慢性间歇缺氧诱导小鼠海马损伤的神经炎症机制及药物干预的研究

发布时间：2018-05-29 16:51

本文选题：睡眠呼吸暂停 + 间歇缺氧　；参考：《华中科技大学》2016年博士论文

【摘要】：目的：睡眠呼吸暂停综合征可引起患者认知障碍,但其具体机制尚不清楚。慢性间歇缺氧是睡眠呼吸暂停最重要的特征,此缺氧模型常用于对睡眠呼吸暂停综合症的研究。P2X7受体是一种常见于多种神经系统疾病的嘌呤受体,在脑组织中广泛表达。本实验拟研究P2X7受体在慢性间歇缺氧导致的神经损伤中的作用及其可能的通路。方法：实验使用8周龄C57BL/6小鼠。为探究P2X7受体表达的变化,每个时间点(第3,7,14,21天)采用8只小鼠,随机分为缺氧组(氧浓度21%～6%,40循环/小时,一天8小时)和常氧组(氧浓度21%,其余条件一致)2组。使用蛋白质印迹法和实时定量荧光PCR检测P2X7受体。之后在为期21天的BBG (P2X7受体拮抗剂)实验中,采用48只小鼠,随机分为慢性间歇缺氧+生理盐水组,慢性缺氧+BBG组,常氧+生理盐水组,常氧+BBG组4组。使用Morris水迷宫实验检测小鼠的认知功能。使用蛋白质印迹法检测NFκB和NOX2水平。使用实时定量荧光PCR测量肿瘤坏死因子α (TNF-α),白介素-1β(IL-1β), IL-18, IL-6的水平。使用相应试剂盒检测丙二醛和超氧化物歧化酶水平。用苏木精-伊红染色法和TUNEL染色法检测神经细胞损伤和凋亡。结果：实验中发现P2X7受体mRNA于间歇缺氧3天后开始增高,持续到21天,P2X7受体蛋白水平于间歇缺氧第7天开始增高持续到21天。在BBG实验中,慢性间歇缺氧小鼠在Morris水迷宫的表现差于其他组小鼠,表现为潜伏期更长,在平台的穿越次数更少,目标象限停留时间更少。BBG处理后认知功能改善。慢性间歇小鼠中NFκB和NOX2升高,使用BBG后其水平下降。慢性间歇小鼠中TNF-α, IL-1β, IL-18, IL-6的水平均高于正常组。BBG处理CIH组炎症因子的水平下降。慢性间歇小鼠中氧化应激水平均高于正常组。BBG可使MDA下降,SOD增高。慢性间歇缺氧小鼠的海马神经细胞出现细胞肿胀,核聚缩等细胞退化,细胞凋亡的改变,BBG使细胞受损改善。结论：拮抗P2X7受体可以改善间歇缺氧导致的神经炎症、氧化应激、神经损害和空间认知能力损伤,提示P2X7受体可能为睡眠呼吸暂停综合征所致认知缺损的新靶点,为今后药物的研究提供更多的选择。目的：海马神经损伤为睡眠呼吸暂停导致的认知损害的重要原因。阿托伐他汀是一种广泛使用的降脂药物,有文献报导其能改善认知损伤。TLR4受体是一种参与天然免疫的Toll受体,近期发现其在阿尔茨海默病等多种神经疾病中发挥作用。本实验拟研究阿托伐他汀是否能改善慢性间歇缺氧所致小鼠海马神经细胞损伤,以及TLR4及其下游通路是否参与其中。方法：实验使用40只8周龄C57BL/6小鼠。小鼠随机分为慢性间歇缺氧(氧浓度21%-6%,40循环/小时,一天8小时)组,慢性间歇缺氧+阿托伐他汀组,常氧组,常氧+阿托伐他汀组4组。使用蛋白质印迹法和实时定量荧光PCR检测TLR4受体及其下游通路蛋白MYD88和TRIF。使用酶联免疫吸附测定法(ELISA)检测肿瘤坏死因子a (TNF-a),白介素-1β(IL-β)。使用相应试剂盒检测丙二醛和超氧化物歧化酶水平。使用用苏木精-伊红染色法和TUNEL染色法检测神经细胞损伤和凋亡。结果：慢性间歇小鼠中TLR4受体,MYD88和TRIF水平升高,阿托伐他汀的使用降低TLR4受体及下游通路蛋白的表达。慢性间歇缺氧海马和血清中TNF-α, IL-1β水平均高于正常组,阿托伐他汀处理使炎症因子水平下降。慢性间歇小鼠中氧化应激水平高于正常组。阿托伐他汀可使SOD增高,MDA下降。慢性间歇缺氧小鼠的海马神经细胞出现细胞肿胀,核聚缩等细胞退化的改变,神经细胞凋亡增加,阿托伐他汀使细胞受损改善。结论：阿托伐他汀可以改善慢性间歇缺氧所致海马神经损伤,其作用可能涉及TLR4/MYD88和TLR4/TRIF通路。目的：海马神经损伤为睡眠呼吸暂停导致认知损害的主要原因。TLR2受体是-种在脑缺血等多种神经疾病中发挥重要作用的I型受体,在OSAS患者中表达亦有增高。邻香草醛为新近发现的TLR2通路抑制剂,且具有抗氧化等作用。本实验拟研究邻香草醛是否能通过抑制TLR2通路改善慢性间歇缺氧所致小鼠海马神经细胞损伤。方法：实验使用40只8周龄C57BL/6小鼠。小鼠随机分为慢性间歇缺氧(氧浓度21%-6%,40循环/小时,一天8小时)组,慢性间歇缺氧+邻香草醛组,常氧组,常氧+邻香草醛组4组。使用免疫共沉淀和蛋白质印迹法检测TLR2受体与其下游通路蛋白MYD88相互作用,使用蛋白质印迹法检测NFκB激活状态。使用酶联免疫吸附测定法检(ELISA)测肿瘤坏死因子α (TNF-α),白介素-1β(IL-β)。使用相应试剂盒检测丙二醛和超氧化物歧化酶水平。使用用免疫组织化学方法检测小胶质细胞的表达,使用TUNEL染色法检测细胞凋亡。结果：慢性间歇小鼠中TLR2/MYD88通路被激活,邻香草醛的使用抑制TLR2通路激活及下游NFkB的表达。慢性间歇缺氧海马和血清中TNF-α, IL-1β水平均高于正常组,邻香草醛的处理使炎症因子水平下降。慢性间歇小鼠中氧化应激水平高于正常组。邻香草醛可使MDA下降,SOD增高。慢性间歇缺氧小鼠的海马小胶质细胞聚集增加,活化程度较高,神经细胞出现细胞肿胀,核聚缩等细胞退化的改变,神经细胞凋亡增加,邻香草醛可以减少小胶质细胞的聚集,减轻神经细胞凋亡。结论：邻香草醛可以改善慢性间歇缺氧所致海马神经损伤,其作用涉及TLR2/MYD88通路。
[Abstract]:Objective: sleep apnea syndrome can cause cognitive impairment, but its specific mechanism is not clear. Chronic intermittent hypoxia is the most important feature of sleep apnea. This anoxia model is commonly used in the study of sleep apnea syndrome. The.P2X7 receptor is a purinine receptor common in a variety of nervous system diseases and in the brain tissue. This experiment aims to study the role and possible pathway of P2X7 receptor in the nerve injury caused by chronic intermittent hypoxia. Methods: the experiment used 8 weeks old C57BL/6 mice. To explore the changes of the expression of P2X7 receptor, 8 mice were used at each time point (day 3,7,14,21) and randomly divided into hypoxia group (oxygen concentration 21% ~ 6%, 40 circulation / small) In the 2 groups, 8 hours a day) and the normal oxygen group (the oxygen concentration 21%, the rest of the conditions). The P2X7 receptor was detected by Western blot and real-time quantitative fluorescence PCR. In the 21 day BBG (P2X7 receptor antagonist) experiment, 48 mice were randomly divided into chronic intermittent oxygen deficiency + physiological saline group, chronic hypoxia +BBG group, and normal oxygen + physiological saline group. The 4 groups of the normal oxygen +BBG group. The cognitive function of mice was detected by the Morris water maze test. The level of NF kappa B and NOX2 was detected by Western blot. The level of tumor necrosis factor alpha (TNF- a), interleukins -1 beta (IL-1 beta), IL-18, IL-6 were measured by real-time quantitative fluorescent PCR. The level of malondialdehyde and superoxide dismutase was detected by the corresponding kit. Results: the hematoxylin eosin staining and TUNEL staining were used to detect nerve cell damage and apoptosis. Results: in the experiment, the P2X7 receptor mRNA began to increase after 3 days of intermittent hypoxia, and the P2X7 receptor protein level began to increase to 21 days at seventh days of intermittent hypoxia. In the BBG experiment, the chronically intermittent anoxic mice were in the Morris water maze table. It is worse than the other groups, showing longer latency, fewer crossing times on the platform, less time for the target quadrant and less.BBG treatment. The level of NF kappa B and NOX2 in chronic intermittent mice and the decrease in BBG level. The level of TNF- alpha, IL-1 beta, IL-18 and IL-6 in chronic intermittent mice is higher than that of.BBG CIH in the normal group. The level of inflammatory factors decreased in the group of chronic intermittent mice. The average oxidative stress in the chronic intermittent mice was higher than that of the normal group.BBG, and the MDA decreased and the SOD increased. The hippocampal neurons in the chronic intermittent hypoxia mice showed cell swelling, the degeneration of nuclear polycondensation, the change of cell apoptosis, and the improvement of the cell damage by BBG. Conclusion: the antagonistic P2X7 receptor can improve the interval. Anoxia induced neuroinflammation, oxidative stress, nerve damage, and spatial cognitive impairment suggest that P2X7 receptor may be a new target for cognitive impairment induced by sleep apnea syndrome, and provides more options for future research on drugs. Atorvastatin is a widely used lipid lowering drug. It has been reported that it can improve the cognitive impairment.TLR4 receptor is a kind of Toll receptor involved in natural immunity. It has recently been found to play a role in a variety of neurodiseases such as Alzheimer's disease. Nerve cell injury, and whether TLR4 and its downstream pathways were involved. Methods: 40 8 weeks old C57BL/6 mice were used in the experiment. The mice were randomly divided into groups of chronic intermittent hypoxia (oxygen concentration 21%-6%, 40 circulation / hour, 8 hours a day), chronic intermittent hypoxia + atorvastatin group, normooxy group, and oxygen + atorvastatin group 4 groups. Detection of TLR4 receptor and its downstream pathway protein MYD88 and TRIF. using enzyme linked immunosorbent assay (ELISA) for detection of tumor necrosis factor A (TNF-a) and interleukin -1 beta (IL- beta) by real-time quantitative fluorescence PCR. Detection of malondialdehyde and superoxide dismutase by corresponding kits. The use of hematoxylin eosin staining and TUNEL staining Results: the levels of TLR4 receptor, MYD88 and TRIF increased in chronic intermittent mice. The use of atorvastatin reduced the expression of TLR4 receptor and downstream pathway protein. The level of TNF- alpha and IL-1 beta in the hippocampus and serum of chronic intermittent hypoxia was higher than that in the normal group. The level of inflammatory factors decreased in the treatment of atorvastatin. The level of oxidative stress in the mice was higher than that in the normal group. Atorvastatin increased the SOD and MDA. The hippocampal neurons in the chronic intermittent hypoxia mice showed cell swelling, the degeneration of nuclear polycondensation, the increased apoptosis of the nerve cells, and the improvement of the atorvastatin. Conclusion: atorvastatin could improve the chronic intermittent deficiency. The effects of oxygen induced hippocampal nerve damage may involve TLR4/MYD88 and TLR4/TRIF pathways. Objective: hippocampal nerve injury is the main cause of cognitive impairment caused by sleep apnea, the.TLR2 receptor is a type of I receptor that plays an important role in a variety of neurological diseases such as cerebral ischemia. The expression is also elevated in OSAS patients. The recent discovery of TLR2 pathway inhibitor and antioxidant activity. This experiment is to investigate whether ortho vanillin can improve the damage of hippocampal neurons in mice induced by chronic intermittent hypoxia by inhibiting the TLR2 pathway. Methods: 40 8 weeks old C57BL/6 mice were used. The mice were randomly divided into chronic intermittent hypoxia (oxygen concentration 21%-6%, 40 cycle / hour). 8 hours a day) group, chronic intermittent hypoxia + ortho vanillin group, normooxy group, normoxic Oxal group 4 groups. The interaction between TLR2 receptor and downstream pathway protein MYD88 was detected by immunoprecipitation and Western blot, and the activation state of NF kappa B was detected by Western blot. The cause of cancer death was measured by enzyme linked immunosorbent assay (ELISA). Zi (TNF- alpha), interleukin -1 beta (IL- beta). Use the corresponding kits to detect the level of malondialdehyde and superoxide dismutase. The use of immunohistochemistry to detect the expression of microglia and the detection of apoptosis by TUNEL staining. Results: the TLR2/ MYD88 pathway was activated in chronic intermittent mice and the use of ortho vanillin inhibited the TLR2 pathway. The level of TNF- A and IL-1 beta in the hippocampus and serum of chronic intermittent hypoxia was higher than that in the normal group. The treatment of ortho vanillin decreased the level of inflammatory factors in chronic intermittent hypoxia. The level of oxidative stress in chronic intermittent mice was higher than that in the normal group. The MDA decreased and the SOD increased. The microglia in the hippocampus of mice with slow intermittent hypoxia could accumulate microglia. The increase of the set, the higher activation degree, the cell swelling of the nerve cells, the degeneration of the nuclear polycondensation, the increase of the apoptosis of the nerve cells, the reduction of the aggregation of microglia and the reduction of the apoptosis of the nerve cells. Conclusion: ortho vanillin can improve the damage of the hippocampal nerve caused by chronic intermittent oxygen deficiency, and its effect involves the TLR2/MYD88 pass. Road.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R766

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