创伤应激早期CRH对大鼠下丘脑CRH分泌的超短反馈调节机制研究
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摘要
应激反应的主要特征是以下丘脑—垂体—肾上腺皮质轴(HPA)的激活为核心的生理和心理反应。HPA轴激活的中枢控制是十分复杂的，在这一系列神经内分泌调节过程中，下丘脑合成和释放的促肾上腺皮质激素释放激素(CRH)起着关键的作用，它控制着HPA轴的兴奋水平。1985年，Ono等提出在应激反应中可能存在超短正反馈调节机制，调节下丘脑神经元分泌CRH。研究发现，在下丘脑含CRH的轴突可与合成CRH的神经元形成突轴联系，且下丘脑神经元可表达CRH受体，从形态学方面推测可能存在超短反馈调节机制。
CRH能否激活下丘脑神经元，以及作用机制如何，仍不十分清楚。本研究通过大鼠严重创伤模型，用免疫组织化学、Western blot、RT-PCR等方法对下丘脑神经元CRH、CRHⅠ型受体(CRH_1R)的分布及含量进行检测；应用CRH刺激原代培养的胎鼠下丘脑神经元，用Fura—2／AM比率荧光成像系统、免疫组织化学、Western blot等方法对分析培养神经元的单个神经元内游离钙浓度([Ca~(2+)]i)、cAMP、PKA、CREB、CRH、CRH_1R的分布及含量进行检测。用培养的下丘脑神经元和大鼠严重创伤动物模型，较系统的探讨了CRH对下丘脑神经元表达CRH的调节作用及相关信号转导机制，为调控创伤性应激反应提供理论和实验依据。
本课题的主要结果和结论
1．建立了大鼠严重胸部撞击伤伴单侧股骨骨折的严重创伤应激动物模型。该模型具有：可精确控制撞击参数、撞击部位准确、伤情稳定、重复性好等优点，是研究严重创伤后应激反应较好的模型。
2．建立了较稳定的胎鼠下丘脑神经元培养方法。神经元培养1d—3d生长最快，培养7d—10d神经元胞体大小和突起长度均达高峰，生长状
态最好，同时可见神经元间突轴联系，此时是进行神经元形态和机能测
试的最佳时期。
3．CRH刺激培养的下丘脑神经元后，［Cak］i迅速升高。应用L型CaZ”
通道阻滞剂Ni诉0 PKA阻滞剂H89可显著抑制CRH引起的K／勺；增加，说
明 L型o“通道和 PKA信号通路在CRH刺激＊ak］变化中可能起重要作
用。
4．正常情况下PKA主要分布于神经元的胞浆。CRH刺激后，PKA除
分布于胞浆外，核膜周围阳性着色加深，细胞核也明显深染，活化PKA
含量显著增加。提示CRH通过与下丘脑神经元CRHIR结合后可激活PKA
信号通路。
5．CRH可引起下丘脑神经元磷酸化CREB含量明显增加，运用
CP154526、H89、Nif可显著抑制磷酸化CREB的生成，结合CRH可引起
下丘脑神经元活化PKA生成增多和 ［Ca卜1水平升高，提示此两种信号传导途
径都可使CREB磷酸化含量增加，以调控下丘脑神经元转录活性。
6．CRH可引起培养的下丘脑神经元CRH含量显著增加，运用
CP 54526、H89、Nif可明显抑制CRH含量增加，说明PKA信号通路及从
L型Cak通道内流的Cah在CRH通过与CRH以结合而刺激下丘脑神经元引
起其自身基因表达中起重要作用。提示CRH可通过PKA和［Cayv信号通
路调节下丘脑神经元表达CRH。
7．严重创伤后下丘脑室旁核oVN）和视上核侣ON兀RH和CRHIR含量
明显增多，而CP—154526可显著抑制CRH和CRHIR的增加，说明严重创
伤后增多的CRH与CRHIR结合后进一步引起下丘脑PVN神经元CRH和
CRH人表达增加。提示在严重创伤应激反应中，CRH对下丘脑神经元
CRH的表达可能存在超短正反馈调节机制。
The main characteristics of stress response are a series of physiological and psychological changes from activation of hypothalamus-pituitary-adrenal (HPA) axis. It is very complicated to control the activation of HPA axis in central nervous system. Synthesis and release of corticotropin-releasing hormone (CRH) from hypothalamus plays a key role in the regulation of the HPA axis. CRH controls the stimulating level of HPA axis. The existence of ultrashort feedback control of CRH secretion during stress was first suggested by Ono et al in 1985. CRH-containing axon may contact with CRH-producing neurons in synapse formation. CRH type 1 receptor (CRH1R) is expressed in hypothalamic neurons. It is suggested from morphology that there is a possibility of feedback regulation of CRH receptors in CRH-producing neurons.
In this study the model of traumatic stress in rats was established, the changes of CRH, CRH1R and c-fos distribution and expression were observed and determined with immunocytochemistry, western blotting , RT-PCR in vivo; Hypothalamic neurons from fatal rats were and irritated with CRH. Using Fura-2/AM ratio fluorescence imaging analysis was applied to detect intracelluar Ca2+, immunocytochemistry and western blotting the distribution and content of PKA, CREB, CRH in virto.
The main results and conclusions are as follows:
1. The model of impact injury of right lateral superior chest of rats with unilateral femur fracture was established. The model has the
advantages of simple manning, controllable physical parameters, accuracy and repeatability of impacting site and constant severity of wound.
2. Hypothalamic neurons in vitro was well and stables cultured. The neurons grew most fast during days 1-3, and reached confluent monolayer and grew up synapse during days 7-10.
3. CRH caused an increased intracellular concentration of Ca2+ with marked difference between every expermental group and control group. The L-type Ca2+ channel inhibitor nifedipine or the PKA inhibitor H89 can reduce the CRH-induced increase in cytosolic Ca +, indicating that L-type Ca2+ channel and PKA signal pathway may play an impoatant role in CRH-induced [Ca2+]i.
4. Normally PKA is mainly distributed in the cytoplasm of neuron. After stimulation of CRH, however, it appeared also around the nucleal membrane, and even in the nucleus. The concentration of PKA increased, indicating that binding of CRH to its cognate receptors was associated with the activation of PKA signal pathway.
5. CRH may cause the increase of phosphorylated CREB in the neuron of hypothalamus, while CP-154526, H98, Nif can significantly inhibit the production of phosphorylated CREB. Combining with CRH CP-154526, H89 and Nif may cause the increase of activated PKA production and [Ca2+]i in the neuron of hypothalamus, indicating both of these two signal transduction pathways may increase the content of phosphorylated CREB , thus regulate transcription activation of the neuron in hypothalamus.
6. CRH may cause macked increase of CRH content in cultured hypothalamic neuron, while CP-154526, H89 and Nif may greatly inhibit the increase of CRH content, indicating PKA signal pathway and Ca2+ pathway play an important role in combination of CRH with CRH1R and stimulating hypothalamic neuron to cause its self gene expression.
7. Traumatic stress induced an increased intracellular concentration of CRH and CRH1R in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) of rat hypothalamus, while CP-154526, H89 or nifedipine significantly inhibit the expression of CRH and CRH1R.
CRH能否激活下丘脑神经元，以及作用机制如何，仍不十分清楚。本研究通过大鼠严重创伤模型，用免疫组织化学、Western blot、RT-PCR等方法对下丘脑神经元CRH、CRHⅠ型受体(CRH_1R)的分布及含量进行检测；应用CRH刺激原代培养的胎鼠下丘脑神经元，用Fura—2／AM比率荧光成像系统、免疫组织化学、Western blot等方法对分析培养神经元的单个神经元内游离钙浓度([Ca~(2+)]i)、cAMP、PKA、CREB、CRH、CRH_1R的分布及含量进行检测。用培养的下丘脑神经元和大鼠严重创伤动物模型，较系统的探讨了CRH对下丘脑神经元表达CRH的调节作用及相关信号转导机制，为调控创伤性应激反应提供理论和实验依据。
本课题的主要结果和结论
1．建立了大鼠严重胸部撞击伤伴单侧股骨骨折的严重创伤应激动物模型。该模型具有：可精确控制撞击参数、撞击部位准确、伤情稳定、重复性好等优点，是研究严重创伤后应激反应较好的模型。
2．建立了较稳定的胎鼠下丘脑神经元培养方法。神经元培养1d—3d生长最快，培养7d—10d神经元胞体大小和突起长度均达高峰，生长状
态最好，同时可见神经元间突轴联系，此时是进行神经元形态和机能测
试的最佳时期。
3．CRH刺激培养的下丘脑神经元后，［Cak］i迅速升高。应用L型CaZ”
通道阻滞剂Ni诉0 PKA阻滞剂H89可显著抑制CRH引起的K／勺；增加，说
明 L型o“通道和 PKA信号通路在CRH刺激＊ak］变化中可能起重要作
用。
4．正常情况下PKA主要分布于神经元的胞浆。CRH刺激后，PKA除
分布于胞浆外，核膜周围阳性着色加深，细胞核也明显深染，活化PKA
含量显著增加。提示CRH通过与下丘脑神经元CRHIR结合后可激活PKA
信号通路。
5．CRH可引起下丘脑神经元磷酸化CREB含量明显增加，运用
CP154526、H89、Nif可显著抑制磷酸化CREB的生成，结合CRH可引起
下丘脑神经元活化PKA生成增多和 ［Ca卜1水平升高，提示此两种信号传导途
径都可使CREB磷酸化含量增加，以调控下丘脑神经元转录活性。
6．CRH可引起培养的下丘脑神经元CRH含量显著增加，运用
CP 54526、H89、Nif可明显抑制CRH含量增加，说明PKA信号通路及从
L型Cak通道内流的Cah在CRH通过与CRH以结合而刺激下丘脑神经元引
起其自身基因表达中起重要作用。提示CRH可通过PKA和［Cayv信号通
路调节下丘脑神经元表达CRH。
7．严重创伤后下丘脑室旁核oVN）和视上核侣ON兀RH和CRHIR含量
明显增多，而CP—154526可显著抑制CRH和CRHIR的增加，说明严重创
伤后增多的CRH与CRHIR结合后进一步引起下丘脑PVN神经元CRH和
CRH人表达增加。提示在严重创伤应激反应中，CRH对下丘脑神经元
CRH的表达可能存在超短正反馈调节机制。
The main characteristics of stress response are a series of physiological and psychological changes from activation of hypothalamus-pituitary-adrenal (HPA) axis. It is very complicated to control the activation of HPA axis in central nervous system. Synthesis and release of corticotropin-releasing hormone (CRH) from hypothalamus plays a key role in the regulation of the HPA axis. CRH controls the stimulating level of HPA axis. The existence of ultrashort feedback control of CRH secretion during stress was first suggested by Ono et al in 1985. CRH-containing axon may contact with CRH-producing neurons in synapse formation. CRH type 1 receptor (CRH1R) is expressed in hypothalamic neurons. It is suggested from morphology that there is a possibility of feedback regulation of CRH receptors in CRH-producing neurons.
In this study the model of traumatic stress in rats was established, the changes of CRH, CRH1R and c-fos distribution and expression were observed and determined with immunocytochemistry, western blotting , RT-PCR in vivo; Hypothalamic neurons from fatal rats were and irritated with CRH. Using Fura-2/AM ratio fluorescence imaging analysis was applied to detect intracelluar Ca2+, immunocytochemistry and western blotting the distribution and content of PKA, CREB, CRH in virto.
The main results and conclusions are as follows:
1. The model of impact injury of right lateral superior chest of rats with unilateral femur fracture was established. The model has the
advantages of simple manning, controllable physical parameters, accuracy and repeatability of impacting site and constant severity of wound.
2. Hypothalamic neurons in vitro was well and stables cultured. The neurons grew most fast during days 1-3, and reached confluent monolayer and grew up synapse during days 7-10.
3. CRH caused an increased intracellular concentration of Ca2+ with marked difference between every expermental group and control group. The L-type Ca2+ channel inhibitor nifedipine or the PKA inhibitor H89 can reduce the CRH-induced increase in cytosolic Ca +, indicating that L-type Ca2+ channel and PKA signal pathway may play an impoatant role in CRH-induced [Ca2+]i.
4. Normally PKA is mainly distributed in the cytoplasm of neuron. After stimulation of CRH, however, it appeared also around the nucleal membrane, and even in the nucleus. The concentration of PKA increased, indicating that binding of CRH to its cognate receptors was associated with the activation of PKA signal pathway.
5. CRH may cause the increase of phosphorylated CREB in the neuron of hypothalamus, while CP-154526, H98, Nif can significantly inhibit the production of phosphorylated CREB. Combining with CRH CP-154526, H89 and Nif may cause the increase of activated PKA production and [Ca2+]i in the neuron of hypothalamus, indicating both of these two signal transduction pathways may increase the content of phosphorylated CREB , thus regulate transcription activation of the neuron in hypothalamus.
6. CRH may cause macked increase of CRH content in cultured hypothalamic neuron, while CP-154526, H89 and Nif may greatly inhibit the increase of CRH content, indicating PKA signal pathway and Ca2+ pathway play an important role in combination of CRH with CRH1R and stimulating hypothalamic neuron to cause its self gene expression.
7. Traumatic stress induced an increased intracellular concentration of CRH and CRH1R in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) of rat hypothalamus, while CP-154526, H89 or nifedipine significantly inhibit the expression of CRH and CRH1R.
引文
1.Lennard DE, Eckert WA, Merchenthaler I. Corticotropin-releasing hormone neurons in the parlventricular nucleus project to the external zzone of the median eminence:a study combining retrograde labeling with immunocytochemistry. J Neuroendocrinol, 1993;5:175-181
2.Kawano H, Daikoku S, Shibasaki T. CRF-containing neurons in the rat hypothalamus: retrograde tracing and immunohistochemical studies. J Comp Neurol, 1988;272:260-268
3.Dunn AJ, Berridge CW. Physiological and behavioral responses to Corticotropin-releasing factor administration: is CRF a mediatir of anxiety or stress responses? Brain Rev, 1990;15:71-100
4.Owens MJ, Nemeroff CB. Physiology and pharmacology of Corticotropin-releasing factor. Pharmacol Rev, 1991;43:425-473
5.Imaki T, Nahan JL, Rivier C,Sawchenko PE, Vale W. Differential regulation of CRH mRNA in rat brain regionsby glucocorticoids and stress. J Neurosci, 1991; 11:585-599
6.Rivest S, Laflamme N.Neuronal activity and neuropeptide gene transcriptoin in the brains of immune-chsllenged tats. J Neuroendocrinol, 1995;7:501-525
7.Makino S, Gokd PW, Schulkin J. Corticosterone effects on corticotropin-releasing hormone mRNA in the central nucleus of the amygdala and the paraventricular nuclrus of the hypothalamus. Brain Res, 1994;640:105-112
8.Itoi K, Horiba N, Tozawa F, Sakai Y, et al. Major role of 3',5'-cyclic adenosine monophosphate-dependent protein kinase A pathway in corticotropin-releasing hormone gene expression in the rat hypothalamus in vivo. Endocrinology, 1996; 137:2389-2396
9. Kraus J, Hollt V. Identification of a cAMP-response elenemt on the human proopiomelanocortin gene upstream promoterl DNA Cell Biol,1995;14:103-110
10. Calogero A, Bernadini R, Margioris AN,et al. Effects of serotinergic agonists and antagonists on corticotropin-releasing hormone secretion by explanted rat hypothalami. Peptides, 1989;10:189-200
11. Benveniste H. Brain microdialysis. J Neurochem, 1989;52:1667-1678
12．王正国，孙立英，刘宝松等．生物撞击机的研制及撞击伤发生机制的研究．中华创伤杂志，1999；15(4)：293—297
13. Tin YS. Corticotropin-releasing hormone mediates the response to cold stress in the neonatal rat without compensatory enhancement of the peptide's gene expression. Endocrinology, 1994; 135:2365-2378
14. Schiller HJ, Reilly PM, Bulkley GB. Tissue perfusion in critical illnesses: Antioxidant therapy. Crit Care Med, 1993,21:s92-103
15. Theoharides TC, Spanos C, Pang X, et al. Stress-induced intracranial mast cell degranulation: A Corticotropin-releasing hormone-mediated effect. Endocrinology, 1995;136:5745-5753
16. Ellis L, Gilston V, Soo CC,et al. Activation of the transcription factor NF-kappaBin in the tat air pouch model of inflammation. Ann Rheum Dis, 2000;59(4):303-307
17．童伟光，刘淑琴、钱国桢．烫伤或外伤骨折对下丘脑神经内分泌细胞的影响k Gomori方法、免疫组化和免疫电镜观察．解剖学杂志，1990；13(2)：89-92
18. Brownstein MJ, Russell JT, Gainer H. Synthesis, transport, and release of posterior pituitary hormones. Science, 1980;207(4429): 373-378]
19. Morris JF. Membrams retrieval at neurosecrotory axon ending.Nature, 1976;261:723-742
20. Brownstein MJ, Russell JT, Gainer H. Synthesis, transport, and
release of posterior pituitary hormones. Science, 1980;207(4429): 373-378]
21.Perlmutter LS, Tweedle CD, Hatton GI. Neuronal/glial plasticity in the supeaoptic dendritic zone: dendritic bundling and double synapse formation at parturition. Neuroscience, 1984; 13(3):769-779
22.Matsumoto A, Arnold AP, Micevych PE. Gap junctions between lateral spine motoneurons in the rat. Brain Res, 1989;495:362-366
2.Kawano H, Daikoku S, Shibasaki T. CRF-containing neurons in the rat hypothalamus: retrograde tracing and immunohistochemical studies. J Comp Neurol, 1988;272:260-268
3.Dunn AJ, Berridge CW. Physiological and behavioral responses to Corticotropin-releasing factor administration: is CRF a mediatir of anxiety or stress responses? Brain Rev, 1990;15:71-100
4.Owens MJ, Nemeroff CB. Physiology and pharmacology of Corticotropin-releasing factor. Pharmacol Rev, 1991;43:425-473
5.Imaki T, Nahan JL, Rivier C,Sawchenko PE, Vale W. Differential regulation of CRH mRNA in rat brain regionsby glucocorticoids and stress. J Neurosci, 1991; 11:585-599
6.Rivest S, Laflamme N.Neuronal activity and neuropeptide gene transcriptoin in the brains of immune-chsllenged tats. J Neuroendocrinol, 1995;7:501-525
7.Makino S, Gokd PW, Schulkin J. Corticosterone effects on corticotropin-releasing hormone mRNA in the central nucleus of the amygdala and the paraventricular nuclrus of the hypothalamus. Brain Res, 1994;640:105-112
8.Itoi K, Horiba N, Tozawa F, Sakai Y, et al. Major role of 3',5'-cyclic adenosine monophosphate-dependent protein kinase A pathway in corticotropin-releasing hormone gene expression in the rat hypothalamus in vivo. Endocrinology, 1996; 137:2389-2396
9. Kraus J, Hollt V. Identification of a cAMP-response elenemt on the human proopiomelanocortin gene upstream promoterl DNA Cell Biol,1995;14:103-110
10. Calogero A, Bernadini R, Margioris AN,et al. Effects of serotinergic agonists and antagonists on corticotropin-releasing hormone secretion by explanted rat hypothalami. Peptides, 1989;10:189-200
11. Benveniste H. Brain microdialysis. J Neurochem, 1989;52:1667-1678
12．王正国，孙立英，刘宝松等．生物撞击机的研制及撞击伤发生机制的研究．中华创伤杂志，1999；15(4)：293—297
13. Tin YS. Corticotropin-releasing hormone mediates the response to cold stress in the neonatal rat without compensatory enhancement of the peptide's gene expression. Endocrinology, 1994; 135:2365-2378
14. Schiller HJ, Reilly PM, Bulkley GB. Tissue perfusion in critical illnesses: Antioxidant therapy. Crit Care Med, 1993,21:s92-103
15. Theoharides TC, Spanos C, Pang X, et al. Stress-induced intracranial mast cell degranulation: A Corticotropin-releasing hormone-mediated effect. Endocrinology, 1995;136:5745-5753
16. Ellis L, Gilston V, Soo CC,et al. Activation of the transcription factor NF-kappaBin in the tat air pouch model of inflammation. Ann Rheum Dis, 2000;59(4):303-307
17．童伟光，刘淑琴、钱国桢．烫伤或外伤骨折对下丘脑神经内分泌细胞的影响k Gomori方法、免疫组化和免疫电镜观察．解剖学杂志，1990；13(2)：89-92
18. Brownstein MJ, Russell JT, Gainer H. Synthesis, transport, and release of posterior pituitary hormones. Science, 1980;207(4429): 373-378]
19. Morris JF. Membrams retrieval at neurosecrotory axon ending.Nature, 1976;261:723-742
20. Brownstein MJ, Russell JT, Gainer H. Synthesis, transport, and
release of posterior pituitary hormones. Science, 1980;207(4429): 373-378]
21.Perlmutter LS, Tweedle CD, Hatton GI. Neuronal/glial plasticity in the supeaoptic dendritic zone: dendritic bundling and double synapse formation at parturition. Neuroscience, 1984; 13(3):769-779
22.Matsumoto A, Arnold AP, Micevych PE. Gap junctions between lateral spine motoneurons in the rat. Brain Res, 1989;495:362-366
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