格列本脲在脑出血后神经保护效应及机制研究

发布时间：2018-06-09 07:07

本文选题：脑出血 + 磺酰脲类受体1　；参考：《第三军医大学》2017年硕士论文

【摘要】：脑水肿是脑出血(intracerebral hemorrhage,ICH)后继发脑损伤的关键环节,而血脑屏障(blood-brain barrier,BBB)的破坏是ICH后脑水肿重要的始动因素。研究保护血脑屏障完整性的有效措施对减轻临床脑出血后的继发脑损伤具有重要意义。格列本脲(Glibenclamide,GLC)为磺酰脲类药物,被临床用于口服降糖已有数十年;磺酰脲类药物都是通过阻断磺酰脲类受体1(sulfonylurea receptor 1,Sur1)发挥药理作用。磺酰脲类受体1参与构成并调节Sur1-Kir6.2和Sur1-Trpm4两种离子通道。Sur1-Kir6.2通道和Sur1-Trpm4通道有相反的生理学作用,Sur1-Kir6.2通道开放细胞超激化,Sur1-Trpm4通道开放时细胞去激化,细胞超激化和去激化参与不同的生理过程;Sur1-Trpm4通道去极化具有减少病理性钙离子通过电压门控通道内流的作用,但如果失去正常调节,过度开放阳离子会通过该通道内流引起细胞毒性水肿和细胞坏死;Sur1-Kir6.2通道开放细胞超激化具有减少钙离子通过电压门控通道内流的重要作用,但如果过度开放,ATP耗竭引起神经元能量代谢紊乱并减慢小胶质细胞对外界刺激的反应速度。最近格列本脲通过阻断磺酰脲类受体1在缺血性卒中、脊髓损伤、颅内转移瘤、蛛网膜下腔出血等中枢神经系统疾病模型研究中发挥神经保护效应而被广泛关注,糖尿病合并卒中患者回顾性研究分析发现:卒中后磺酰脲类药物的使用可获得更好的预后。课题组前期研究成果及相关研究报道基质金属蛋白酶(metalloproteinases,MMPs)在脑出血后血脑屏障完整性破坏中起重要作用,格列本脲能减少基质金属蛋白酶的表达或降低其活性。然而,格列本脲在脑出血中的作用目前仍不清楚。本课题假设格列本脲能通过阻断Sur1-Trpm4通道抑制基质金属蛋白酶表达、保护血脑屏障紧密连接蛋白从而发挥潜在的神经保护效应;课题选用大鼠自体血注射脑出血模型进行实验研究,观察脑出后神经功能缺损、血脑屏障破坏、脑水肿的变化特征;并以血肿周围脑组织Sur1表达上调为前提,观察探索格列苯脲对大鼠脑出血后脑水肿、血脑屏障、神经功能的干预效应和相关机制。为此,将课题分成了两个研究部分来验证上述假设:第一部分,建立成年SD大鼠脑出血模型,观察出血损伤对大鼠脑组织磺酰脲类受体1表达的影响;第二部分,建立大鼠脑出血模型后,给予格列本脲干预,观察格列本脲对脑出血模型后脑水含量、神经功能恢复、血脑屏障完整性、基质金属蛋白酶表达的影响。一、大鼠脑出血后血肿周围Sur1表达目的通过自体血注入方法建立大鼠ICH模型,动态观察出血后Sur1及相关离子通道表达变化,评估出血损伤对血肿周围脑组织Sur1表达的调节。方法实验动物随机分成ICH组和Sham组;ICH组将自体血(取自实验大鼠股动脉)100μL立即缓慢(约10μL/min)注入右侧基底神经节;对照组与ICH组进行同样的手术过程,但不注入自体血液。于术后12h、24h、48h收集脑标本行PCR,术后24h收集脑标本行Western blot检测、免疫荧光染色。结果大鼠实验性自体血脑出血模型血肿周围脑组织Sur1表达明显上调,但Kir6.2通道蛋白未探及表达增加。因此推断:出血性损伤后,血肿周围脑组织Sur1表达上调主要是伴随Sur1-Trpm4通道的表达上调。结论出血性损伤后,血肿周围脑组织Sur1-Trpm4通道表达上调。二、大鼠脑出血后格列本脲的干预效应目的建立成年SD大鼠脑出血模型,给予格列本脲干预;观察大鼠ICH后的神经功能缺损、脑水肿等情况以及运用格列本脲干预的神经保护效应,探讨格列本脲神经保护效应的可能机制。方法将实验动物随机分为对照组(Sham组)、ICH+Vehicle(Vehicle组)、ICH+GLC(GLC组);大鼠脑出血模型建立后CLC组立即给予格列本脲10μg/kg腹腔注射,并皮下植入微量泵以200ng/h的速度持续泵入(建模后15分钟内完成给药);Vehicle组用相同方式给予相同剂量的溶剂。在建模后24小时、72小时测定脑组织水含量;建模后每天行改良神经功能评分(m NSS);建模后23天开始行水迷宫测试训练;建模后24小时行Western blotting免疫印迹分析MMP9表达情况,72小时行Western blotting免疫印迹分析ZO-1、occludin表达情况;建模72小时切片行免疫荧光观察血脑屏障完整性、MMP9的表达;建模后72小时切片观察伊文思蓝(Evans-blue)渗漏;建模后12小时、24小时、48小时收集标本行PCR检测MMP2、MMP9、MMP12 m RNA表达变化。结果1.格列本脲干预能减轻ICH后脑水含量,促进ICH后神经功能(改良的神经功能评分)恢复。2.格列本脲干预减少ICH后血肿周围脑组织伊文思蓝渗漏。3.格列本脲干预能改善ICH模型实验大鼠空间学习记忆能力(通过水迷宫实验测试)中的表现。4.格列本脲干预保护ICH后血脑屏障的完整性。5.格列本脲干预能减少ICH后血肿周围脑组织MMPs的表达。结论在大鼠脑出血模型实验中观察到:格列本脲能保护血脑屏障、改善脑水肿、改善神经功能,并且这种神经保护作用可能与格列本脲减少MMPs的表达有关。
[Abstract]:Cerebral edema is the key link of brain injury after intracerebral hemorrhage (ICH), and the destruction of blood-brain barrier (BBB) is an important starting factor for the cerebral edema after ICH. The effective measures to protect the integrity of the blood brain barrier are of great significance to reduce the secondary brain injury after clinical cerebral hemorrhage. Glibenclamide, GLC) is a sulfonylurea drug that has been clinically used for oral hypoglycemic for decades; sulfonylurea has been involved in pharmacological action by blocking sulfonylurea receptor 1 (sulfonylurea receptor 1, Sur1). Sulfonylurea receptor 1 participates in the formation and regulation of Sur1-Kir6.2 and Sur1-Trpm4 two ion channels.Sur1-Kir6.2 channels and Sur1-Trpm The 4 channel has the opposite physiological function, the Sur1-Kir6.2 channel open cell overintensification, the Sur1-Trpm4 channel opening when the opening is open, the cell excates, the cell overintensification and the intensification participates in the different physiological processes; the Sur1-Trpm4 channel depolarization can reduce the role of the pathological calcium ion through the voltage gated channel, but if the normal regulation is lost, it is excessive. Open cations can cause cytotoxic edema and cell necrosis through the flow of the channel. The overactivation of Sur1-Kir6.2 channel open cells has an important role in reducing the flow of calcium ions through the voltage-gated channel. However, if overopen, the depletion of ATP causes the energy metabolism disorder of neurons and slows the response of microglia to external stimuli. Neuroprotective effects of sulfonylurea receptor 1 in ischemic stroke, spinal cord injury, intracranial metastases, subarachnoid hemorrhage and other central nervous system disease models are widely concerned. A retrospective study of diabetes combined with stroke patients has been found: the use of sulfonylureas after stroke Metalloproteinases (MMPs) plays an important role in the integrity destruction of the blood brain barrier after intracerebral hemorrhage. Glibenclamide can reduce the expression of matrix metalloproteinase or reduce its viability. However, the role of glibenclamide in cerebral hemorrhage is present. It is still unclear. It is hypothesize that glibenclamide can inhibit the expression of matrix metalloproteinase by blocking the Sur1-Trpm4 channel and protect the close connexin of blood brain barrier and thus play a potential neuroprotective effect. The changes of brain edema and the up-regulation of Sur1 expression in the brain tissue around the hematoma were used to observe the intervention effects and related mechanisms of glibenclamide on cerebral edema, blood brain barrier and nerve function in rats after intracerebral hemorrhage. To this end, the subjects were divided into two parts to verify the above hypothesis: the first part was to establish the brain of adult SD rats. Blood model, observe the effect of bleeding injury on the expression of sulfonylureas receptor 1 in rat brain tissue; the second part, after establishing rat model of cerebral hemorrhage, the effect of glibenclamide on brain water content, nerve function recovery, blood brain barrier integrity and expression of matrix metalloproteinase after cerebral hemorrhage model. The expression of Sur1 in the posterior hematoma was established by autologous blood injection to establish the rat ICH model. The changes of Sur1 and related ion channel expression after bleeding were dynamically observed and the expression of Sur1 in the brain tissue around hematoma was regulated by bleeding injury. Methods the experimental animals were randomly divided into ICH and Sham groups, and the ICH group took the autologous blood (from the femoral artery of experimental rats) 100 L was injected into the right basal ganglia immediately and slowly (about 10 L/min); the control group performed the same procedure with the ICH group, but did not infuse the autologous blood. After the operation, 12h, 24h, 48h collected the brain line PCR, and the 24h collection was collected by Western blot detection, immunofluorescence staining. The expression of Sur1 was obviously up-regulated, but the expression of Kir6.2 channel protein was not detected and expressed. Therefore, after hemorrhagic injury, the up regulation of Sur1 expression around the hematoma was mainly associated with the up regulation of the expression of the Sur1-Trpm4 channel. Conclusion the expression of Sur1-Trpm4 channel in the brain tissue around hematoma is up to up after hemorrhagic injury. Two, glibenclamide after intracerebral hemorrhage in rats. Objective to establish the cerebral hemorrhage model of adult SD rats and give glibenclamide intervention, observe the nerve function defect, brain edema after ICH and the neuroprotective effect of glibenclamide, and explore the possible mechanism of neuroprotective effect of glibenclamide. Methods the experimental animals were randomly divided into control group (group Sham), ICH+Ve Hicle (group Vehicle), ICH+GLC (group GLC); after the rat model of cerebral hemorrhage was established, the CLC group was given an injection of glipezourea 10 u g/kg, and the micropump was implanted subcutaneously for 200ng/h at the rate of 200ng/h, and the Vehicle group was given the same dose of solvent in the same way. The brain group was determined at 24 hours after modeling and 72 hours after modeling. Water content was improved after modeling (m NSS) every day after modeling; water maze test training was conducted 23 days after modeling. MMP9 expression was analyzed by Western blotting immunoblotting at 24 hours after modeling, ZO-1 with Western blotting immunoblotting was performed at 72 hours, and occludin table was reached, and the blood brain was observed by immunofluorescence for 72 hours of modeling. Barrier integrity, MMP9 expression, 72 hours after modeling, observation of Evans blue (Evans-blue) leakage; after modeling 12 hours, 24 hours, 48 hours, collection of specimens, PCR MMP2, MMP9, MMP12 m RNA expression changes. Results 1. glibenclamide intervention can reduce the content of the brain water after ICH, promote ICH neural function (improved neural function score) recovery.2. The intervention of glibenclamide intervention to reduce the eevith blue leakage of the peripheral brain tissue around the hematoma after ICH to improve the spatial learning and memory ability of the ICH model rats (through the water maze test) the performance of.4. glibenclamide interfered with the integrity of the blood brain barrier after the protection of ICH by.5. glibenclamide intervention to reduce the brain tissue around the hematoma after ICH. MMPs expression. Conclusion glibenclamide can protect the blood brain barrier, improve brain edema and improve the function of nerve, and this neuroprotective effect may be related to the expression of glibenclamide to reduce the expression of MMPs.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R743.34

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