【作者】 王涛；

【导师】 毕建忠；

【作者基本信息】 山东大学 ， 神经病学（专业学位）， 2015， 博士

【摘要】 半胱胺对大鼠蛛网膜下腔出血后早期脑损伤的保护作用[目的] 动脉瘤性蛛网膜下腔出血(aneurysmal subarachnoid hemorrhage)是一种灾难性的卒中亚型,因其常导致幸存患者永久性的神经功能障碍,发病率和死亡率值得关注。前期研究表明,早期脑损伤(early brain injury, EBI)即蛛网膜下腔出血后最初72小时内发生的全脑急性损伤,包括颅内压升高,脑灌注压下降,微循环紊乱,脑水肿形成,氧化应激和迟发性脑血管痉挛以及微血管系统损伤。尤其是氧化应激和神经元凋亡在蛛网膜下腔出血后早期脑损伤的病理机制中发挥重要作用。本研究目的在于评估半胱胺能否通过其抗氧化和抗凋亡作用抑制蛛网膜下腔出血后的氧化应激损伤。[方法]建立SD大鼠枕大池二次注血法的蛛网膜下腔出血模型。实验分为假手术组(n=24),安慰剂治疗蛛网膜下腔出血组(n=24)和半胱胺治疗蛛网膜下腔出血组；在半胱胺治疗蛛网膜下腔出血组中,剂量为(0.1 ml,20mg/kg/天)的半胱胺从首次枕大池注血后腹腔注射给药。安慰剂治疗组蛛网膜下腔出血的大鼠在相应的时间点经腹膜腔注射等体积生理盐水(0.1 ml)。经过神经功能评估,在蛛网膜下腔出血后48小时处死大鼠。每组6只大鼠取脑用于分子生物学和生物化学检验。每组6只大鼠检测血脑屏障损伤。每组6只大鼠检测脑组织含水量。剩余每组6只大鼠用于免疫组织学研究。观察如下指标的变化：1.早期脑损伤指标(包括神经行为缺损、干湿重法检测脑水肿、伊文思蓝(EB, Evans blue)染料溢出法检测血脑屏障通透性、原位末端标记(TUNEL)及免疫组织化学染色法评估皮层区神经元凋亡)；2.脑组织中的活性氧族(reactive oxygen species, ROS)、丙二醛(malondialdehyde, MDA)、还原型谷胱甘肽(glutathione, GSH)含量检测,乳酸脱氢酶(lactate dehydrogenase, LDH)活性和谷胱甘肽过氧化物酶(Glutathione peroxidase, GSH-Px)活性测定；3.酶联免疫吸附测定3-硝基酪氨酸(3-nitrotyrosine,3-NT)8-羟基脱氧鸟苷浓度(8-hydroxydeoxyguanosine,8-OHDG)以及脑源性神经生长因子(brain-derivedneurotrophic factor, BDNF)水平。[结果]半胱胺能显著地减轻早期脑损伤(包括神经行为缺损、脑水肿、血脑屏障通透性升高和皮层区神经元凋亡),同时明显地降低的脑组织中的活性氧族、丙二醛、3-硝基酪氨酸和8-羟基脱氧鸟苷浓度,还能增加大鼠蛛网膜下腔出血后的脑组织的谷胱甘肽过氧化物酶活力,还原型谷胱甘肽以及脑源性神经生长因子水平也观察到明显升高。[结论]实验数据表明,腹腔内注射给予半胱胺(20mg/kg/天)能够通过抑制氧化应激和上调体内BDNF表达,从而对大鼠SAH诱导的早期脑损伤提供保护。在半胱胺治疗后,动物的神经功能缺损得到改善,脑水肿和血脑屏障渗出减轻,氧化应激和皮层凋亡被抑制。以上发现提示半胱胺可能成为SAH后早期脑损伤的备选药物治疗方案。鸦胆子苦素A与酮康唑对细胞色素P450 (CYP) 3A4的潜在抑制作用对比研究[目的]脑出血是一种发生于脑组织中的对人体健康损害严重的颅内出血性疾病,可能继发于脑外伤,有时是自发的出血性卒中。寻找治疗脑出血的有效药物是一项艰巨的任务。针对脑出血需要以下治疗策略：(1)抗高血压治疗,用于降低血压水平；(2)降低颅内压；(3)抑制脑出血后继发炎症反应损伤和细胞凋亡。鸦胆子苦素a (Bruceine A)具有抗肿瘤的细胞毒性的潜力和对NF-κB p65的抑制作用,是一种潜在的脑出血治疗药物。本研究目的是明确鸦胆子苦素a对代谢绝大多数临床药物的细胞色素P450 (CYP) 3A4的潜在抑制作用。[方法]从蛋白质数据库获取3A4晶体结构。从3A4晶体结构列表中,选择活性中心与酮康唑结合的构象。应用化学办公套件中的化学绘图软件绘制鸦胆子苦素a的二维结构,其结构依据标准键长和角度绘制。根据化学得分函数的分值排序,我们只从前五名的构象中选择最合适的构型。位于苯环中的羰酰基团是代谢位点,与CYP3A4舌性中心的距离为1.98A。在CYP3A4蛋白序列Ile301氨基酸与鸦胆子苦素a之间形成强氢键。通过分子对接技术实现鸦胆子苦素a对接至CYP3A4活性中心的步骤。共对接法用于比较鸦胆子苦素a与酮康唑结合潜能的高低。[结果]鸦胆子苦素a表现出比酮康唑更强的与活性中心结合的能力。提示监测鸦胆子苦素a与CYP3A4催化代谢底物之间药物相互作用的重要性。[结论]不同于体外筛选方法,生物信息学指导的分子对接法具有更快速的筛选能力。这种方法已被用于筛选CYP3A4抑制剂。本研究中,鸦胆子苦素a被证明是CYP3A4的一种强抑制剂,本研究也证实分子对接预测在代谢行为预测中的价值。更多还原

【Abstract】 Part ONE The neuron protective effects of cysteamine on the early brain injury in a rat subarachnoid hemorrhage model[Objective] Aneurysmal subarachnoid hemorrhage is a disastrous stroke subtype with significant morbidity and mortality, and often results in lasting neurological deficits for survivors. Previous studies have indicated that early brain injury, which refers to the acute injuries to the whole brain within the first 72 h after SAH, including increased intracranial pressure, decreased cerebral perfusion pressure, disturbed microcirculation, brain edema formation, oxidative stress, and delayed cerebral vasospasm and damage to the microvascular system. In particular, oxidative stress and neuron apoptosis plays an important role in the pathogenesis of early brain injury following subarachnoid hemorrhage. The aim of this study was to assess whether cysteamine prevents post-SAH oxidative stress injury via its antioxidative and anti-apoptotic effects.[Methods] Experimental subarachnoid hemorrhage was induced in Sprague-Dawley rats using double blood injection into cisterna magna method. The experimental groups consisted of sham-operated group (n=24), vehicle-treated SAH group (n= 24), and cysteamine-treated SAH group (n=24). In cysteamine-treated SAH group, dose (0.1 ml,20 mg/kg/day) of cysteamine was administered after first blood injection. Rats of vehicle-treated SAH group received equal volumes of 0.9% saline intraperitoneal administration (0.1 ml) at corresponding time points. After the neurological assessment, all the rats were killed at 48h after SAH. Six rats in each group were decollated, then the brain sample was removed for molecular biological and biochemical experiments. Six rats in each group were for detecting blood-brain barrier impairment. Six rats in each group were for detecting brain water content. Six rats in each group were for immunohistological staining study. The following indicators were observed:1. Indicators for early brain injury (including neurobehavioral defects, assessment of brain edema according to the wet/dry method, blood-brain barrier permeability assessed by Evans blue dye extravasation, TUNEL and immunohistological staining for the evaluation of cortical neuron apoptosis).2. The reactive oxygen species level, the concentration of malondialdehyde and glutathione were detected; and the activity of lactate dehydrogenase and glutathione peroxidase determination was measured using commercial assay kit.3. The concentration of 3-nitrotyrosine,8-hydroxydeoxyguanosine and brain-derived neurotrophic factor was measured with commercial enzyme-linked immunosorbent assay kit for rats.[Results] It was observed that intraperitoneal administration of cysteamine (20 mg/kg/day) could significantly alleviate EBI (including neurobehavioral deficits, brain edema, blood-brain barrier permeability, and cortical neuron apoptosis) after SAH in rats. Meanwhile, cysteamine treatment reduced post-SAH elevated the reactive oxygen species level, the concentration of malondialdehyde,3-nitrotyrosine, and 8-hydroxydeoxyguanosine and increased the glutathione peroxidase enzymatic activity, the concentration of glutathione and brain-derived neurotrophic factor in brain cortex at 48 h after SAH.[Conclusion] These results indicated that intraperitoneal administration of cysteamine (20 mg/kg/day) confers protection against SAH-induced EBI in rats through the inhibition of oxidative stress and the promotion of BDNF production in vivo. Neurobehavioral deficit was improved; brain edema and blood-brain barrier permeability were attenuated; oxidative stress and cortical apoptosis were reduced after cysteamine treatment. These finding suggest that cysteamine may be a candidate treatment for EBI after SAH.Part TWO The inhibition potential of bruceine a and ketoconazole towards cytochrome P450 3A4[Objective] Cerebral haemorrhage, a kind of intracranial hemorrhage occurring in the brain tissue, severely damages the health of human body. It can be induced by brain trauma, and sometimes occurs spontaneously with hemorrhagic stroke. Searching efficient medicines for cerebral haemorrhage is an important and necessary task. The following therapeutic regimens are needed:1) antihypertensive therapy is employed to lowerthe blood pressure; 2) drugs to reduce elevated intracranial pressure.3) inhibition of secondary inflammatory response injury and apoptosis after cerebral hemorrhage. Bruceine a, with the potential of antitumor cytotoxic and inhibiting the nuclear factor-kappa B p65, is a potential therapeutic drug for cerebral haemorrhage. The present study aims to determine the inhibition potential of bruceine a towards cytochrome P450 (CYP) 3A4 which catalyzed most of clinical drugs.[Methods] Protein data bank is the source to get the crystal structure of CYP3A4. Among the list of CYP3A4 crystal structures, we select the crystal structure for which the activity center was bound with the inhibitor ketoconazole. Chemdraw software in the ChemOffice package was used to draw the two-dimensional structure of bruceine a, and the structure was drawn using standard bond lengths and angles. Ranked by Chemscroe values, we just selected the most suitable binging conformation among the top five poses. The carbonyl group located in the benzene ring was the metabolic site which has 1.98 A distance towards the catalytic center of CYP3A4. Strong hydrogen bond was formed between the structure of bruceine a and amino acid Ile301 in the protein sequence of CYP3A4. The docking process of bruceine a into the activity cavity of CYP3A4 was performed using Surflex-Dock. Co-docking method was used to compare the binding potential between bruceine a and ketoconazole.[Results] Bruceine a exerted stronger binding potential than ketoconazole towards the activity center, indicating the importance to monitor the drug-drug interaction between bruceine a and substrates mainly undergoing CYP3A4-catalyzed metabolism.[Conclusion] Different from the in vitro screening method, bioinformatics-guided molecular docking method has more rapid screening capability. This method has also been employed to screen the inhibitors of CYP3A4. In this study, bruceine a was demonstrated to be a strong inhibitor of CYP3A4. The present study also demonstrates the power of molecular docking prediction in the metabolic behavior prediction.更多还原