【作者】 王基野；

【导师】 骆文静； 柯涛；

【作者基本信息】 第四军医大学 ， 劳动卫生与环境卫生学， 2013， 博士

【摘要】 我国高原面积广阔，具有重要军事及经济意义的青藏高原平均海拔在4000米以上。空气稀薄、低温干燥等特殊的高原环境对进入人群的身心健康产生严重威胁。大脑对低温、低氧刺激尤为敏感，以脑水肿为病理基础的急性高原病在以往得到了广泛研究。近年来，高原低氧环境引起的记忆能力下降，反应速度下降，错误判断增加和感觉异常等认知能力损伤成为特殊环境医学关注的焦点。认知能力的损伤严重影响高原地区官兵与居民的作业效能与生命健康，如何防护高原低氧环境引起的学习记忆能力下降成为亟待解决的重要问题。目前高原低温低氧损伤学习记忆的特点与医学防护研究仍处于起步阶段，有关高原环境损伤学习记忆的相关基础与应用研究主要集中在动物模型水平，人群研究主要为模拟环境下的实验室研究，并不能有效反应现实条件下的暴露情况及生物学效应。此外，现有研究往往只关注低压低氧单一因素对认知功能产生的影响，而忽略了高原地环境区的另一主要因素——环境低温，高原低温低氧复合环境对认知功能造成的影响有待探讨。在药物防护方面，神经兴奋药物与激素类药物如安非他命、地塞米松等对高原低氧认知功能损伤具有一定的保护作用，但由于其副作用较大，无法作为常规预防药物。最近的研究发现，中药银杏叶的多种成分对脑功能具有良好的保护作用，其标准提取物EGb-761能够调节脑血流，降低氧化损伤，并被认为具有促进高原习服的作用。乙酰唑胺作为预防急性高原反应（AMS）的首选药物，能够加快机体低氧适应。但银杏叶提取物EGb-761及乙酰唑胺对高原认知损伤的预防作用尚不明确。因此，乙酰唑胺及银杏叶提取物EGb-761对高原低温低氧记忆损伤的预防效果及作用机制有待证实。目的通过人群现场研究及动物模型研究，观察高原低温低氧复合环境对学习记忆能力的影响，初步评价乙酰唑胺及银杏叶提取物EGb-761对急进高原人群学习记忆能力的影响，探讨银杏叶提取物活性成分单体银杏内酯B（GKB）对小鼠低温低氧暴露学习记忆损伤的防护作用，阐明GluR1蛋白磷酸化水平在GKB防护低温低氧学习记忆能力损伤中的作用，为急进高原引起的学习记忆损伤药物防护提供实验依据和理论基础。方法1.受试者分组、药物干预及试验流程招募年龄17至20岁的男性志愿者进行人群现场研究，随机分为两组，在夏季与冬季分别进行研究。每组按药物干预不同随机分为乙酰唑胺组（125mgBID）、银杏叶组（120mg BID）和安慰剂组。药物干预在进入高原前3天开始，共持续4天。研究对象乘飞机在3小时内从海拔396m上升到海拔3658m。试验地点为西藏拉萨市，平均气温为：夏季18-21℃，冬季0-3℃。2.受试者记忆能力评价利用记忆能力测试评价受试者在夏季及冬季急进高原前后记忆水平的变化情况。主要测试包括瞬时回忆测试（瞬时单词回忆测试、瞬时图形回忆测试）、定速视觉相加测试（Paced visual serial addition test，PVSAT）、数符转换测试（Digital symbolsubstitution test，DSST）和延迟回忆测试（延迟单词回忆测试、延迟图形回忆测试）。3.受试者AMS发病情况利用Lake Louise评分标准进行评价，其中Lake Louise评分标准包括自我评分（Self-reported score, S-LLS）和临床评分（Clinical assessment score, C-LLS）两部分。4.低温低氧暴露模型建立雄性C57J小鼠（18-24g）经不同海拔低温低氧暴露后，以5000m/4℃暴露2h为暴露条件建立低温低氧暴露模型。动物随机分为对照组、GKB预处理组、低温低氧暴露组和GKB预处理低温低氧暴露组。GKB通过腹腔注射方式于暴露开始前1h给药，剂量为40mg/kg bodyweight。5.低温低氧暴露小鼠学习记忆测试通过新物体识别测试（Novel object recognition test）及回避离开平台实验（Step-dwoninhibitoryavoidance task）观察小鼠短期记忆与工作记忆能力。主要观察小鼠在新物体识别实验中对新物体的探索时间与鉴别指数（DI）及回避离开平台实验中小鼠离开平台的潜伏期。6.低温低氧暴露小鼠记忆相关蛋白表达的检测通过westernblot方法检测NMDA、GluR1、Akt、GSK、PKC等蛋白的表达与活化水平。7.低温低氧暴露小鼠海马蛋白激酶A（PKA）活性的检测通过ELISA试剂盒方法检测小鼠海马组织PKA活性水平。结果1.低氧环境对学习记忆的影响及药物的防护效果（1）低氧环境对各组AMS发生情况的影响各组受试对象在高原停留期间均有AMS发生，乙酰唑胺组S-LLS评分（1.5）显著低于安慰剂组（3.0）和银杏叶组（2.8）（p<0.05）。（2）低氧环境对学习记忆的影响各组学习测试成绩在急进高原后与平原水平相比均呈现下降趋势，并以急进高原后8h最为显著（p<0.05）。（3）乙酰唑胺对低氧环境学习记忆能力的影响在高原3天的测试中，乙酰唑胺组在DSST、PVSAT及延迟回忆测试中的成绩显著低于安慰剂组（p<0.05），通过对急进高原8h后测试成绩的进一步比较后我们发现，乙酰唑胺组在瞬时回忆及PVSAT测试中的成绩显著低于安慰剂组（p<0.05）。（4）银杏叶对低氧环境学习记忆能力的影响在高原3天的测试中，银杏叶组在DSST、PVSAT及延迟回忆测试中的成绩显著高于安慰剂组（p<0.05）。通过对急进高原8h后测试成绩的进一步比较后我们发现，银杏叶组受试对象在延迟回忆测试中的成绩显著高于安慰剂组（p<0.05）。（5）AMS对学习记忆的影响各药物干预组中发生AMS的受试对象测试成绩与未发生AMS的受试对象相比无显著差别。2.低温低氧复合环境对学习记忆的影响及药物的防护效果（1）低温低氧复合环境对AMS发生情况的影响各组受试对象在高原停留期间均有AMS发生，乙酰唑胺组S-LLS评分（2.2）显著低于安慰剂组（3.9）和银杏叶组（3.3）（p<0.05）。（2）低温低氧复合环境对学习记忆的影响各组学习测试成绩在急进高原后与平原水平相比均呈现下降趋势，并以急进高原后8h最为显著（p<0.05）。（3）乙酰唑胺对低温低氧复合环境学习记忆能力的影响在高原3天的测试中，乙酰唑胺组在PVSAT测试中的成绩显著低于安慰剂组（p<0.05），通过对急进高原8h后测试成绩的进一步比较后我们发现，乙酰唑胺组受试对象在DSST及PVSAT测试中的成绩显著低于安慰剂组（p<0.05）。（4）银杏叶对低温低氧复合环境学习记忆能力的影响在高原3天的测试中，银杏叶组受试对象在PVSAT及延迟回忆测试中的成绩显著高于安慰剂组（p<0.05）。通过对急进高原8h后测试成绩的进一步比较后我们发现，银杏叶组在延迟回忆测试中显著高于安慰剂组（p<0.05）。（5）AMS对学习记忆能力的影响各药物干预组中发生AMS的受试对象测试成绩与未发生AMS的受试对象相比无显著差别。（6）低温低氧复合环境与低氧环境学习记忆能力的比较通过对夏季和冬季两组急进高原人群安慰剂组测试成绩的比较后发现，冬季低温低氧暴露组在瞬时/延时回忆、DSST和PVSAT测试中的成绩显著低于夏季低氧暴露组（p<0.05）。3. GKB在低温低氧损伤学习记忆中的作用及机制（1）低温低氧暴露对小鼠记忆能力的影响在新物体识别测试和回避离开平台测试中我们发现，在新物体识别测试中，5000m海拔暴露组小鼠与对照组相比，对新物体的探索时间及鉴别指数（DI）显著降低（p<0.05）；在回避离开平台测试中，5000m海拔暴露组小鼠与对照组相比，离开平台的潜伏期显著缩短（p<0.05）。（2）GKB对低温低氧暴露损伤小鼠学习记忆的影响在新物体识别测试中，GKB能够显著提高低温低氧暴露后小鼠对新物体的探索时间（p<0.05）及鉴别指数（DI）（p<0.05）。GKB注射组小鼠在回避离开平台测试中离开平台的潜伏期显著多于生理盐水注射低温低氧暴露组（p<0.05）。（3）低温低氧暴露及GKB干预对学习记忆相关蛋白的影响通过western blot检测我们发现小鼠海马组织Akt、GSK-3β、Erk的磷酸化水平及PKCα/β、NMDA的表达水平在低温低氧暴露后无明显变化趋势，而GluR1蛋白ser831/845位点磷酸化水平及PKA活性在暴露后显著低于对照组（p<0.05）。GKB能够缓解低温低氧暴露引起的GluR1蛋白磷酸化水平及PKA活性的下降，在低温低氧暴露后，GKB组GluR1蛋白ser831/845位点磷酸化水平及PKA活性显著高于生理盐水组（p<0.05）。结论本研究通过人群现场研究发现急进高原能够引起学习记忆能力下降，而环境低温能够显著加重这种损伤效应。乙酰唑胺对低氧及低温低氧复合环境引起的学习记忆能力下降无防护效果，并能够加重学习记忆能力的损伤，但乙酰唑胺能有效预防AMS的发生。银杏叶提取物EGb-761在急进高原情况下能显著改善急进高原引起的学习记忆能力下降，但对预防AMS无明显效果。银杏叶单体GKB可能在银杏叶提取物EGb-761保护学习记忆水平下降过程中起到关键作用，其作用机制可能是通过调节小鼠海马组织PKA活性及GluR1蛋白磷酸化水平实现的。更多还原

【Abstract】 The high-altitude areas cover a quarter of land area in China. The special natureenvironment of cold and hypoxia in Qinghai-Tibet Plateau, which has an average elevationof4000m, seriously endangers people’s physical and mental health. Brain is one of themost sensitive organs to cold and hypoxia. Previous studies paid much attention aboutacute mountain sickness (AMS) which was caused by cerebral edema during acuteexposure of altitude. However, the impairments of higher-order brain functions caused by cold and hypoxia complex environment were rarely reported. Recently, the cognitiveability of learning and memory, reaction capacity, reasoning and judgments were reportedto be impaired during acute altitude exposure, which was becoming the focus in thisresearch field. The impairment of cognition may exert widespread impacts on the lifequality and working efficacy of peoples who were living in the high land or taking part inthe military assignments. So, how to prevent people from cognitive impairment duringacute cold and hypoxia exposure was one of the most important questions.The mechanisms leading to cognitive impairment and specific chemoprophylaxisduring acute cold and hypoxia exposure remain largely unknown. As its limitedexperimental conditions of temperature and atmospheric pressure control, most studiesabout altitude exposure using hypobaric chamber for animal and human experiments.However, the results from such studies under simulated environment may not provide awhole picture of actual situation because there are many more factors that may haveeffects on cognitive performance in high-altitude areas. It was reported that some analepticand steroids such as amphetamine and dexamethasone could improve cognitiveperformance during altitude exposure. However, in view of the side effects of these drugs,they could not be used as conventional medications. As preferred prophylactic drug inprevention of AMS, acetazolamide could promote hypoxia adaptation and relievesymptoms of AMS. Recently, it has been reported that the extract of ginkgo biloba,EGb-761, could protect brain from impairment through its regulative effects on cerebralblood flow during hypoxia exposure. Though may have some beneficial effects, theprotective effects of the two drugs mentioned above remain to be clarified during cold andhypoxia exposure. We hypothesize that acetazolamide and EGb-761might protect peoplefrom impairment of learning and memory during acute cold and hypoxia exposure.Objective.The objectives of our study are to determine the impairment effects of acute cold andhypoxia exposure on learning and memory, and assess the protective effects of acetazolamide and EGb-761in human field tests. In animal experiments, we will furtherexplore the protective and regulative effects of Ginkgolide B (GKB), monomer of ginkgobiloba extract, on prevention of learning and memory impairment during acute cold andhypoxia exposure.Methods.1. Subjects and experimental procedure.Male volunteers aging17-20were recruited. And they were randomized into twogroups that ascent to Lhasa in summer and winter separately. And subjects in each groupwere treated with acetazolamide (125mg BID), EGb-761(120mg BID) and placeborandomly. The medication was started3days before ascent and lasted for4days. All thesubjects were lifted from Xianyang (396m) to Lhasa (3658m) where the cognitive testswere performed. The ambient temperature in Lhasa was18-21°C in summer and0-3°Cin winter。2. Incidence of acute mountain sicknessLake Louise scoring system was used to assess the incidence of AMS, which isconsisted of the self-reported score (S-LLS) and clinical assessment score (C-LLS).3. Assessment of learning and memory capacityNeurocognitive test battery was used to assess the effects of acute altitude exposureon learning and memory in summer and winter. The test battery was consists of immediaterecall (immediate word recall/immediate figure recall), PVSAT (paced visual serialaddition task), DSST (digit symbol substitution test) and delayed recall (delayed wordrecall/delayed figure recall).4. Cold and hypoxia exposure modelMale C57J mice weighing18-24g was used and exposed to5000m/4°C for2hours.Animals were randomized into control group (Norm), GKB treated control group (Norm+GKB), cold and hypoxia exposure group (Hypo) and GKB treated cold and hypoxiaexposure group (Hypo+GKB). GKB (40mg/kg body weight) was injectedintraperitoneally (i.p.)1hour before the onset of exposure. 5. Behavior testsNovel object recognition test and step-down inhibitory avoidance test were used toassess the impact of cold and hypoxia exposure on learning and memory.6. Molecular biological studyThe expression and activation level of NMDA, GluR1, Akt, GSK-3β and PKC wasdetected by western blot.Results.1. Effects of acetazolamide and EGb-761on learning and memory during acutehypoxia exposure(1) Incidence of AMS among groups during acute hypoxia exposureDuring the stay in Lhasa, all the drug treatment groups were reported to have AMS,but the mean score of S-LLS in acetazolamide group (1.5) was significantly lower thanthat in the placebo (3.0) and EGb-761(2.8) group (p<0.05).(2) Effects of acute hypoxia exposure on learning and memoryThe capacity of learning and memory was severely impaired8h after exposure andgot to be recovered in the following time.(3) Effects of acetazolamide on learning and memory during hypoxia exposureDuring the stay in Lhasa, the subjects in acetazolamide group performed significantl yworse in the delayed recall, DSST and PVSAT tests when compared with placebo group(p<0.05). And further analysis showed that the subjects in acetazolamide group performedsignificantly worse in the immediate recall and PVSAT tests after8h of exposure whencompared with placebo group (p<0.05).(4) Effects of EGb-761on learning and memory during hypoxia exposureDuring the stay in Lhasa, the subjects in EGb-761group performed significantl ybetter in delayed recall, PVSAT and DSST tests when compared with placebo group(p<0.05). And further analysis showed that the subjects in EGb-761group performedsignificantly better in delayed recall test after8h of exposure when compared withplacebo group (p<0.05). (5) Effects of AMS on learning and memory during hypoxia exposureThere were no significant differences between the subjects with and without AMSwhen compared of their scores in all the neurocognitive tests, no matter what drug theywere treated with.2. Effects of acetazolamide and EGb-761on learning and memory during acutecold and hypoxia exposure(1) Incidence of AMS among groups during acute cold and hypoxia exposureDuring the stay in Lhasa, all the groups were reported to have AMS, but the meanscore of S-LLS in acetazolamide group (2.2) was significantly lower than that in theplacebo (3.9) and group EGb-761(3.3)(p<0.05).(2) Effects of acute cold and hypoxia exposure on learning and memoryThe capacity of learning and memory was severely impaired8h after exposure andgot to be recovered as the exposure time prolonged in all the groups. After8hours ofhypoxia exposure, subjects in each group performed significantly worse in theimmediate/delayed recall, DSST and PVSAT tests (p<0.05).(3) Effects of acetazolamide on learning and memory during cold and hypoxiaexposureDuring the stay in Lhasa, the subjects in acetazolamide group performed significantl yworse in PVSAT test when compared with placebo group (p<0.05). And further analysisshowed that the subjects in acetazolamide group performed significantly worse in theDSST and PVSAT tests after8h of exposure when compared with placebo group(p<0.05).(4) Effects of EGb-761on learning and memory during cold and hypoxia exposureDuring the stay in Lhasa, the subjects in EGb-761group performed significantl ybetter in PVSAT and delayed recall tests when compared with placebo group (p<0.05).And further analysis showed that subjects in EGb-761group performed significantlybetter in delayed recall test when compared with placebo group (p<0.05).(5) Effects of AMS on learning and memory during hypoxia exposureThere were no significant differences between the subjects with and without AMS when compared of their scores in all the neurocognitive tests, no matter what drug theywere treated with.(6) Effects of cold temperature on the impairment effects of hypoxia exposure onlearning and memoryThe cognitive performances of the subjects in placebo group in the summer trail andwinter trial were compared. And we found that the performance of subjects in the wintertrail was significantly worse than those in the summer trail (p<0.05).3. Effects of GKB on learning and memory performance of mouse after coldand hypoxia exposure(1) Effects of acute cold and hypoxia exposure on learning and memory performanceThe time spent in exploring of the novel object as well as the discrimination indexwere significantly reduced in the novel object recognition test (p<0.05), and the latency ofstep down of the platform was significantly shorter in the step-down inhibitory avoidancetask (p<0.05) after2h of cold and hypoxia exposure at5000m.(2) Effects of GKB on learning and memory performance after cold and hypoxiaexposureGKB significantly increased the time spent in exploring of the novel object as well asthe discrimination index (p<0.05), and it also significantly increased the latency of stepdown of the platform (p<0.05) when compared with those treated with saline.(3) Effects of cold and hypoxia exposure and GKB on the expression andphosphorylation level of proteins relative with learning and memoryThe expression level of NMDA and PKCα/β and the phosphorylation level of Akt,GSK-3β and Erk were not affected by cold and hypoxia exposure. However, thephosphorylation level of GluR1at ser831/845and the activity of PKA decreasedsignificantly after2h of cold and hypoxia exposure at5000m. This change could bereversed by GKB treatment1h before exposure.Conclusion.1. Acute hypoxia exposure could impair learning and memory, and cold temperature could aggravate the impairment effects.2. Acetazolamide impaired learning and memory during hypoxia as well as cold andhypoxia exposure. However, it could prevent people from AMS.3. EGb-761could potently protect people from impairment of learning and memory.But it has no protective effects on AMS.4. GKB might be the key component of EGb-761in prevention of learning andmemory impairment by regulating the phosphorylation of GluR1during cold andhypoxia exposure.更多还原