【作者】 孔繁平；

【导师】 杜继曾； 陈学群；

【作者基本信息】 浙江大学 ， 神经生物学， 2013， 博士

【摘要】 我国西部的青藏高原,最突出的自然环境特征就是低氧,随着西部大开发战略的实施,日益增多的援建移民、旅游、探险、军事换防等,使得每年有数百万人进入青藏高原,面临高原低氧损伤的危险。急性高原反应和急性高原病(AMS)是人体快速暴露于高原低氧环境后产生的各种生理反应和低氧性疾病,常见的症状有头痛,恶心呕吐,全身乏力,头晕和睡眠障碍等。急性高原病是人类到达高原的主要挑战,AMS的发病率随着所到达的高度升高而显著升高,在1850米到2750米时有25%的发病率,在3000米时有42%的发病率,而在6000米的时候发病率高达75%。如果AMS得不到及时正确的治疗,可能会发展成为严重的危及生命的高原肺水肿(HAPE)和脑水肿(HACE)。AMS极大地限制了人们在高原的活动。研究AMS的发病机制,预警预测AMS发生的风险具有重大的现实意义,然而目前AMS的发病机制尚未完全清楚,人们试图研究探索快速有效地预测AMS,提出低氧通气反应,心率变异率,最大运动后的氧饱和度的变化等指标来预测AMS的发生,但都不能有效地预测其发生。我们实验室以前的研究发现高原低氧促进神经肽促肾上腺皮质激素释放激素(CRH)在下丘脑的分泌及其1型受体(CRHR1)的过度表达,在脑-内分泌-免疫网络低氧损伤调节过程中起核心主导的作用,所以我们提出CRH可能成为预测急性高原低氧应激损伤的生物标志分子,证明血浆和唾液CRH水平过度增高与发生AMS相关,通过检测唾液CRH水平变化程度,可以预测人们到达高原后可能发生AMS的潜在风险性。本研究分为实验动物研究和人类研究两部分。在动物实验中,成年大鼠被置于低压氧舱中分别模拟2,5,和7km不同海拔高度高原低氧8小时以及海拔7km高原低氧2,8和24小时不同时间,研究大鼠下丘脑和外周血液CRH和皮质酮水平的相应变化,分析下丘脑和血浆CRH线性相关性。还研究了大鼠脑CRHR1和CRHR2受体基因的表达变化模式,探讨了CRHR1在低氧脑细胞损伤中的作用。在以人类为对象的研究中,令志愿者进行自行车功量仪运动和登高跑运动两种平原耗竭运动模拟低氧,检测运动前后血浆和唾液中CRH和皮质醇水平,测定心率,氧饱和度,以及血清和唾液中的乳酸脱氢酶(LDH)水平；此外,志愿者进入3600和5500海拔的青藏高原现场,结合Lake Louise Score AMS标准,进行AMS问卷调查,根据临床AMS症状和发病率以及血浆、唾液CRH水平的相关变化,计算分析了CRH和发生AMS风险间的相关性。提出了应用检测唾液CRH水平预测发生高原AMS风险的方法。研究结果1低氧强度和时间依赖性地增加大鼠脑PVN区CRH的分泌低氧显著性降低大鼠脑PVN和ME处CRH蛋白水平,升高大鼠垂体CRH水平,这个变化呈现低氧强度和时间变化依赖的关系。低氧显著性增加大鼠脑PVN区CRH mRNA的表达,随着低氧强度的增加,CRH mRNA的表达也增加。2低氧强度和时间依赖性地升高大鼠血浆CRH和皮质酮水平及相关性分析低氧诱导大鼠血浆CRH水平升高,低氧后CRH水平的变化呈现低氧强度和时间依赖的关系。低氧诱导大鼠血浆皮质酮水平升高,皮质酮水平的变化同样呈现低氧强度和时间依赖的变化。3低氧升高大鼠血浆CRH与中枢CRH分泌增加的相关性分析低氧升高大鼠血浆CRH水平和低氧刺激PVN区CRH释放相关,下丘脑CRH水平与血浆CRH水平呈负相关。血浆CRH水平与皮质酮水平呈正相关。4低氧刺激大鼠不同脑区和垂体CRHR1和CRHR2受体的不同表达模式低氧后大鼠皮层、海马、杏仁中央核、PVN和蓝斑区CRHR1mRNA表达升高,在7km高原低氧8小时达到峰值,低氧24小时后开始回落。大鼠垂体CRHR1mRNA表达模式相同。大鼠皮层、海马、杏仁中央核、蓝斑区和垂体CRHR2mRNA表达降低,而下丘脑PVN区CRHR2mRNA的表达与CRHR1mRNA的表达模式相似的升高。CRHR1mRNA和CRHR2mRNA在不同脑区的相对表达水平不同,其中在皮层,杏仁核,蓝斑CRHR1mRNA的表达水平是CRHR2mRNA的3-5倍,而海马,PVN和垂体中CRHR1和CRHR2mRNA的表达水平相近。低氧后,CRHR1和CRHR2mRNA表达水平差异显著,7km海拔低氧8小时后最为明显,其中在皮层,杏仁中央核和蓝斑区两型受体表达水平相差8倍,而海马,PVN区和垂体两型受体相差2倍。5低氧过度激活大鼠脑皮层CRHRl与皮层细胞凋亡相关大鼠皮层促凋亡基因(bim, bnip3, bax) mRNA表达水平在5km高原低氧8小时和7km高原低氧2和8小时后显著升高,而抗凋亡基因bcl-2mRNA在2km高原低氧8小时后升高,5和7km高原低氧8小时后下降。凋亡蛋白caspase-3的活性在7km高原低氧8和24小时后都有显著升高。HE和TUNEL染色发现,7km高原低氧8小时后,大鼠皮层细胞肿胀,间隙加大,凋亡细胞显著增加。而预先使用CRHR1拮抗剂CP154,526处理后,能显著降低低氧诱导的凋亡基因bim和bax的表达升高,下调凋亡蛋白caspase-3的活性,降低凋亡细胞数目。6急性耗竭运动诱导人体低氧生理反应志愿者登高跑运动后,心率由86±10/分钟升高到177±16/分钟,唾液LDH活性由199.7±112.7U/L升高到468.7±279.9U/L,而血清LDH活性没有显著变化,同时动脉血氧饱和度从98%±1%降低到93%±3%。自行车功量仪运动后心率从82±11/min升高到166±18/min,唾液LDH活性从156.7±92.5U/L升高到294±187.6U/L,而血清LDH活性没有明显变化,动脉血氧饱和度同样从98%±1%降低到94%±1%。7急性耗竭运动和高原低氧诱导人体唾液和血液CRH水平升高检测健康人体唾液CRH和皮质醇水平存在明显的昼夜节律,唾液CRH水平在清晨7：00最高,中午11：00下降,在下午15：00和19：00保持一个稳定的水平,夜间23：00时降低到最低水平,唾液皮质醇的变化趋势和唾液CRH相同。耗竭运动低氧诱导唾液和血浆CRH和皮质醇水平显著升高,在登高跑运动后,唾液和血浆CRH水平分别从25.25±6.41pg/ml和39.82±7.87pg/ml升高到35.78±8.51pg/ml和54.37±9.31pg/ml,唾液和血浆皮质醇水平分别从1.62±0.54ng/ml和6.5±1.51ng/ml升高到3.52±1.22ng/ml和10.8±2.58ng/ml.自行车功量仪运动后,唾液CRH和皮质醇水平分别从5.68±6.15pg/ml和1.96±0.53ng/ml升高到40.21±10.1pg/ml和4.11±1.12ng/ml。志愿者在快速到达青藏高原3.6和5.5km后,唾液和血浆CRH分别从17.97±4.46pg/ml和43.71±9.1pg/ml升高到53.04±16.06pg/ml和106.7±31.7pg/ml,唾液和血浆皮质醇分别从1.87±0.51ng/ml和11.43±2.76ng/ml升高到3.84±0.99ng/ml和1.72±5.81ng/ml。急性耗竭运动和高原低氧暴露前后,唾液和血浆CRH都有很好的线性相关性。在耗竭运动前后,唾液和血浆CRH水平的相关系数分别是0.9336和0.9086。而高原现场低氧暴露前后,唾液和血浆CRH水平的相关系数分别是0.8410和0.8142。此外,在急性耗竭运动后CRH水平和皮质醇水平也有很好的相关性,其相关系数是0.9228和0.9437,而志愿者暴露于高原环境低氧后,CRH水平和皮质醇水平无明显相关性。8高原低氧AMS和血液、唾液高CRH水平相关人类志愿者分别从平原快速到达海拔3.6km和5.5km高度的青藏高原,到达高原后,使用AMS评分量表(Lake Louise Score)测定了个体AMS症状(AMS症状：包括头痛,恶心呕吐,全身无力,头昏和失眠)。在3.6km组,AMS的发病率是28.6%(63人中有18人发病),其中50.7%的志愿者出现头疼,6.2%出现恶心呕吐,41.2%出现全身无力,49.2%出现头昏,17.4%的志愿者出现失眠。而在5.5km组,AMS发病率是50%(48人中有24人发病),其中头痛,恶心呕吐,全身无力,头昏和失眠的发生率分别是50%,37.5%,41.7%,39.6%和21.7%。根据AMS评分量表将志愿者分为AMS发病组和AMS未发病组,其中AMS发病组的血浆和唾液CRH水平比AMS未发病组高40%,而血浆皮质醇没有显著差异。9唾液CRH基础水平可预测进入高原后发生AMS的风险性无论3.6km组中唾液和血浆基础CRH,还是5.5km组的血浆基础CRH都可以预测AMS的发生,ROC曲线下的面积(Area under curve,AUC)可以用来衡量预测的准确性,3种样本的AUC分别是0.7292,0.6和0.7236。从ROC曲线上我们也得出了最佳预测诊断阈值。基础血浆CRH水平大于或等于50.33pg/ml的志愿者中有83%(12人当中有10人)在到达3.6km海拔高度的高原环境后发生AMS；而唾液中CRH水平的阈值为21pg/ml,血浆和唾液中CRH水平存在线性相关,血浆CRH在50.33pg/ml时对应的唾液CRH值为20.8pg/ml.在另一组到达5.5km海拔高度的高原环境的志愿者中,基础血浆CRH水平在大于或等于49.16pg/ml有81%(16人当中有13人)发生AMS。结论高原低氧高度和时间依赖性地刺激大鼠下丘脑PVN区CRH合成和分泌,上调不同脑区和垂体CRHR1mRNA的表达,抑制CRHR2mRNA的表达,激活皮层细胞促凋亡基因bnip3, bim, bax的表达和caspase-3活性的增加,当轻度低氧时,促进抗凋亡基因bcl-2的表达,严重低氧则抑制bcl-2的表达,诱导皮层细胞凋亡增加,CRHR1拮抗剂可逆转低氧诱导的细胞凋亡,提示低氧脑损伤与CRHR1的过度激活有关。低氧强度和时间依赖性的增加大鼠血浆CRH的水平,而与脑CRH水平呈负相关,与大鼠血浆皮质酮水平呈正相关,提示血浆CRH来源于下丘脑中枢的分泌,CRH以及CRHR1是低氧应激下脑损伤HPA的标志分子。人体耗竭运动诱导机体低氧,升高血浆和唾液CRH及皮质醇水平,唾液CRH水平和血浆CRH水平有很好的线性相关,由于动物实验证明唾液腺不含CRH神经元,提示唾液CRH来源于血液循环。此外,高原低氧暴露后血浆和唾液CRH同时升高,发生AMS的志愿者中具有高的CRH水平,提示高水平的CRH可能与AMS发生相关。唾液和血浆CRH可作为中枢CRH/CRHR1激活的标志分子和AMS的发生密切相关,提示特异的CRHR1抑制剂可能成为治疗或预防AMS的一种选择。此外,由于CRH和CRHR1过度激活与脑损伤有关,可被视为监视AMS的一种生物标志分子,提示通过检测唾液中CRH的水平,可以预测个体应对低氧应激损伤的能力,并预测AMS的风险。更多还原

【Abstract】 Hypoxia is the most significant geographical feature of the Qinghai-Tibet Plateau in west China. Every year, millions of people enter the Qinghai-Tibet Plateau with the demand of travel, adventure, plateau search and rescue, and national defense. Acute mountain sickness (AMS) is a common feature of acute altitude illness while people rapid ascent to altitudes above3000m without sufficient time to acclimatize, and common symptoms includes headache, nausea, vomiting, fatigue, dizziness, and sleep disorders. Acute mountain sickness is the major challenge for people to reach the plateau, and the incidence of AMS correlates with the arrival altituede. At1850m to2750m, the incidence of AMS is25%,42%at3000m, and up to75%at6000m. If AMS is treated improper, life threaten high altitude cerebral edema (HACE) could occur. AMS can sharply limit recreation and work at high altitude, and have brought great difficulties to the staff in Yushu earthquake rescue operations. Understand AMS pathogenesis to early warn and forecast the risk of AMS occurred is very important, however, the pathogenesis of AMS is currently not yet entirely clear, and a lot of research have done on AMS prediction, hypoxic ventilatory response, heart rate variability rate, the change of the oxygen saturation after maximum workload exercise were proposed to predict the occurrence of AMS, but the results are not particularly desirable. Base our previous work on modulation mechanism of brain-endocrine-immune network under hypoxia, here we report the corticotrophin releasing factor (CRH) and its type1receptors (CRHR1), as biomarkers, are associated with AMS and could be used for prediction of AMS through monitoring salivary CRH levels. In animal studies adult rats were put in a hypobaric chamber to simulate high-altitude hypoxia at2,5, and7km for8hours and7km for2,8and24hours. The brain and plasma samples from rats were collected for evaluation of CRH, CRHR1mRNA, CRHR2mRNA and corticosterone levels respectively. And the plasma and saliva were collected for evaluation of CRH and cortisol level. The CRH and CRHR1mRNA, and cortisol/corticosterone were assayed with specific kits and real-time qPCR respectively. Volunteers are arranged to do exercise by bicycle and climbing up stairways in the Zhejiang University campus as and are ascent from lowland to high-altitude (3.6and5.5km), CRH levels were tested in plasma and saliva and AMS questionnaires were tested in all of these volunteers before and after these exercise. The LDH and O2saturation in both serum and saliva were evaluated. The linear correlation coefficients for levels of CRH among the brain, plasma, and saliva were analyzed. Double-blunted analysis was applied.Results1Hypoxia stimulated CRH release and CRH mRNA expression via intensity and time dependent mannersWe found CRH levels in PVN and ME significantly decreased while those in pituitary significantly increased in a dose and time dependent manner. CRH mRNA expression in PVN was significantly increased with ascent at altitude of2,5and7km and peaked at8hours, then returned to control level for24hours at altitude of7km hypoxia.2Hypoxia elicited increase of CRH and corticosterone levels in rat plasma via intensity and time dependent mannersHypobaric hypoxia enhances CRH and corticosterone levels in plasma in a dose dependent manner. Plasma CRH significantly increased with ascent to altitude of5and7km for8hours, while plasma corticosterone increased at altitude of2,5and7km. Extreme altitude (7km) increased plasma CRH and corticosterone levels reached the highest levels at24hours.3Elavation of CRH under hypoxia correlated with increase release of PVN CRH The increase of plasma CRH was positively correlated with increased plasma corticosterone, but the plasma CRH level negatively correlated with PVN CRH contents.4Hypobaric hypoxia activates CRHR1mRNA expression in various brain regions and pituitaryCRHR1mRNA expressions were sharply and dose dependently enhanced with ascent to altitude of5and7km in cortex, hippocampus, CeA, PVN, and LC as well as in pituitary, in the meantime, CRHR2mRNA expressions were decreased in cortex, CeA, and pituitary, while unchanged in hippocampus and LC, but increased in PVN. All increased or decreased expressions were removed24hours later except for in pituitary.There were different expression amount of CRHR1mRNA and CRHR2mRNA in brain regions. In cortex, CeA and LC, the expression amounts of CRHR1mRNA were about four times larger than CRHR2mRNA, but in hippocampus, PVN and pituitary, the expression amounts of CRHR1mRNA were close to CRHR2mRNA. After7km hypoxia for8hours, the expression amounts of CRHR1mRNA in cortex, CeA and LC were about eight folds higher than CRHR2mRNA, and the ratio in hippocampus, PVN and pituitary was about two.5Over-activated CRHR1was involved in hypoxia induced rat cortex cell apoptosisThe pro-apoptosis genes bnip3, bax and bim significantly increased in cerebral cortex at altitude of7km for8hours but not at altitude of2or5km. Moreover, anti-apoptosis gene bcl-2was significant increased only at2km for8hours.The severe hypoxia (7km) caused peak expression of pro-apoptotic genes bnip3, bim, and bax mRNA at8hours following turn down, while bcl-2mRNA was kept unchanged at all time points. Moreover, cell swelling in cerebral cortex were presented by HE staining, and increased numbers of apoptotic cells in cortex were also presented by TUNEL staining.Hypoxia up-regulated bax and bim mRNA expression at altitude of7km for8and24hours were reversed by the CP-154,526treatment, while hypoxia-induced bnip3mRNA expression did not. The bcl-2mRNA expression was not up-regulated by altitude of7km and also not affected by CP-154,526. As result, caspase-3activity in cerebral cortex was increased at altitude of7km for8and24hours and the increase was abolished by pretreatment of CP-154,526.6Acute exercises led to temportary hypoxia in humansWe found that in climbing stairs group, HR was significantly increased from86±10/min to177±16/min, salivary LDH activity was significantly increased from199.7±112.7U/L to468.7±279.9U/L, but serum LDH activity was unchanged. The saturated O2was significantly decreased from98%±1%to93%±3%. In cycling group, HR and salivary LDH activity were increased from82±11/min and156.7±92.5U/L to166±18/min and294±187.6U/L, however serum LDH activity was unchanged, while the saturated O2was decreased from98%±1%to94%±1%.7Intensive exercise and high altitude hypoxia both elevated CRH and cortisol levels in plasma and salivaWe first determined the circadian rhythm of CRH and cortisol release through measuring salivary CRH and cortisol in volunteers at regular life status. Saliva samples were collected once per～4hours intervals setting at7:00,11:00,15:00,19:00, and23:00hours. The CRH circadian rhythm was shown that the peak reached at7:00am and the nadir occurred at23:00pm as well as a similar level of CRH kept at11:00,15:00, and19:00pm.The CRH and cortisol levels in saliva and plasma both significantly elevated after exercises. In climbing stairs group, CRH in saliva and plasma increased from25.25±6.41pg/ml and39.82±7.87pg/ml to35.78±8.51pg/ml and54.37±9.31pg/ml, respectively; cortisol in saliva and plasma increased from from1.62±0.54ng/ml and6.5±1.51ng/ml to3.52±1.22ng/ml and10.8±2.58pg/ml, respectively. Similarly in cycling exercise, saliva CRH and cortisol increased from25.68±6.15pg/ml and1.96±0.53ng/ml to40.21±10.1pg/ml and4.11±1.12pg/ml, respectively.After the volunteers fast ascended to Lhasa at altitude of3.6km, CRH in plasma and salivary were significantly increased from43.71±9.1pg/ml and17·97±4·46pg/ml to106.7±31.7pg/ml and53.04±16.06pg/ml, respectively. Cortisol level also increased from11.43±2.76ng/ml to21.72±5.81ng/ml in plasma and from1.87±0.51ng/ml to3.84±0.99ng/ml in saliva, respectively.8High CRH level is associated with occurrence of AMS at high-altitude Two groups of volunteers were ascended to altitude of3.6km in Lhasa or5.5km respectively. In3.6km group, AMS developed in28.6%(18in63) volunteers, while50.7%(24in48) in5.5km group. We divide volunteers into AMS goup and No AMS group. AMS group has a high level of CRH in plasma and saliva, and also much higher amplify rate of CRH increase compared with No AMS group at high-altitude of3.6km, while cortisol level change was not statistically significant. The CRH level in plasma and saliva was also positively correlated with AMS score at altitude of3.6km, respectively; however, the basal CRH level in plasma and saliva was not correlated. Furthermore, there was no correlation between AMS score and cortisol level was shown either at sea level or at altitude of3.6km.9Basal CRH could be used to predict risk of AMSWe are able to predict individual risk of AMS when arrive at high altitude from their basal CRH level. In volunteers who had above50.33pg/ml plasma CRH at sea level,83%(10out of12) of them developed AMS after ascent to3.6km high altitude, and when monitoring in saliva, the threshold value was21pg/ml. In another group volunteers who ascended to5.5km high altitude,81%(13out of16) of them who had above49-16pg/ml basal plasma CRH level developed AMS at5.5km.ConclusionsIn rat study, hypobaric altitude-hypoxia produced a lowered O2level-dependent increase of CRH release and CRH mRNA expression in the PVN and upregulation of CRHR1mRNA expression in various brain region and pituitary as well as activation of apoptotic genes mRNA of binip3, bim, bax, and caspase-3activity but biphase of bcl-2in cortex. All these were associated with activation of CRHRl. This hypoxia enhanced plasma CRH levels were negatively correlated with brain CRH, but positively with plasma corticosterone in rats. In human, the exercises mimic hypoxia increased CRH and corticosterone levels in both plasma and saliva. This increased plasma CRH correlated with increased salivary CRH. In addition, volunteers with high plasma CRH at lowland developed the AMS by rapid ascent to Tibet high-altitude. The CRH level in both plasma and saliva reflect an activation of CRH neurons in the brain during high-altitude hypoxia that correlates with occurrence of AMS and acute brain injury through the CRHRl pathway, a specific CRHR1antagonist, therefore, may be a choice for therapy or prevention of AMS. Additionally, since over-activated CRH is associated with brain injury, it could be treated as a biomarker for AMS to monitor and predict an individual susceptibility to hypoxia and for AMS by salivary CRH level test.更多还原