, saline control group: 12 hours after brine pretreatment, MCAO ischemia 2 hours, reperfusion 12 hours; CSD preconditioning group: MCAO ischemia for 2 hours after CSD preconditioning, and reperfusion for 12 hours respectively; 3-MA group: CSD prelocation The autophagic inhibitor 3-MA (200 nmol, i.c.v.) was given 30 minutes before treatment, and MCAO ischemia was performed for 2 hours after 12 hours of pretreatment and reperfusion for 12 hours. The solvent control group was given the same volume of solvent (i.c.v.) 30 minutes before CSD preconditioning. After 12 hours of pretreatment, MCAO ischemia was performed for 2 hours and reperfusion for 12 hours, and TTC staining was used to calculate each group of infarct bodies. Product, Longa method was used to evaluate neural function defect and to carry out statistical analysis. (4) the mechanism of neuroprotective effect of autophagy in ischemia-reperfusion injury was preliminarily discussed in.30 male Wistar rats randomly divided into 5 groups: sham operation group (Sham), MCAO group, CSD+MCAO group, 3-MA+CSD+MCAO group, and solvent +CSD+MCAO group. Reverse transcriptase-PCR, Western blo Tting detection of cortical apoptosis related protein Bcl-2, Caspase12, Caspase12, endoplasmic reticulum stress related protein GRP78, XBP1, m RNA level and protein level of gap connexin Connexin 36 after MCAO ischemia reperfusion injury; TUNEL and immunofluorescence double labeling staining showed the ratio of apoptotic cells and cell types. Results: (1) cortex was given 1 mol/L chlorination The potassium solution could cause a continuous decrease in the amplitude of the electroencephalogram of the induced lateral cortex, from 117.25 + 3.51 V to 78.36 + 3.46 mu V (P0.01), but the frequency and waveform did not change obviously, the EEG activity was suppressed, the electrophysiological change was negative reversal of the DC field potential, which proved that the model of CSD was established successfully, while the control group did not have the corresponding electroencephalogram and the control group. Electrophysiological changes. (2) the effect of CSD pretreatment on autophagy and AMPK-m TOR pathway: Western blotting shows that CSD can increase the LC3-II, Beclin-1 level and p-AMPK (Thr172) /AMPK ratio in the pretreated lateral cortex of rats, and reduce the level of P62 and Thr172. This change reaches the peak after 12 hours of preconditioning. The ratio of LC3-II, p-AMPK (Thr172) /AMPK, P62 and p-P70S6K (Thr389) was up-regulated in the naughty layer. Immunofluorescence showed that the number of LC3-II positive cells in the pretreated group was significantly higher than that of the control group (P0.01), and was mainly distributed in the neuron cells (87.2%) and in the glial cells (2.89%). The positive cells were used after 3-MA (P0.01) and CC. Transmission electron microscopy showed that the number of autophagic bodies in the pretreated group was significantly higher than that of the control group (P0.05). (3) CSD preconditioning significantly reduced the volume of cerebral infarction after MCAO and improved the functional score, and the protective effect reached its peak at 12 hours after preconditioning (P0.05), and the application of autophagy inhibitor 3-MA (200 nmol, i.c.v.) could reduce C The effect of SD preconditioning on infarct volume and nerve function score (P0.05). (4) CSD preconditioning can reduce the level of apoptosis related markers Caspase12 protein and m RNA in the semi dark zone of ischemia reperfusion injury, increase the Bcl-2 protein and m RNA levels of anti apoptotic markers, and TUNEL staining shows that CSD pretreatment significantly reduces the deity of the dark zone zone. The ratio of cell apoptosis (P0.01), CSD can reduce the protein level of the endoplasmic reticulum stress marker and the level of M RNA, reduce the level of XBP-1 protein, and increase the level of M RNA. The application of autophagy inhibitor 3-MA can reverse the above changes of the protein and M levels of the main gap junctional connexin 36 of the neuron surface after the pretreatment of.CSD. Significant changes (P0.05). Conclusion: (1) the continuous administration of 1 mol/L potassium chloride solution in the cortex for 2 hours can cause the inhibition of electroencephalogram activity in the pretreated lateral cortex, the decrease of the amplitude of electroencephalogram, the induction of negative direct current DC wave, and the successful establishment of the CSD preconditioning model, while 1 mol/L Sodium Chloride Solution does not cause the above changes. (2) CSD can be induced by AMPK-m TOR pathway. Autophagy is activated by autophagy, and the activation of autophagy is mainly distributed in neuron cells and in glial cells. (3) CSD preconditioning has a clear neuroprotective effect, reduces infarct volume, improves neurological function score, and reaches a peak at 12 hours after preconditioning. (4) CSD preconditioning induced autophagy can reduce the area of ischemia reperfusion injury in rats The neuronal apoptosis in the cortex inhibits the overactivation of endoplasmic reticulum stress and produces neuroprotective effects, but has no effect on the main gap connexin Connexin 36 on the surface of the neuron.

【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R743.3

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