褪黑素对大鼠蛛网膜下腔出血后继发性脑损伤及认知功能影响机制的实验研究

发布时间：2018-01-17 19:31

本文关键词：褪黑素对大鼠蛛网膜下腔出血后继发性脑损伤及认知功能影响机制的实验研究　出处：《苏州大学》2014年硕士论文　论文类型：学位论文

【摘要】：第一部分褪黑素对大鼠蛛网膜下腔出血后继发性脑损伤及认知功能的影响目的：研究褪黑素（Melatonin,MT）对大鼠蛛网膜下腔出血（subarachnoidhemorrhage,SAH）后继发性脑损伤及认知功能障碍的影响。方法：任选80只成年雄性SD大鼠，随机分为以下四个实验组：正常组、SAH组、安慰剂治疗组和褪黑素治疗组，将SD大鼠股动脉非肝素化动脉血通过1.0ml注射器注入大鼠视交叉前池，从而建立实验性蛛网膜下腔出血动物模型。在褪黑素治疗组，模型建立后的第2小时、24小时及36小时，腹腔注射褪黑素溶液（150mg/kg）。在安慰剂治疗组，模型建立后的第2小时、24小时及36小时，腹腔注射生理盐水。观察SD大鼠SAH后神经行为功能的变化，同时进行神经行为功能评分。SAH动物模型建立完毕第48小时处死部分大鼠，取出大鼠额颞底皮层脑组织做标本，采用TUNEL荧光染色与FLUORO-JADE B荧光染色检测大鼠额颞底皮层脑组织神经元细胞凋亡及坏死比率，剩余未处死SD大鼠在SAH动物模型建立完毕第48小时开始进行Morris水迷宫试验，检测SD大鼠的认知功能障碍。结果：1.与正常组相比，SAH组SD大鼠的神经认知功能水平降低，活动能力及饮食能力降低（P0.05）；褪黑素组SD大鼠与安慰剂组SD大鼠相比，神经认知功能明显改善，食欲及活动能力明显好转（P0.01）；SAH组SD大鼠和安慰剂组SD大鼠之间无统计学意义（P0.05）。2. TUNEL染色结果显示：与正常正常组比较，SAH组SD大鼠神经元细胞的凋亡百分显著增高（P0.01），褪黑素治疗组SD大鼠神经元细胞凋亡百分比明显低于安慰剂治疗组SD大鼠（P0.01），而安慰剂治疗组与SAH组SD大鼠神经元细胞的凋亡百分比比较无统计学差异（P0.05）。3. Fluoro-Jade B荧光染色检测各组大鼠神经元细胞的坏死情况，结果显示：与正常组相比，SAH组 SD大鼠神经元细胞坏死的百分比显著增高（P0.01），褪黑素治疗组SD大鼠神经元细胞坏死的百分比显著低于安慰剂治疗组SD大鼠（P0.05），而安慰剂治疗组SD大鼠与SAH组SD大鼠神经元细胞坏死的百分比无统计学差异（P0.05）。5.在Morris水迷宫试验测试中，与正常组相比，SAH组SD大鼠及安慰剂组SD大鼠的空间学习记忆功能明显下降（P0.01），且SAH组SD大鼠及安慰剂组SD大鼠之间无统计学差异（P0.05）。褪黑素组SD大鼠与安慰剂组SD大鼠相比，SD大鼠的空间学习记忆能力有显著提高（P0.01）。结论：褪黑素（Melatonin,MT）能够降低SD大鼠蛛网膜下腔出血后继发性神经元细胞凋亡及坏死水平，同时明显改善SD大鼠SAH后的空间学习等认知功能障碍。第二部分褪黑素对大鼠蛛网膜下腔出血后TLR4介导的炎性信号通路相关蛋白表达影响的研究目的：建立大鼠蛛网膜下腔出血（SAH，subarachnoid hemorrhage）模型，观察褪黑素治疗后，TLR4介导的炎性信号通路相关蛋白表达的变化，如高迁移率族蛋白B1（HMGB1），Toll样受体4（TLR4），核转录因子（NF-κB），髓样分化因子88（MyD88），白介素-1β（IL-1β），肿瘤坏死因子α（TNF-α），白介素-6（IL-6），和诱导型一氧化氮合酶等。方法：任选80只成年雄性SD大鼠，随机分为以下四个实验组：正常组、SAH组、安慰剂治疗组和褪黑素治疗组，将SD大鼠股动脉非肝素化动脉血通过1.0ml注射器注入大鼠视交叉前池，从而建立实验性蛛网膜下腔出血动物模型。在褪黑素治疗组，模型建立后的第2小时、24小时及36小时，腹腔注射褪黑素溶液（150mg/kg）。在安慰剂治疗组，模型建立后的第2小时、24小时及36小时，腹腔注射生理盐水。观察SD大鼠SAH后神经行为功能的变化，同时进行神经行为功能评分。SAH动物模型建立完毕第48小时处死各组大鼠，取出大鼠额颞底皮层脑组织做标本，用Western blot、EMSA、PCR、ELISA及免疫组化等方法，分析测定TLR4介导的炎性信号通路相关蛋白表达的变化。结果：Western blot结果显示：高迁移率族蛋白B1（HMGB1），Toll样受体4蛋白（TLR4），核转录因子（NF-κB P65，NF-κB P50），髓样分化因子88（MyD88），，诱导型一氧化氮合酶（iNOS）在正常组样本中呈低水平表达。与正常组相比，SAH组样本上述蛋白的表达水平明显增加（P0.01），但是在单纯SAH组的表达水平与安慰剂治疗组并无明显差别（P0.05）。在褪黑素治疗组，上述蛋白的表达水平明显低于安慰剂治疗组（P0.05）。EMSA结果显示：通过凝胶射线显迹显示，正常组NF-κB结合活性水平较低，而SAH组及安慰剂治疗组NF-κB结合活性水平较正常组明显上升（P0.01）。同时在褪黑素治疗组，NF-κB结合活性水平较安慰剂治疗组明显下调（P0.05）。免疫组化结果：正常组中表达TLR4、NF-κB和iNOS的阳性细胞比率很低。SAH组及安慰剂治疗组中表达TLR4、NF-κB和iNOS的阳性细胞比率明显上升（P0.01）。褪黑素治疗组TLR4、NF-κB和iNOS的阳性细胞比率较安慰剂治疗组明显下降（P0.05）。实时定量PCR结果：正常组TNF-α，IL-1β，IL-6的mRMA表达水平较低，而SAH组及安慰剂治疗组上述蛋白mRNA表达水平明显增高（P0.01），且SAH组与安慰剂治疗组上述蛋白mRNA表达水平无明显差异（P0.05）。同时褪黑素治疗组上述蛋白mRNA表达水平较安慰剂治疗组有明显下降（P0.01）。ELISA结果显示：正常组IL-1β，TNF-α和IL-6浓度较低，SAH组及安慰剂治疗组IL-1β，TNF-α和IL-6因子浓度大幅上升（P0.01），而褪黑素治疗组IL-1β，TNF-α和IL-6因子浓度有相当程度的降低（P0.05）。结论：褪黑素（Melatonin,MT）在大鼠蛛网膜下腔出血模型中显著降低TLR4介导的炎性反应信号通路相关蛋白如HMGB1、TLR4、NF-κB、MyD88、IL-1β、TNF-α、IL-6和iNOS的表达及转录水平。
[Abstract]:The effect of melatonin on secondary brain injury and cognitive function after subarachnoid hemorrhage in rats
Objective: To study the effects of Melatonin (MT) on secondary brain injury and cognitive impairment after subarachnoid hemorrhage (subarachnoidhemorrhage, SAH) in rats.
Methods: select 80 adult male SD rats were randomly divided into four experimental groups: normal group, SAH group, placebo group and melatonin treatment group, SD rats femoral artery non heparinized arterial blood pool before crossing rat optic by 1.0ml injection, so as to establish the experimental animal model of subarachnoid space subarachnoid hemorrhage in the treatment group. Melatonin, second hours after the establishment of the model, 24 hours and 36 hours, intraperitoneal injection of melatonin solution (150mg/kg). In the placebo group, second hours after the establishment of the model, 24 hours and 36 hours, intraperitoneal injection of saline. Changes of neurobehavioral function in SD rats after SAH were observed. At the same time the neurobehavioral function evaluation.SAH animal model has been established. Some rats were sacrificed forty-eighth hours, remove the rat frontotemporal bottom cerebral cortex specimens, using TUNEL staining and FLUORO-JADE B staining were used to detect the rat frontal and temporal cortex at the end The rate of apoptosis and necrosis of neurons in brain tissue was not detected in SD rats. After forty-eighth hours of establishment of SAH animal model, Morris water maze test was carried out to detect cognitive dysfunction in SD rats.
Results: 1. compared with the normal group, SAH group, SD rats the level of cognitive function decreased, decreased activity and eating ability (P0.05); melatonin group SD rats compared with the placebo group SD rats, neurocognitive function improved appetite and activity improved significantly (P0.01); there was no statistically significant between group SAH and SD the placebo group rats and SD rats (P0.05) and.2. TUNEL staining showed that: compared with normal group, SAH group of neurons of SD rats significantly increased the apoptosis percentage (P0.01), melatonin treatment group SD rat neuronal apoptosis percentage was significantly lower than the placebo group SD rats (P0.01). But no significant differences between the placebo group and the percentage of apoptotic neurons in SAH SD rats (P0.05) necrosis neurons of rats were detected.3. Fluoro-Jade B fluorescent staining, the results showed: compared with normal group, S AH group
The percentage of necrosis of neuron cells in SD rats increased significantly (P0.01), and the neurons of SD rats in the melatonin treatment group
The percentage of cell necrosis was significantly lower than that of the placebo group SD rats (P0.05), but no significant difference between the placebo treatment group the percentage of SD rats and SAH rats group SD neuron necrosis (P0.05).5. in the Morris water maze test, compared with the normal group, SAH group, SD rats and placebo group SD in the spatial learning and memory function decreased significantly (P0.01), and between SAH group and placebo group SD rats SD rats showed no significant difference (P0.05). Melatonin group SD rats compared with the placebo group SD rats, SD rats spatial learning notes
The memory ability was improved significantly (P0.01).
Conclusion: Melatonin (MT) can reduce the secondary neuronal apoptosis and necrosis level after subarachnoid hemorrhage in SD rats, and significantly improve spatial learning and cognitive dysfunction in SD rats after SAH.
The effect of the second part of melatonin on the expression of TLR4 mediated inflammatory signaling pathway related protein after subarachnoid hemorrhage in rats
Objective: to establish a rat model of subarachnoid hemorrhage (SAH subarachnoid hemorrhage) model, to observe the effect of melatonin treatment, changes in the expression of inflammatory signal pathway mediated by TLR4, such as high mobility group protein B1 (HMGB1), Toll like receptor 4 (TLR4), a nuclear transcription factor (NF- K B), myeloid differentiation factor 88 (MyD88), interleukin -1 beta (IL-1 beta), tumor necrosis factor alpha (TNF- alpha), interleukin -6 (IL-6), and inducible nitric oxide synthase.
Methods: select 80 adult male SD rats were randomly divided into four experimental groups: normal group, SAH group, placebo group and melatonin treatment group, SD rats femoral artery non heparinized arterial blood pool before crossing rat optic by 1.0ml injection, so as to establish the experimental animal model of subarachnoid space subarachnoid hemorrhage in the treatment group. Melatonin, second hours after the establishment of the model, 24 hours and 36 hours, intraperitoneal injection of melatonin solution (150mg/kg). In the placebo group, second hours after the establishment of the model, 24 hours and 36 hours, intraperitoneal injection of saline. Changes of neurobehavioral function in SD rats after SAH were observed. At the same time the neurobehavioral function evaluation.SAH animal model has been established. Rats were killed forty-eighth hour, remove the rat frontotemporal bottom cerebral cortex specimens with Western, blot, EMSA, PCR, ELISA and immunohistochemistry method, determination of TLR4 mediated Changes in the expression of inflammatory signaling pathway related proteins.
Results: Western blot showed that high mobility group protein B1 (HMGB1), Toll like receptor 4 protein (TLR4), a nuclear transcription factor (NF- - B P65, NF- P50 K B), myeloid differentiation factor 88 (MyD88), inducible nitric oxide synthase (iNOS) in normal group was the low expression level. Compared with the normal group, the expression level of SAH protein significantly increased the sample group (P0.01), but the expression level in SAH group and placebo treatment groups had no significant difference (P0.05). The treatment group in the expression level of melatonin, the protein was significantly lower than that in the placebo group (P0.05).EMSA results by ray tracing gel showed normal NF- kappa B binding activity of low level, while the SAH group and the placebo group NF- kappa B binding activity level increased significantly compared with the normal group (P0.01). At the same time in the treatment group, melatonin, NF- kappa B binding activity was significantly lower than that of placebo group (P0.05 ). The results of immunohistochemistry: the expression of TLR4 in the normal group, the expression of TLR4 positive cell ratio of NF- kappa B and iNOS low.SAH group and placebo group, positive cell ratio of NF- kappa B and iNOS was significantly increased (P0.01). Melatonin treatment group TLR4, positive cell ratio of NF- kappa B and iNOS were the placebo treatment group decreased significantly (P0.05). Real time quantitative PCR results: in normal group, TNF- alpha, IL-1 beta, IL-6 mRMA expression level was low, while the SAH group and the placebo group the protein expression level of mRNA was significantly higher (P0.01), and SAH group and the placebo group showed no significant difference between the mRNA protein expression level (P0.05). At the same time melatonin treatment group the protein expression level of mRNA compared with the placebo treatment group decreased significantly (P0.01).ELISA results showed: normal group IL-1 beta, TNF- alpha and low concentrations of IL-6, SAH group and placebo group IL-1 beta, TNF- alpha and IL-6 factor concentration increased significantly (P0. 01), while the concentration of IL-1 beta, TNF- alpha and IL-6 in the melatonin treatment group decreased to a considerable degree (P0.05).
Conclusion: Melatonin (MT) significantly reduces the expression and transcription level of TLR4 mediated inflammatory response related proteins such as HMGB1, TLR4, NF- kappa B, MyD88, IL-1, beta, TNF-, IL-6 and TNF- in rat subarachnoid hemorrhage models.

【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R651.15

【共引文献】