NLRP3介导的神经炎症反应在颅脑创伤中的作用及机制研究

发布时间：2018-01-04 23:19

本文关键词：NLRP3介导的神经炎症反应在颅脑创伤中的作用及机制研究　出处：《南京医科大学》2017年博士论文　论文类型：学位论文

【摘要】：第一部分线粒体自噬对颅脑外伤后神经炎症反应和神经元凋亡的调控作用目的:研究颅脑外伤后线粒体自噬的发生情况和具体机制,探讨其和神经炎症反应之间的关系。方法:运用临床标本,以癫痫患者的脑组织为对照组,通过电镜和western blotting等方法检测颅脑损伤后脑组织的自噬及线粒体自噬发生情况;以WT和GFP-LC3转基因Sprague Dawley老鼠为研究对象,运用控制性皮层损伤方法,构建颅脑损伤的动物模型;通过免疫荧光、western blotting、电镜等检测线粒体自噬的发生情况;使用BAF和3-MA两种不同的自噬抑制剂,检测其对颅脑外伤后线粒体自噬的影响;依据外伤后1、3、6以及24小时,取脑组织检测线粒体自噬和线粒体释放Cyt C的情况,研究线粒体自噬和细胞死亡在时间上的关系;检测正常状态下和颅脑外伤后心磷脂在线粒体上的分布情况;抑制PLS3或者CLS,干扰心磷脂的重新分布,检测其对颅脑外伤后线粒体自噬的影响;使用线粒体自噬抑制剂mdivi-1,Sprague Dawley大鼠分为sham组,TBI组和TBI+mdivi-1组;TUNEL和western blotting检测损伤后细胞凋亡情况;脑缺损面积实验,检测脑组织损伤情况;平衡木实验和水迷宫实验,探讨外伤后神经功能恢复情况。结果:1,电镜显示颅脑外伤后大量损伤的线粒体被双层膜包裹,提示显著增加的线粒体自噬;2,western blotting显示自噬的标志蛋白LC3-Ⅱ明显升高,p62下降,线粒体的标志蛋白(COXIV,MnSOD,TOM40)下降;3,免疫荧光显示GFP-LC3和线粒体共定位明显增加;4,线粒体自噬发生早于Cytc从损伤线粒体释放;5,心磷脂由线粒体内膜翻转至外膜,与LC3结合,阻止心磷脂外翻,抑制颅脑外伤后线粒体自噬;6,抑制线粒体自噬加剧颅脑损伤后神经炎症反应;7,抑制线粒体自噬使脑缺损面积增加;8,干扰大鼠神经功能恢复,影响平衡木实验和水迷宫实验。结论:1,颅脑损伤后,脑组织线粒体自噬显著增加;2,线粒体自噬早于细胞凋亡的发生;3,心磷脂充当识别损伤线粒体的作用,调节线粒体自噬;4,线粒体自噬起神经保护作用,抑制线粒体自噬会加剧神经炎症反应,阻碍神经功能恢复。第二部分NLRP3炎症小体在颅脑外伤中的重要作用目的:研究颅脑外伤后神经炎症反应的发生情况,探讨NLRP3在其中的重要调节作用,及与预后的关系。方法:运用临床标本,以癫痫患者的脑组织为对照组,通过免疫组化和western blotting等方法检测颅脑损伤后脑组织的神经炎症发生情况;运用siRNA敲低NLRP3后,western blotting检测敲低效果,研究其对颅脑外伤后神经炎症反应的影响;以WT、及NLRP3-/-caspase-1-/-、IL-1R-/-转基因小鼠为研究对象,构建控制性皮层损伤模型,平衡木实验和水迷宫实验,探讨颅脑外伤后肌力、运动、学习、记忆、认知等神经功能恢复情况。结果:1,免疫组化显示颅脑外伤后NLRP3、IL-1β和IL-18表达增加,提示显著增强的神经炎症反应;2,western blotting结果显示caspase-1(P20)和分泌型IL-1β明显增加;3,运行siRNA敲低nlrp3,使nlrp3低表达,后续western blotting结果显示caspase-1(P20)和IL-1β明显减少;4,在平衡木实验和水迷宫实验中,与WT小鼠相比,NLRP3-/-caspase-1-/-、IL-1R-/-转基因小鼠的外伤后神经功能恢复更好。结论:1,颅脑损伤后,NLRP3表达增加,神经炎症反应明显增强;2,敲低NLRP3,显著减少颅脑外伤引起的神经炎症反应;3,敲除NLRP3,或者caspase-1,或者IL-1R有利于颅脑外伤后神经功能恢复。第三部分Omega-3通过调节NLRP3介导的炎症反应减少颅脑外伤后细胞死亡和改善预后目的:探讨omega-3如何抑制颅脑外伤后神经炎症反应,及改善颅脑损伤后神经功能缺陷。方法:合笼后第1天,雌鼠被随机分配接受标准食物(EPA 20:5 n-3 0.02%和DHA22:6 n-3 0.02%),或高 co-3 Fas 的饮食(EPA5.3%和 DHA23.8%),或者高ω-6 Fas 的饮食;术前 6 周予以 co-3(DHA,22:6,#D2534),或者 ω-6(oleic acid,18:2,#62160),或者 ω-9(linolenic acid,18:1,#01008),或者生理盐水灌胃,2 次/周;通过ELISA、western blotting等检测不同组的颅脑外伤后神经炎症反应;使用GPR40受体拮抗剂GW1100或者siRNAGPR40,抑制ω-3 Fas的抗炎的作用;通过免疫共沉淀检测ARRB2与NLRP3结合情况;分离线粒体和胞浆蛋白,检测NLRP3和线粒体共定位情况;TNUNEL检测颅脑外伤后细胞死亡情况,同时使用western blotting检测cleaved-caspase-3表达水平;使用脑水肿实验和脑缺损面积,检测脑损伤情况;使用平衡木实验和水迷宫实验探讨外伤后神经功能恢复情况。结果:1,与sham组相比,ELISA结果显示co-3Fas抑制颅脑外伤诱导的IL-1β、IL-18 和 IL-6;2,omega-6 或-9 不能抑制 caspase-1 和 IL-1β的激活;3,siRNA GPR40或者GW1100能够显著抑制ω-3Fas的抵抗炎症作用;4,免疫共沉淀结果显示ω-3Fas促进GPR40下游蛋白ARRB2与NLRP3相结合;5,分离线粒体蛋白和胞浆蛋白,后续western blotting结果显示ω-3Fas抑制NLRP3与损伤的线粒体结合,从而减少cleaved-caspase-1和IL-1β的表达;6,ω-3Fas可显著减少颅脑外伤引起的细胞死亡;7,ω-3Fas减少颅脑外伤引起的皮层缺损和脑水肿,促进运动和认知能给恢复;8,GW1100抑制ω-3Fas对于颅脑外伤的神经保护作用。结论:1,ω-3Fas改善脑外伤引起的神经炎症反应和神经功能行为缺陷;2,ω-3Fas通过GPR40促进ARRB2与NLRP3相结合,并抑制NLRP3与损伤的线粒体结合,从而减少创伤性神经炎症反应;3,ω-3Fas通过GPR40/ARRB2/NLRP3信号通路有效减轻脑水肿和脑皮层缺损,促进颅脑外伤后神经功能恢复。
[Abstract]:The first part of the regulation of mitochondrial autophagy and apoptosis of nerve inflammation after traumatic brain injury Objective: the occurrence and the specific mechanism of mitochondrial autophagy after traumatic brain injury, to investigate the relationship between inflammatory reaction and nerve. Methods: the clinical specimens in the brain of epileptic patients as the control group, the incidence of brain tissue by electron microscopy western and blotting were used to detect brain injury and mitochondrial autophagy autophagy; WT and Sprague Dawley in GFP-LC3 transgenic mice as the research object, using the control method of cortical injury, animal model of brain injury; by immunofluorescence, Western blotting, electron microscopy detection of mitochondrial autophagy occurs; the use of BAF and 3-MA in two different autophagy inhibitor, testing its impact on mitochondrial autophagy after traumatic brain injury; on the basis of 1,3,6 and 24 hours after injury, brain tissue Detection of mitochondrial autophagy and mitochondrial release of Cyt and C, to study the relationship between mitochondrial autophagy and cell death in time; the distribution of cardiolipin in mitochondrial detection on normal condition and after traumatic brain injury; inhibition of PLS3 or CLS, the redistribution of interference of cardiolipin, testing its effect on mitochondrial autophagy after traumatic brain injury; the use of mitochondrial autophagy inhibitor mdivi-1, Sprague Dawley rats were divided into sham group, TBI group and TBI+mdivi-1 group; TUNEL and Western blotting to detect cell apoptosis after brain injury; defect detection experiments, damage of brain tissue; the balance beam test and water maze test, to study the recovery of nerve function after injury. Results: 1. Electron microscopy showed that large amounts of damage to mitochondria after brain trauma was encapsulated in a double membrane, suggesting that mitochondrial autophagy significantly increased; 2, Western blotting LC3- II protein markers of autophagy increased significantly High p62 decreased mitochondrial protein markers (COXIV, MnSOD, TOM40) decreased; 3, immunofluorescence showed GFP-LC3 and mitochondrial colocalization increased significantly; 4, mitochondrial autophagy occurred earlier than Cytc from the mitochondrial release; 5, mitochondrial cardiolipin by turning to the outer membrane, combined with LC3, preventing cardiolipin valgus inhibition of mitochondrial autophagy after traumatic brain injury; 6, inhibition of mitochondrial autophagy exacerbated brain nerve injury in inflammatory reaction; 7, inhibition of mitochondrial autophagy enables the brain defect area increased; 8, the interference effect of neural functional recovery of rats, beam balance test and water maze test. Conclusion: 1, brain injury, brain mitochondrial autophagy significantly increased; in the early 2, mitochondrial autophagy apoptosis; 3, cardiolipin acts as the damage identification of the role of mitochondria in regulating mitochondrial autophagy; 4, mitochondrial autophagy plays a role in neuroprotection, inhibition of mitochondrial autophagy exacerbates neuroinflammation, Hinder the recovery of nerve function. The second part of the important role of NLRP3 inflammasome in Brain Injury Objective: incidence of nerve inflammation after traumatic brain injury research of NLRP3, in which an important role, and the relationship with prognosis. Methods: the clinical specimens in epileptic patients with brain tissue as the control group, the incidence of nerve inflammation through brain tissue by immunohistochemistry and Western blotting methods to detect brain injury; using siRNA knockdown NLRP3, Western detection of blotting knockdown effect, to study its effect on nerve inflammation after traumatic brain injury; WT, and NLRP3-/-caspase-1-/-, IL-1R-/- transgenic mice as the research object, the construction control of cortex injury model. Balance beam test and water maze test, to investigate the brain injury after muscle strength, exercise, learning, memory, cognitive and neural functional recovery. Results: 1. Immunohistochemistry showed that the skull After traumatic brain injury NLRP3, IL-1 beta and IL-18 increase the expression of nerve inflammation prompted significantly enhanced; 2, Western blotting results showed that caspase-1 (P20) and secretory IL-1 beta increased significantly; 3, run siRNA knockdown of NLRP3, the low expression of NLRP3, the subsequent Western blotting results showed caspase-1 (P20) and IL-1 beta in 4, significantly reduced; the balance beam test and water maze test, compared with WT mice, NLRP3-/-caspase-1-/-, better functional recovery in IL-1R-/- transgenic mice after injury. Conclusion: 1, after craniocerebral injury, increase the expression of NLRP3, inflammatory reaction was enhanced obviously; 2, knockdown of NLRP3 significantly reduced brain injury of nerve inflammation by 3; knockdown of NLRP3 or caspase-1, or IL-1R, is conducive to the recovery of nerve function after traumatic brain injury. The third part Omega-3 to reduce the inflammatory reaction by regulating brain injury after NLRP3 mediated cell death and improve the prognosis of objective Study how to suppress the inflammatory response of omega-3 nerve after traumatic brain injury, and improve the defect of nerve function after brain injury. Methods: first days after mating, female rats were randomly assigned to receive standard food (EPA 20:5 n-3 0.02% and DHA22:6 n-3 0.02%, co-3) or high Fas diet (EPA5.3% and DHA23.8%), or high Omega -6 the Fas diet; 6 weeks before surgery to be co-3 (DHA, 22:6, #D2534), or -6 (oleic acid, w 18:2, #62160), or -9 (linolenic acid, w 18:1, #01008), or saline, 2 times per week; through ELISA, nerve inflammation brain injury Western blotting detection of different groups; the use of GPR40 receptor antagonists GW1100 or siRNAGPR40 inhibited Omega -3 Fas anti-inflammatory effect; by immunoprecipitation assay of ARRB2 and NLRP3 combination; separation of cytosolic and mitochondrial proteins, the detection of NLRP3 and mitochondria co localization; TNUNEL detection after craniocerebral trauma Cell death, while using Western blotting to detect cleaved-caspase-3 expression level; brain edema and brain defects using experimental area to detect brain injury; use the balance test and water maze test to investigate the recovery of nerve function after injury. Results: 1, compared with the sham group, ELISA results showed that co-3Fas inhibited brain injury induced by IL-1 beta. IL-18 and IL-6; 2, the activation of omega-6 or -9 could not inhibit caspase-1 and IL-1 beta; 3, GPR40 or GW1100 siRNA resist inflammation can significantly inhibit -3Fas; 4, results showed that Omega -3Fas promotes GPR40 downstream protein ARRB2 and NLRP3 combined immunoprecipitation; 5, separation of mitochondrial and cytoplasmic protein, follow-up western blotting results showed that -3Fas inhibited NLRP3 and Omega damage mitochondrial binding thereby reducing the expression of cleaved-caspase-1 and IL-1 beta; 6, Omega -3Fas can significantly reduce brain injury caused by Cell death; 7, Omega -3Fas reduce craniocerebral trauma caused by the cortical defect and brain edema, promote the movement and cognition to recovery; 8, GW1100 inhibited the Omega -3Fas protective effect on nerve craniocerebral injury. Conclusion: 1, nerve inflammation and nerve function Omega -3Fas caused by traumatic brain injury can improve the behavior of defects; 2. -3Fas through the promotion of GPR40 ARRB2 combined with NLRP3, and inhibit the binding of NLRP3 and the damage of mitochondria, thereby reducing traumatic nerve inflammation; 3, Omega -3Fas via GPR40/ARRB2/NLRP3 pathway effectively reduce brain edema and cerebral cortex defect, promote the recovery of neurological function after cerebral trauma.

【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R651.15

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