本文選題：富馬酸二甲酯　切入點：早期腦損傷　出處：《蘇州大學》2014年碩士論文

【摘要】：第一部分富馬酸二甲酯對大鼠蛛網膜下腔出血后早期腦損傷保護的研究 目的：通過建立大鼠蛛網膜下腔出血（subarachnoid hemorrhage，SAH）后腦損傷早期模型，初步探討富馬酸二甲酯（Dimethylfumarate，DMF）對SD大鼠蛛網膜下腔出血后早期腦損傷是否具有神經保護作用。 方法：成年健康雄性SD大鼠96只，隨機分為兩部分（48只/部分），一部分觀察行為功能評分和水腫指數(shù)，另一部分單純觀察血腦屏障。每部分分為4組，對照組（n=12）、SAH組（n=12）、安慰劑組（n=12）、DMF組（n=12）。經大鼠自體非肝素化股動脈動脈血注入視交叉前池，建立蛛網膜下腔出血后腦損傷早期模型。建立模型后1h、12h、24h和36h經胃管灌注DMF（15mg/kg），48h觀察大鼠行為評分和功能變化，立即處死大鼠，取大鼠顳底皮層做標本，測定血腦屏障和腦水腫變化，探討富馬酸二甲酯對大鼠蛛網膜下腔出血后早期腦損傷是否具有神經保護作用。 結果：通過對比對照組大鼠，SAH組大鼠食欲、認知功能和活動能力明顯降低，血腦屏障破壞明顯，建模后48h測定大鼠皮層組織伊文氏藍（EB）含量達到21.93ng/mg，腦組織含水量明顯增加，48h水腫百分數(shù)達到82.45%；相比SAH組，富馬酸二甲酯治療組神經功能明顯改善，48h測定血腦屏障和腦水腫指數(shù)分別為：14.12ng/mg、80.72%，并有效改善血腦屏障和降低腦水腫指數(shù)；安慰劑組與SAH組相對改善不明顯。 結論：1.建立SD大鼠SAH模型后，觀察到大鼠SAH后48h神經行為學功能評分明顯降低，血腦屏障破壞明顯和腦組織水腫含量增加顯著，說明大鼠SAH后存在早期腦損傷（EBI）。2.通過胃管灌注DMF后，改善大鼠SAH后神經行為功能、血腦屏障和降低腦水腫，進而說明DMF對大鼠SAH后EBI具有一定神經保護作用。 第二部分富馬酸二甲酯對大鼠SAH后大腦皮層Keap1-Nrf2-ARE氧化應激傳導通路的調控作用 目的：觀察大鼠SAH后大腦皮層Keap1-Nrf2-ARE氧化應激傳導通路及其下游抗氧化酶基因表達變化及富馬酸二甲酯對Keap1-Nrf2-ARE氧化應激傳導通路影響，探討富馬酸二甲酯對SAH后早期腦損傷神經保護作用機制。 方法：健康成年雄性SD大鼠48只，隨機化分為：對照組(n=12), SAH組(n=12),安慰劑組(n=12)和DMF組(n=12)。采用向視交叉前池注血技術，構建大鼠SAH模型，，對照組開骨窗后不注入動脈血，DMF組給予胃管灌注DMF治療，每次注射劑量15mg/kg，分別在SAH后1h、12、24h和36h胃管灌注，共灌注4次。安慰劑+SAH組注射等容量溶劑（生理鹽水，15mg/kg）。48小時后斷頭處死大鼠，立即取大鼠顳底皮層腦組織后做標本檢測，采用RT-PCR法測定NQO1、GST-α1和HO-1的mRNA表達，酶活性檢測GST-α1和NQO1量；采用免疫組化方法檢測HO-1、Nrf2和Keap1表達；Western blot法測定Nrf2、Keap1和HO-1變化；Fluoro-jade B和TUNEL染色方法檢測神經細胞凋亡情況；EMSA法檢測Nrf2的DNA結合情況和結合活性；ELISA法測定IL-1β、IL-6和TNF-α炎癥介質的表達。 結果：RT-PCR結果顯示對照組NQO1、GST-α1和HO-1的mRNA表達量較低，和對照組相比，SAH組NQO1、GST-α1和HO-1的mRNA表達水平明顯增高（P 0.01），安慰劑組與SAH組對比無明顯差異（P0.05），在DMF治療組中NQO1、GST-α1和HO-1的mRNA表達水平明顯高于安慰劑組和SAH組（P 0.01）；酶活性檢測結果提示安慰劑組和SAH組的GST-α1、NQO1酶活性明顯高于對照組（P 0.01），而安慰劑組和SAH組對比無明顯差異（P0.05），DMF組中GST-α1、NQO1酶活性明顯高于SAH組（P 0.01，P 0.05）；免疫組化法檢測結果（Nrf2、Keap1和HO-1免疫反應性主要在腦組織中神經元細胞內，陽性：細胞質棕色黃染）顯示對照組Nrf2、Keap1和HO-1陽性率較低，與對照組相比，SAH組和安慰劑組的Nrf2、Keap1和HO-1陽性率明顯增高（P 0.01），安慰劑組與SAH組對比無明顯差異（P0.05），DMF治療組中Nrf2、Keap1和HO-1陽性率高于SAH組及安慰劑組（P 0.01）；Western blot結果顯示對照組Nrf2、Keap1和HO-1蛋白水平較低，和對照組相比，SAH組Nrf2、Keap1和HO-1蛋白水平明顯增高（P 0.01），安慰劑組與SAH組對比無明顯差異（P0.05），DMF組中Nrf2、Keap1和HO-1蛋白水平明顯高于SAH組和安慰劑組（P 0.01）；TUNEL和Fluoro-jade B染色技術檢測結果表明，在對照組有少量凋亡細胞，而安慰劑組和SAH組大鼠腦組織凋亡率高于對照組（P 0.01），安慰劑組與SAH組對比無明顯差異（P0.05），DMF治療組中大鼠皮層腦組織凋亡率明顯低于安慰劑組與SAH組（P0.01）；EMSA法檢測結果顯示，DMF治療Nrf2的DNA結合情況和結合活性明顯高于對照組、安慰劑組和SAH組（P 0.01），安慰劑組與SAH組對比無顯著差異（P0.05）；ELISA法測定結提示DMF治療組的IL-1β、IL-6和TNF-α炎癥介質的表達量明顯低于安慰劑組和SAH組（P 0.01），安慰劑組與SAH組對比無顯著差異（P0.05）。 結論：1.在SD大鼠SAH后48h能夠激活Keap1、Nrf2通路蛋白及下游抗氧化酶基因表達，產生抗氧化作用，減輕免疫炎癥，進而保護神經細胞。2.富馬酸二甲酯可能通過激活和穩(wěn)定Keap1-Nrf2-ARE氧化應激傳導通路，達到對大鼠SAH后EBI神經細胞保護。
[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.
緇撴灉錛歊T-PCR緇撴灉鏄劇ず瀵圭収緇凬QO1,GST-偽1鍜孒O-1鐨刴RNA琛ㄨ揪閲忚緝浣

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