【作者】 陈少军；

【导师】 杜继曾； 陈学群；

【作者基本信息】 浙江大学 ， 生理学， 2010， 博士

【摘要】 我国西部的青藏高原,最大的自然地理特征就是低氧,低氧不仅影响人们的健康,严重时还可导致高原病。高原低氧脑水肿和肺水肿,是最危险的急性高原病,常发生在严重低氧时,处理不当或不及时会危及生命,然而其发病机制,至今尚不十分清楚。阐明其作用机理,在理论和临床实践方面均有重要意义。低氧是一种非特异性应激原,它可诱导机体产生应激反应。下丘脑-垂体-肾上腺皮质轴(Hypothalamus-pituitary-adrenal axis, HPA)是机体重要的体液调节系统和应激反应系统,对各种应激做出应答反应,以新的稳态调节,适应变化了的不利环境。下丘脑促肾上腺皮质激素释放激素(Corticotropin releasing factor, CRF)肽是HPA轴的脑中枢调节者,它调节神经和体液两大系统。我们实验室以往研究发现,低氧激活下丘脑PVN区CRF, CRF mRNA和CRFR1受体表达,进而刺激HPA轴级联反应。CRFR1属G-蛋白偶联受体家族,在中枢神经系统和外周组织广泛分布。我们还发现,CRFR1参与低氧应激生理功能调节,对神经-内分泌-免疫网络系统中重要低氧靶基因和蛋白起核心主导调节作用。水通道蛋白(Aquaporins, AQPs)是细胞膜上一类特异性通道蛋白,介导不同类型细胞的水分子跨膜转运。AQP4是脑组织中分布最多的水通道蛋白,广泛存在于星状胶质细胞的终足上。AQP4在正常生理状态下与脑水平衡调节相关,在病理损伤时参与脑水肿形成。因此,我们设想,严重低氧应激时,通过刺激脑CRF分泌,激活CRFR1受体,驱动细胞内信号通路,增加AQP水通道功能,参与脑水肿形成,或低氧时肺内CRFR1参与肺水肿形成。本文阐述CRFR1受体参与低氧诱导脑水肿与肺水肿的形成以及CRFR1受体激活后的细胞内信号调控机制。本研究以雄性SD大鼠为研究对象,将其置于低气压舱内暴露于7000m(7.8%O2)低氧8小时,模拟急性高原低氧脑水肿和肺水肿,研究CRFR1参与机制。利用核磁共振技术检测脑组织水分子表观弥散系数(ADC)；干湿重法检测脑和肺组织含水量；Western blot法检测前额叶皮层水通道蛋白4(AQP4)蛋白和肺组织中AQP5蛋白表达变化；ELISA法检测前额叶皮层内皮素(ET-1)和血浆皮质酮的变化；RIA法检查肺组织内皮素ET-1的变化；激光共聚焦免疫荧光法等方法检测CRFR1和AQP4在原代培养的大鼠皮层星状胶质细胞和大鼠前额叶皮层中的共表达；免疫共沉淀检测AQP4蛋白磷酸化状态；荧光标记法记录星状胶质细胞内[Ca2+]i变化和细胞膜通透性的改变；比色法检测肺组织NO表达和NOS酶活性的变化。采用相关药盒,检测cAMP/PKA和PKC/Ca2+通路,揭示CRFR1受体激活后第二信使通路参与脑水肿和肺水肿发生的细胞内信号调节机制。研究结果第一部分1 G-蛋白偶联CRFR1受体参与模拟高原急性低氧诱导大鼠脑水肿的形成模拟7000m高原低氧8小时,可以诱导大鼠脑含水量增加,明显升高脑ADC值,形成脑水肿(P<0.05)。低氧前,预先腹腔注射30mg/kg CRFR1受体拮抗剂CP154,526,可以阻断低氧诱导的脑含水量增加和ADC值的升高(P<0.05 vs.hypoxia).2 G-蛋白偶联CRFR1受体对模拟高原急性低氧诱导大鼠大脑皮层AQP4和ET-1蛋白的调节作用免疫荧光激光共聚焦结果显示,CRFR1和AQP4共存于大鼠前额叶皮层中。Western blot实验结果显示：低氧诱导大鼠前额叶皮层AQP4表达升高(P<0.01),低氧前预先腹腔注射CRFR1受体拮抗剂CP154,526,可以阻断这种作用(P<0.05 vs. hypoxia),而预先腹腔注射NF-kB抑制剂PDTC,或CP154,526和PDTC复合注射,对这种作用无明显影响。与常氧组比较,该低氧诱导大鼠前额叶皮层ET-1表达明显升高(P<0.001),低氧前预先腹腔注射CRFR1拮抗剂CP 154,526、NF-kB抑制剂PDTC,或CP154,526+PDTC复合注射,都可以阻断这种作用(P<0.001 vs. hypoxia)。与常氧组比较,低氧促使大鼠血浆皮质酮水平明显升高(P<0.01),低氧前预先腹腔注射CRFR1拮抗剂CP154,526,或CP154,526+PDTC复合注射都可以阻断这种作用,而预先注射NF-kB抑制剂PDTC则部分阻断这种作用。拮抗剂CP154,526、抑制剂PDTC,或CP154,526+PDTC复合注射对常氧下血浆皮质酮水平无明显作用。3 G-蛋白偶联CRFR1受体关联的胞内二信使通路,对星状胶质细胞水通道AQP4磷酸化的调节免疫荧光实验显示,CRFR1受体与AQP4水通道蛋白在大鼠皮层原代星状胶质细胞中共存。体外培养原代大鼠星状胶质细胞,与外源CRF共温育,细胞内钙离子[Ca2+]i浓度迅速升高,细胞膜通透性明显增加,此作用可被CRFR1受体拮抗剂所拮抗。在原代培养的大鼠星状胶质细胞,与CRF共温育10分钟或30分钟可明显升高胞内cAMP水平,该作用在与CRFR1拮抗剂预温育30分钟而阻断；与CRF温育30分钟可导致AQP4蛋白PKA位点磷酸化表达,温育10分钟无明显影响；与CRF共温育30分钟,导致AQP4蛋白PKC位点磷酸化表达,该作用可以被PKC拮抗剂Bisindolylmaleimide预温育30分钟阻断；此外,与CRF共温育10分钟或30分钟对AQP4蛋白PKG位点磷酸化表达无影响,PKG拮抗剂KT-5823预温育30分钟对该作用无明显影响。原代培养的大鼠星状胶质细胞,暴露于低氧(1% O2)8小时,胞内cAMP水平无明显改变(P>0.05)。与10nM CRF温育时,再暴露于低氧(1% O2)8小时,胞内cAMP水平明显升高(P<0.001),而预先与CRFR1拮抗剂CP154,526共温育,则拮抗了CRF诱导的cAMP水平升高。然而与CRF温育8小时或低氧温育8小时,对AQP4蛋白PKA位点磷酸化表达没有明显影响。Western blot结果显示常氧下或低氧下10nM CRF作用8h都可以促进胶质细胞AQP4蛋白量表达增加。第二部分1 G-蛋白偶联CRFR1受体及NF-kB对模拟高原急性低氧诱导肺水肿的作用NF-kB参与模拟高原低氧诱导的肺水肿。模拟7000m高原急性低氧8小时,可明显升高大鼠肺组织的含水量(P<0.01 vs. control),该作用可被预先腹腔注射NF-kB抑制剂PDTC阻断(P<0.01 vs. hypoxia),但不被预先注射CRFR1受体拮抗剂CP154,526,也不被CP154,526+PDTC复合注射所阻断。2 G蛋白偶联CRFR1受体及NF-kB对模拟高原急性低氧诱导肺组织ET-1水平的作用NF-kB参与模拟高原低氧诱导肺组织ET-1水平升高。模拟7000m高原低氧8小时,导致大鼠肺组织中ET-1水平明显升高(p<0.05),此作用不被预先腹腔注射15mg/kg CRFR1拮抗剂CP154,526所拮抗,而预先注射30mg／kg CP154,526则导致低氧增高的肺ET-1水平进一步增高(P<0.05 vs. hypoxia);而预先注射NF-kB抑制剂PDTC,则可抑制这一低氧效应(P<0.05 vs. hypoxia),但预先腹腔注射复合拮抗剂CP154,52+PDTC,也导致低氧诱导的大鼠肺组织ET-1表达进一步升高(P<0.01 vs.hypoxia)。3 G-蛋白偶联CRFR1受体对模拟高原急性低氧降低肺组织NO含量和NOS酶活力的作用模拟7000m高原低氧8小时,明显降低大鼠肺组织中NO水平(P<0.01),此作用在预先腹腔注射CRFR1拮抗剂(30mg／kg)后,NO水平进一步降低(P<0.001),15mg/kg CRFR1拮抗剂无明显作用。该低氧还降低总一氧化氮合酶(tNOS)活力水平,CRFR1拮抗剂预注射,不论15mg／kg或30mg／kg均导致低氧降低的总一氧化氮合酶活力进一步降低。该低氧和CRFR1拮抗剂均不影响诱导型一氧化氮合酶(iNOS)酶活力水平。4 G-偶联蛋白CRFR1受体不参与低氧对肺组织AQP5蛋白表达的调节模拟7000m高原低氧8小时,诱导大鼠肺组织AQP5蛋白表达升高(P<0.05),预先腹腔注射CRFR拮抗剂,对大鼠肺组织AQP5表达无明显影响。结论：1模拟7000m高原急性低氧8小时,导致大鼠脑水肿,这与低氧激活脑皮层CRFR1受体相关。CRFR1受体激活,介导皮层ET-1蛋白(NF-kB也介导ET-1的高表达)和AQP4蛋白表达上调。2离体培养大鼠原代星状胶质细胞,可见AQP4蛋白与CRFR1受体蛋白共存现象；CRF刺激星状胶质细胞上的CRFR1受体,升高胞内钙离子[Ca2+]i浓度、激活cAMP/PKA和PKC通路,使AQP4蛋白PKA、PKC位点磷酸化,改变AQP4通道水通透性,促成脑细胞水肿。3模拟7000m高原急性低氧,诱导大鼠肺组织含水量增加,形成肺水肿。低氧上调肺组织ET-1表达,下调NO水平和总NOS酶活力,增加水通道AQP5表达,此与肺水肿形成相关。核转录因子NF-kB通过上调ET-1表达参与低氧诱导肺水肿。提示CRFR1受体可能通过NO/NOS途径和ET-1表达影响肺水肿形成。促肾上腺皮质激素释放激素；促肾上腺皮质激素释放激素1型受体；内皮素-1；高原；低氧；核因子KB；一氧化氮；一氧化氮合酶；蛋白激酶A；蛋白激酶C；肺水肿；大鼠本文创新点：1发现G蛋白偶联CRFR1受体,通过上调ET-1和水通道AQP4活动,参与了模拟7000m高原急性低氧8小时诱导的大鼠脑水肿形成。NF-kB通过上调ET-1表达参与低氧脑水肿。2发现在离体培养大鼠原代皮层胶质细胞中,AQP4蛋白与CRFR1受体蛋白共存更多还原

【Abstract】 The Qinghai-Tibet Plateau in west China, the most significant geographical feature is the low oxygen level. Hypoxia not only affects people health, but also leads to serious altitude sickness. High altitude cerebral edema (HACE) and high altitude pulmonary edema (HAPE), the most serious altitude sickness, often occur in severe hypoxia environment, which would endanger life if improper handling. Its pathogenesis remains obscure, and the mechanism would be important in both theory and clinical practice.As a non-specific stressor, hypoxia can induce stress response generated. The hypothalamus-pituitary-adrenal axis (HPA axis) is the humoral regulation system and the stress response system, making a variety of stress response to adapt the harmful environment into a new homeostasis. Hypothalamic corticotropin releasing factor (CRF) peptide is the central regulator of HPA axis, which regulates both body fluid system and nervous system. Previous studies in our lab found that hypoxia activated CRF release, CRF mRNA and CRFR1 receptor express in hypothalamic PVN, which led to stimulate HPA axis cascade. Corticotropin releasing factor receptor-1 is a member of G-protein coupled receptor family, widely distribute in the central nervous system and periphery system. We also found that CRFR1 participate hypoxia-induced stress responses, playing a central role in regulation of important hypoxia target genes and proteins in neuro-endocrine-immune network system. Aquaporins (AQPs) are a family of membrane channels that serve as selective pores through which water flow cross the plasma membranes in many cell types. AQP4 is the major water channel in brain tissue, strongly enriched in astrocytes end-feet. AQP4 plays an important role both in the cerebral water balance in normal physiological condition and in the formation of brain edema during pathological injury. Therefore, we assume that:severe hypoxia stress could stimulate CRF secretion from hypothalamus to activate CRFR1, driving intracellular signaling pathways to increase AQP4 water channel permeability, involving in the formation of brain edema at last. Furthermore, CRFR1 and transcriptor NF-kB may play a role in hypoxia-induced pulmonary edema. Therefore, the paper is to explore whether CRFR1 involves in HACE and HAPE as well as what is the intracellular signaling mechanism.In this study, we developed a hypoxia model for brain edema and pulmonary edema. The rats were exposed to hypoxia (7.8% O2, equal to 7000m altitude) in a hypobaric chamber for 8 hours. Magnetic resonance imaging was applied to measure the ADC value for brain cell water. The water content in brain and lung was assessed by dry/wet ratio. The AQP4 and AQP5 protein expressed in rat brain cortex and in lung tissue respectively were determined by Western blot. ET-1 in cortex and corticosterone in plasma was detected by ELISA kit. ET-1 in lung was detected by RIA. Coexistence of CRFR1 with AQP4 protein was showed by confocal immunofluorescence in brain cortex and in primary cortex astrocytes cell. The AQP4 and AQP5 phosphorylation was detected by protein immunoprecipitation. Intracellular [Ca2+]i was determined by Fluo3/AM calcium indicator. The water permeability was measured using laser scanning confocal microscopy. And the level of nitric oxide and nitric oxide synthase active unit in the pulmonary tissue were measured by colorimetric method.This study will address whether the CRFR1 contributes to cerebral and pulmonary edema, and what the intracellular signaling pathway driven from CRFR1 is linked to AQPs, making its phosphorylation and altering its permeability to water, then induced cell swelling and the brain and pulmonary edema. Part One1 G-protein coupled CRFR1 mediates acutely simulated altitude hypoxia-induced rat brain edemaAcutely simulated hypoxia (7.8% O2,8 h) significantly increased water content in brain and the ADC value for water permeability, leading brain edema, and the effects were blocked by i.p. preinjection with CRFR1 antagonist CP154,526 (30mg/kg).2 G-protein coupled CRFR1 mediates AQP4 and ET-1 expression induced by acutely simulated altitude hypoxia in rat brainConfocal immunofluorescence image showed coexistence of CRFR1 protein and AQP4 channel protein in rat prefrontal cortex.Western blot results showed that AQP4 channel protein was enhanced in the hypoxic rat prefrontal cortex (P<0.01). The effect was significantly blocked by i.p. preinjection of CRFR1 antagonist CP154,526 (P<0.05 vs. hypoxia), but not by i.p. preinjection of NF-kB inhibitor PDTC(pyrrolidinedithio carbamate ammonium), or combination of CP154,526 plus PDTC. These antagonists had no effect on AQP4 expression in control rat.The hypoxia significantly increased ET-1 expression in rat prefrontal cortex, which was blocked by i.p. preinjection of CP154,526, PDTC, or combination of CP154,526 plus PDTC.The hypoxia significantly enhanced plasma corticosterone (P<0.01), which was blocked by i.p. preinjection of CRFR1 antagonist CP154,526 and combination of CP154,526 plus PDTC, partly blocked by PDTC. And these antagonists had no effect on plasma corticosterone.3 Modulations of intracellular pathway driven by G-protein coupled CRFR1 in AQP4 channel protein in primarily cultured rat astrocytes under the hypoxiaConfocal immunofluorescence image showed that coexistence of CRFR1 protein and AQP4 channel protein in the primary cortical astrocytes.In cultured primary astrocytes, CRF induced intracellular [Ca2+]i fast increase, following water permeability enhancement, which was blocked by i.p. preinjection with CRFR1 antagonist CP154,526 for 30min.Incubation of rat primary cortical astrocytes with CRF for 10 or 30min increased cAMP accumulation in the cells, which was blocked by preincubation with CRFR1 antagonist for 30min. In CRF treated astrocytes for 30min, but not 10min, induced AQP4 protein phosphorylation at PKA site of AQP4 channel protein. Again CRF induced for 30 min AQP4 protein phosphorylation occured at PKC site of the AQP4 protein, which was blocked by preincubation with PKC antagonist (Bisindolylmaleimide) for 30min. PKG sites appeared to be not involved in CRF challenged astrocytes, and the PKG antagonist KT-5823 had no effect.In cultured primary astrocytes treated with 10nM CRF for 8h, cAMP levels increased significantly in these cells, which were blocked by preincubation of CRFR1 antagonist. Exposed to CRF for 8h or hypoxia 8h had no effect on AQP4 protein phosphorylation at PKA site. Western blot showed CRF for 8h induced AQP4 protein expression.Part Two1 Effects of G-protein coupled CRFR1 and NF-kB on rat pulmonary edema induced by acutely simulated hypoxiaAcutely simulated hypoxia (7.8% O2,8 h) significantly increased water content in rat lung, which was blocked by i.p. preinjection of NF-kB inhibitor PDTC, but not by i.p. preinjection of CP154,526, or combination of PDTC plus CP154,526.2 Effects of G-protein coupled CRFR1 and NF-kB on rat pulmonary ET-1 upregulation induced by acutely simulated hypoxiaNF-kB mediated rat pulmonary ET-1 upregulation. Acute hypoxia (7.8% O2,8 h) significantly induced ET-1 expression increase in rat lung tissue, the effect was not blocked by i.p. preinjectiont of CRFR1 antagonist CP154,526 (15mg/kg), whereas enhanced by i.p. pretreatment of antagonist CP154,526 (30mg/kg) or combination of PDTC plus CP154,526 (30mg/kg). However the effect was blocked by single pretreatment of PDTC.3 Effects of G-protein coupled CRFR1 on downregulation of rat pulmonary NO and NOS activity induced by acutely simulated hypoxiaCRFR1 antagonist induced further downregultion of the NO content and NOS activity. Acute hypoxia (7.8% O2,8h) induced NO level decrease in rat lung, which was further reduced by i.p. pretreatment of antagonist CP154,526 (30mg/kg), not by 15mg/kg. Acute hypoxia downregulated NOS activity and the effect was further reduced by i.p. pretreatment of antagonist CP154,526 15 or 30mg/kg. But hypoxia or CRFR1 antagonist had no effect on inducible NOS activity.4 G-protein coupled CRFR1 is not involved in the regulation of hypoxia-induced upregulation of AQP5 channel proteinAcute hypoxia (7.8% O2,8h) upregulated the expression of AQP5 channel protein in rat lung, the effect was not blocked by i.p. pretreatment of CRFR1 antagonist CP 154,526.Conclusion:1 Acute simulated hypoxia (7.8% O2,8 h) induces rat brain edema, which is associated with G-protein coupled CRFR1 triggering, CRFR1 triggering mediates hypoxia-induced ET-1 and AQP4 upregulation in the brain. ET-1 upregulation contributes to brain edema via NF-kB transcription2 In the primary cultured rat cortical astrocytes, CRFR1 and AQP4 co-exist. CRF stimulates CRFR1 on the astrocytes, elevating [Ca2+]i concentration, activating the cAMP/PKA and PKC pathways, inducing AQP4 phosphorylation at the PKA and PKC site of AQP4 channel protein which lead to increase AQP4 channel permeability and make the astrocyte swelling.3 The acute hypoxia (7.8% O2,8 h) increased water content in rat lung and pulmonary ET-1 expression, as well as decreased NO level, total NOS activity, and upregulated AQP5 channel expression, which all associate with hypoxia-induced pulmonary edema. Nuclear transcription factor NF-kB may contribute to pulmonary edema by upreguating ET-1 expression. And CRFR1 may potentially be involved in altitude hypoxic pulmonary edema through the NO/NOS pathways and the ET-1 expression.更多还原