富马酸二甲酯对大鼠蛛网膜下腔出血后早期脑损伤保护作用和机制的实验研究

发布时间：2018-03-31 00:13

  本文选题：富马酸二甲酯　切入点：早期脑损伤　出处：《苏州大学》2014年硕士论文

【摘要】：第一部分富马酸二甲酯对大鼠蛛网膜下腔出血后早期脑损伤保护的研究 目的：通过建立大鼠蛛网膜下腔出血（subarachnoid hemorrhage，SAH）后脑损伤早期模型，初步探讨富马酸二甲酯（Dimethylfumarate，DMF）对SD大鼠蛛网膜下腔出血后早期脑损伤是否具有神经保护作用。 方法：成年健康雄性SD大鼠96只，随机分为两部分（48只/部分），一部分观察行为功能评分和水肿指数，另一部分单纯观察血脑屏障。每部分分为4组，对照组（n=12）、SAH组（n=12）、安慰剂组（n=12）、DMF组（n=12）。经大鼠自体非肝素化股动脉动脉血注入视交叉前池，建立蛛网膜下腔出血后脑损伤早期模型。建立模型后1h、12h、24h和36h经胃管灌注DMF（15mg/kg），48h观察大鼠行为评分和功能变化，立即处死大鼠，取大鼠颞底皮层做标本，测定血脑屏障和脑水肿变化，探讨富马酸二甲酯对大鼠蛛网膜下腔出血后早期脑损伤是否具有神经保护作用。 结果：通过对比对照组大鼠，SAH组大鼠食欲、认知功能和活动能力明显降低，血脑屏障破坏明显，建模后48h测定大鼠皮层组织伊文氏蓝（EB）含量达到21.93ng/mg，脑组织含水量明显增加，48h水肿百分数达到82.45%；相比SAH组，富马酸二甲酯治疗组神经功能明显改善，48h测定血脑屏障和脑水肿指数分别为：14.12ng/mg、80.72%，并有效改善血脑屏障和降低脑水肿指数；安慰剂组与SAH组相对改善不明显。 结论：1.建立SD大鼠SAH模型后，观察到大鼠SAH后48h神经行为学功能评分明显降低，血脑屏障破坏明显和脑组织水肿含量增加显著，说明大鼠SAH后存在早期脑损伤（EBI）。2.通过胃管灌注DMF后，改善大鼠SAH后神经行为功能、血脑屏障和降低脑水肿，进而说明DMF对大鼠SAH后EBI具有一定神经保护作用。 第二部分富马酸二甲酯对大鼠SAH后大脑皮层Keap1-Nrf2-ARE氧化应激传导通路的调控作用 目的：观察大鼠SAH后大脑皮层Keap1-Nrf2-ARE氧化应激传导通路及其下游抗氧化酶基因表达变化及富马酸二甲酯对Keap1-Nrf2-ARE氧化应激传导通路影响，探讨富马酸二甲酯对SAH后早期脑损伤神经保护作用机制。 方法：健康成年雄性SD大鼠48只，随机化分为：对照组(n=12), SAH组(n=12),安慰剂组(n=12)和DMF组(n=12)。采用向视交叉前池注血技术，构建大鼠SAH模型，，对照组开骨窗后不注入动脉血，DMF组给予胃管灌注DMF治疗，每次注射剂量15mg/kg，分别在SAH后1h、12、24h和36h胃管灌注，共灌注4次。安慰剂+SAH组注射等容量溶剂（生理盐水，15mg/kg）。48小时后断头处死大鼠，立即取大鼠颞底皮层脑组织后做标本检测，采用RT-PCR法测定NQO1、GST-α1和HO-1的mRNA表达，酶活性检测GST-α1和NQO1量；采用免疫组化方法检测HO-1、Nrf2和Keap1表达；Western blot法测定Nrf2、Keap1和HO-1变化；Fluoro-jade B和TUNEL染色方法检测神经细胞凋亡情况；EMSA法检测Nrf2的DNA结合情况和结合活性；ELISA法测定IL-1β、IL-6和TNF-α炎症介质的表达。 结果：RT-PCR结果显示对照组NQO1、GST-α1和HO-1的mRNA表达量较低，和对照组相比，SAH组NQO1、GST-α1和HO-1的mRNA表达水平明显增高（P 0.01），安慰剂组与SAH组对比无明显差异（P0.05），在DMF治疗组中NQO1、GST-α1和HO-1的mRNA表达水平明显高于安慰剂组和SAH组（P 0.01）；酶活性检测结果提示安慰剂组和SAH组的GST-α1、NQO1酶活性明显高于对照组（P 0.01），而安慰剂组和SAH组对比无明显差异（P0.05），DMF组中GST-α1、NQO1酶活性明显高于SAH组（P 0.01，P 0.05）；免疫组化法检测结果（Nrf2、Keap1和HO-1免疫反应性主要在脑组织中神经元细胞内，阳性：细胞质棕色黄染）显示对照组Nrf2、Keap1和HO-1阳性率较低，与对照组相比，SAH组和安慰剂组的Nrf2、Keap1和HO-1阳性率明显增高（P 0.01），安慰剂组与SAH组对比无明显差异（P0.05），DMF治疗组中Nrf2、Keap1和HO-1阳性率高于SAH组及安慰剂组（P 0.01）；Western blot结果显示对照组Nrf2、Keap1和HO-1蛋白水平较低，和对照组相比，SAH组Nrf2、Keap1和HO-1蛋白水平明显增高（P 0.01），安慰剂组与SAH组对比无明显差异（P0.05），DMF组中Nrf2、Keap1和HO-1蛋白水平明显高于SAH组和安慰剂组（P 0.01）；TUNEL和Fluoro-jade B染色技术检测结果表明，在对照组有少量凋亡细胞，而安慰剂组和SAH组大鼠脑组织凋亡率高于对照组（P 0.01），安慰剂组与SAH组对比无明显差异（P0.05），DMF治疗组中大鼠皮层脑组织凋亡率明显低于安慰剂组与SAH组（P0.01）；EMSA法检测结果显示，DMF治疗Nrf2的DNA结合情况和结合活性明显高于对照组、安慰剂组和SAH组（P 0.01），安慰剂组与SAH组对比无显著差异（P0.05）；ELISA法测定结提示DMF治疗组的IL-1β、IL-6和TNF-α炎症介质的表达量明显低于安慰剂组和SAH组（P 0.01），安慰剂组与SAH组对比无显著差异（P0.05）。 结论：1.在SD大鼠SAH后48h能够激活Keap1、Nrf2通路蛋白及下游抗氧化酶基因表达，产生抗氧化作用，减轻免疫炎症，进而保护神经细胞。2.富马酸二甲酯可能通过激活和稳定Keap1-Nrf2-ARE氧化应激传导通路，达到对大鼠SAH后EBI神经细胞保护。
[Abstract]:The first part of the study on the protection of early brain injury after subarachnoid hemorrhage in rats by two methyl fumarate
Objective: to establish a rat brain injury model after subarachnoid hemorrhage (subarachnoid hemorrhage, SAH), and to preliminarily explore whether neuroprotective effect of Dimethylfumarate (DMF) on early brain injury after subarachnoid hemorrhage in SD rats is two.
Methods: 96 healthy adult male SD rats were randomly divided into two parts (48 / part), a part of observed behavior score and edema index, the other part simply observe the blood brain barrier. Each part is divided into 4 groups, the control group (n=12), SAH group (n=12) and placebo group (n=12), DMF group (n=12). The rat autologous non heparinized femoral artery blood into the prechiasmatic cistern, a model of early brain injury after subarachnoid hemorrhage. After establishing the model of 1H, 12h, 24h and 36h by gastric perfusion of DMF (15mg/kg) 48h, observe the changes of rat's behavior score and function. The rats were sacrificed immediately, from rat cortex temporal base were measured the blood brain barrier and brain edema, two of fumaric acid methyl ester on rats after subarachnoid hemorrhage in early brain injury has neuroprotective effect.
Results: the rats in control group compared with SAH group, the appetite of rats, cognitive function and activity decreased significantly, blood brain barrier damage obviously after modeling 48h determination of rat cortical tissue of Evans blue (EB) content reached 21.93ng/mg, the brain water content increased significantly, 48h 100 edema score of 82.45%; compared with the SAH group. Two methyl fumarate treatment group nerve function improved significantly, 48h determination of blood-brain barrier and cerebral edema index were 14.12ng/mg, 80.72%, and effectively improve the blood brain barrier and reduce cerebral edema index; placebo group and SAH group is no obvious improvement.
Conclusion: 1. to establish a SD rat model of SAH, observed in rats after SAH 48h neurological function score was significantly decreased, blood brain barrier damage and brain edema were significantly increased significantly after SAH in rats, indicating the presence of early brain injury (EBI).2. by gastric perfusion after DMF, improve the neural function of rats after SAH, the blood brain barrier and reduce cerebral edema, and show that DMF has a neuroprotective effect on rats with SAH EBI.
The regulatory effect of second part of two methyl fumarate on the oxidative stress pathway of Keap1-Nrf2-ARE in the cerebral cortex of rats after SAH
Objective: To observe the cerebral cortex of rats after SAH Keap1-Nrf2-ARE oxidative stress pathway and its downstream antioxidant enzymes gene expression changes and oxidative stress of Keap1-Nrf2-ARE methyl fumarate two pathway of fumaric acid methyl ester on two for the protection of early brain injury after SAH neural mechanism.
Methods: 48 adult male SD rats were randomly divided into control group (n=12), SAH group (n=12) and placebo group (n=12) and DMF group (n=12). To use the prechiasmatic cistern blood injection technology, construct the SAH model of rats and the control group after injected into the artery open bone window the blood group DMF were given intragastric infusion of DMF treatment, each dose of 15mg/kg, respectively, after SAH 1H, 12,24h and 36h of gastric tube perfusion, perfusion were 4 times. The placebo +SAH groups received the equal volume of solvent (saline, 15mg/kg).48 hours after the rats were decapitated immediately, from rat brain tissue after cortical temporal base do samples, NQO1 was determined by RT-PCR method. The expression of GST- 1 and HO-1 mRNA, detection of enzyme activity of GST- alpha 1 and NQO1; HO-1 was detected by immunohistochemistry method, the expression of Nrf2 and Keap1; Nrf2 Western blot determination method, Keap1 and HO-1 Fluoro-jade B and TUNEL staining; neuron apoptosis were detected. Method; EMSA method The DNA binding and binding activity of Nrf2 were detected, and the expression of IL-1 beta, IL-6 and TNF- alpha inflammatory mediators was measured by ELISA.
缁撴灉锛歊T-PCR缁撴灉鏄剧ず瀵圭収缁凬QO1,GST-伪1鍜孒O-1鐨刴RNA琛ㄨ揪閲忚緝浣

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