【作者】 陈扬；

【导师】 郭莲军；

【作者基本信息】 华中科技大学 ， 药理学， 2012， 博士

【摘要】 米邦塔仙人掌多糖提取鉴定及对缺血性脑损伤的保护作用研究目的本研究拟通过提取、分离、纯化获得米邦塔仙人掌多糖并进行相应的含量及组分鉴定；同时建立整体脑缺血复灌模型和离体原代培养神经元氧糖剥夺(oxygen-glucose deprivation, OGD)模型,分别从整体和离体水平,观察米邦塔仙人掌多糖对脑缺血诱发的学习记忆能力损害及缺血性脑损伤的保护作用,并从认知相关基因的表达以及对原代培养神经元细胞内氧自由基浓度的变化、钙离子浓度的影响、细胞培养外液中兴奋性氨基酸的含量变化等方面进行系统的药效学及作用机制方面的研究,综合探讨米邦塔仙人掌多糖对缺血性脑卒中保护的作用效果及机制,阐明对缺血性脑损伤保护作用及可能的作用靶点,为开发米邦塔仙人掌多糖的药用价值提供可靠的科学依据,为今后的临床研究提供可靠的实验依据；对于充分利用米邦塔仙人掌的丰富资源,促进米邦塔仙人掌的产业发展,将会产生巨大的社会和经济效益。方法和结果第一部分米邦塔仙人掌多糖提取分离及组分鉴定将米邦塔仙人掌切碎,干燥后,通过水溶醇沉的方法得到粗多糖,然后将粗多糖过DEAE-纤维素分离柱,根据苯酚-硫酸法接取纯化多糖成分及检测多糖的纯度。经气相色谱-质谱联用(GC-MS)法分析米邦塔仙人掌多糖的单糖成分及相应的含量,通过凝胶渗透色谱(GPC)法分析米邦塔仙人掌多糖片段的分子量大小。结果显示：提取纯化的米邦塔仙人掌多糖纯度达到84%,主要单糖成分及含量分别是：甘露糖(6.37%),鼠李糖(14.94%),木糖(1.99%),阿拉伯糖(24.07%),半乳糖(38.25%),核糖(2.63%)及葡萄糖(11.48%),米邦塔仙人掌多糖的GPC谱图主要显示有两个多糖片段,且Mn分别为5.40×105和2270。第二部分米邦塔仙人掌多糖对原代培养神经元氧糖剥夺所致的损伤模型的保护作用及机制研究离体缺血细胞模型采用原代培养大鼠脑皮质、海马神经元氧糖剥夺损伤模型(OGD)。并通过物理缺氧损伤及化学缺氧损伤两种OGD损伤方式检测米邦塔仙人掌多糖的细胞毒性影响；同时通过检测细胞形态,MTT代谢率和乳酸脱氢酶(Lactate dehydrogenase, LDH)活性综合评价米邦塔仙人掌多糖的神经细胞保护作用；采用高效液相色谱(High performance liquid chromatography, HPLC)荧光检测技术测定培养皮层神经元细胞外液中谷氨酸(Glumatic, Glu)的含量；进一步通过检测神经元细胞胞内活性氧(ROS)及钙离子浓度(Intracellular calcium concentrations, [Ca2+]ⅰ)的变化来探讨MAPs的神经保护作用机制。结果显示：MTT和LDH检测发现,米邦塔仙人掌多糖无细胞毒性,且无论原代培养神经元细胞是通过物理OGD损伤还是化学OGD损伤,MAPs均可减轻OGD引起的神经元损伤,根据荧光标记检测实验方法发现OGD会引起胞内ROS及钙离子浓度升高,HPLC检测发现OGD会引起的胞外氨基酸浓度的增高,但给予MAPs处理后均可逆转细胞外液的Glu、细胞内基础氧自由基和钙离子浓度的升高。第三部分米邦塔仙人掌多糖对小鼠/大鼠脑缺血再灌注损伤的保护作用及机制研究采用局灶性脑缺血-复灌损伤(Middle cerebral artery occlusion, MCAO)或双侧颈总动脉结扎-复灌损伤(Permanent occlusion of the bilateral CCA,2VO)所致的脑缺血再灌注模型分析药物的保护及作用机制的研究。实验动物随机分为假手术组、缺血模型组和MAPs治疗组。MCAO模型：术前48 h,24 h,12 h及术后10 min各组分别给予生理盐水和MAPs治疗,运用TTC染色观察脑梗死体积；运用神经行为学评分分析小鼠的脑缺血后神经行为学的改变。2VO模型：术前48 h,24 h,12h及术后10 min各组分别给予生理盐水和相应药物治疗,同时每天给予药物治疗一次,持续20天。运用Morris水迷宫行为学方法检测小鼠空间学习记忆能力；运用事物记忆行为学方法检测小鼠对新事物认知能力；运用HE染色观察组织形态学改变；运用qRT-PCR、Western blot技术分析各实验组之间的NMDAR2B、NMDAR1、BDNF mRNA和BDNF蛋白的表达水平。结果显示：神经行为学评分发现模型组小鼠有明显的神经行为学障碍,脑梗死体积明显增加,Morris水迷宫检测模型组小鼠学习记忆能力受损,与假手术组比较逃避潜伏期延长、空间辨别能力下降；组织学观察模型组小鼠皮层组织结构异常；结扎双侧颈总动脉导致弥漫性前脑缺血损伤的模型组皮层部位NMDAR2B以及BDNF的mRNA和BDNF蛋白表达减少。给予米邦塔仙人掌多糖治疗后能改善缺血引起的神经行为障碍,减少相应脑区梗死面积,改善脑缺血所致的学习记忆障碍,减少神经元丢失,并增加NMDAR2B、NMDAR1和BDNF的表达。结论提取纯化的米邦塔仙人掌多糖的主要单糖成分及含量分别是：甘露糖(6.37%),鼠李糖(14.94%),木糖(1.99%),阿拉伯糖(24.07%),半乳糖(38.25%),核糖(2.63%)及葡萄糖(11.48%),且米邦塔仙人掌多糖经GPC法分析后得出主要有两个多糖片段组成,Mn分别为5.40×105和2270。米邦塔仙人掌多糖可显著改善脑缺血所致的学习记忆能力损害及相关神经行为学改变,同时可以改善OGD引起的神经元损伤,其抗损伤的机制可能涉及以下几方面：减轻自由基损伤,增加认知相关基因NMDAR2B和脑源性神经生长因子BDNF的表达,抑制谷氨酸的兴奋毒性和神经元胞内活性氧的浓度及防止细胞内钙超载现象发生。更多还原

【Abstract】 Opuntia Milpa Alta Polysaccharides Extraction and Structure identification and Protection Activity on Cerebral IschemiaAIMTo extract and isolate polysaccharides from Opuntia Milpa Alta (MAPs) and analysis the polysaccharides composition, further to investigate the effects and mechanisms of MAPs in vivo and in vitro model of cerebral ischemic injury demonstrated that the polysaccharides had potent neuroprotective. We tried to explore the protective effects of MAPs on cerebral ischemia in vivo with ischemia induced by cerebral hypoperfusion after the bilateral common carotid artery (2VO) and middle cerebral artery occlusion (MCAO). In vitro, rat cortical and hippocampal neurons have suffered from oxygen-glucose deprivation (OGD). To analysis the mechanisms of its action, we have systematically investigated the effect of MAPs on the expression of NMDAR2B, NMDAR1 and BDNF, the content of important neurotransmitter amino acid, the concentration of reactive oxygen species and intracellular calcium.METHODS AND RESULTSPartⅠMAPs extraction and structure identificationOpuntia Milpa Alta has been cut up and drying. We extracted polysaccharides from Opuntia Milpa Alta through water extraction and alcohol precipitation method and used DEAE-cellulose-52 column to purification the polysaccharides fragment. The contents of polysaccharides were determined by Phenol sulfuric Acid Method. MAPs composition was analyzed using Gas Chromatography-Mass Spectrometer (GC-MS) method. And the Molecular weight size through the Gel Permeation Chromatography (GPC) detection. Results:MAPs are extracted by our separation method contained and sugar to 84%. Sugar composition analysis revealed that MAPs consisted primarily of Mannose (6.37%), Rhamnose (14.94%), Xylose (1.99%), Arabinose (24.07%), Galactose (38.25%), Ribose (2.63%), Glucose (11.48%). The GPC analysis for Molecular weight MAPs were 5.40×105 and 2270.PartⅡProtective effects of MAPs on cerebral ischemia in vitroCultured rat cortical and hippocampal neurons suffered from oxygen-glucose deprivation (OGD) was served as an in vitro ischemia model. Primary cultures of neurons were exposed to 4 h OGD. MAPs were added 12 h before OGD initiated and maintained during OGD period. The effect of MAPs on cell activity was observed. Thiazoyl blue tetrazolium bromide (MTT) method and lactate dehydrogenase (LDH) release were detected to determined cell activity.2’,7’-dichlorodihydrofluorescin diacetate (DCFH-DA) staining was used to observe ROS in cortical neurons. Concentrations of extracellular amino acid (Glu), intracellular and calcium ([Ca2+]ⅰ) were detected by high performance liquid chromatography (HPLC) and fluorescent Ca2+ sensitive probe fura 2 acetoxylmethyl ester (Fura 2/AM) respectively in cortical neurons.Results:The extracted of MAPs could significantly inhibit the decrease of cell activity in cortical and hippocampal neurons induced by OGD. MAPs could decrease LDH level in cultures of cortical neurons suffered from OGD. At the same time, DCFH-DA staining showed that ROS in injured neurons was decreased after administration of MAPs. MAPs (0.5μg/mL 5μg/mL 50μg/mL effectively depressed of intracellular calcium ([Ca2+]ⅰ) and decrease the extracellular glutamate (Glu) level induced by OGD.PartⅢProtective effects of MAPs on cerebral ischemia in vivoChronic cerebral hypoperfusion in rats or mice was performed by permanent occlusion of the bilateral common carotid artery (2VO) and middle cerebral artery occlusion (MCAO). The MCAO model:Using mouse model of middle cerebral artery occlusion (MCAO) 2 h-reperfusion 24 h. Male mice were divided into three groups: sham-operated group, ischemic group, MAPs (200 mg/kg) treated group. In vivo cerebral infarct area was measured by tetrazolium staining, and neurological functional deficits were assessed at 4 and 24 h after reperfusion respectively. The 2VO model:Male mice were divided into five groups:sham-operated group, ischemic group, MAPs (100 mg/kg,200 mg/kg and 400 mg/kg) treated group. Male rats were divided into three groups:sham-operated group, ischemic group, MAPs (200 mg/kg) treated group. After ischemic impairment by 2VO, MAPs were administrated in treated groups and saline in sham-operated and ischemic groups. Morris water maze and Novel Object Recognition test were used to measure spatial learning and memory performance and animal behavior. Morphological change was examined by hematoxylin-eosin (HE) staining. Expressions of NMDAR1, NMDAR2B and BDNF were measured by real-time reverse transcriptase-polymerase chain reaction (qRT-PCR) and expression of BDNF was measured by western blot analyses.Results:Chronic cerebral hypoperfusion resulted neurological functional deficits and increase cerebral infarct area, depress the cognitive ability, in spatial learning and memory impairments shown by longer escape latency and shorter time spent in the target quadrant. These behavioral dysfunctions were delayed degeneration of neurons, decreases in NMDAR2B and BDNF mRNA and BDNF protein levels. Intraperitoneally administration of MAPs (200 mg/kg) significantly reduced cerebral infarct area, and attenuated neurological functional deficits. Administration of MAPs markedly improved the spatial learning and memory dysfunction and spatial memory function, attenuated neuronal damage and enhanced the expression of NMDAR2B and BDNF.CONCLUSIONSMAPs are extracted by our separation method contained and sugar to 84%. Sugar composition analysis revealed that MAPs consisted primarily of Mannose (6.37%), Rhamnose (14.94%), Xylose (1.99%), Arabinose (24.07%), Galactose (38.25%), Ribose (2.63%), Glucose (11.48%). The GPC analysis for Molecular weight MAPs were 5.40×105 and 2270. In the pharmacological research, results show that MAPs have a significant neuroprotective effects in vivo and in vitro model of cerebral ischemic injury. The neuroprotective effects is partly due to its functions that are as follows:enhancement of NMDAR2B and BDNF expression; prevention of intracellular inhibition of excessive glutamate release, calcium overload and interrupt the cascade of events leading to neuronal injury and death in ischemia.更多还原