蛙皮素对大鼠IFN性发热及脑内AVP含量的影响
摘要
前言
蛙皮素(bombesin，BN)是20世纪70年代初从欧洲铃蟾皮肤中提取出来的一种14肽。此后陆续发现许多与BN结构相似、生物学特性、生理作用相似的物质，统称为蛙皮素样肽。哺乳动物脑和胃肠道内存在大量的蛙皮素样肽，它们有很多的生理作用。本文旨在探讨蛙皮素的降体温及抗热作用机制。
脑内注射蛙皮素可使大鼠、小鼠、兔等多种动物体温降低，还可拮抗PGE2、TRH所致大鼠的发热反应。干扰素-α(α-IFN)是一种内生性致热原，侧脑室注射可引起剂量依赖性体温升高。研究表明，α-IFN可能通过作用于阿片受体，继之引起前列腺素释放，而引起发热的。故推测，蛙皮素也能拮抗α-IFN的致热作用。
精氨酸加压素(AVP)是一种内生解热物质，AVP在体温调节和退热中的最敏感和最先作用点位于脑内的腹中隔区(VSA)。脑室注射BN能引起室旁核(PVN)中AVP含量升高。而隔区AVP纤维主要来自于室旁核、视上核和终纹床核。故推测BN可引起VSA中AVP含量升高。关于BN能否引起VSA中AVP增加，及BN降温及解热作用是否与AVP相关，还未见报道。本研究观察侧脑室注射BN对大鼠α-IFN性发热反应及下丘脑、VSA中AVP含量的影响。
材料与方法
1．动物和体温测量
健康WSthe大鼠，72只，雄性，体重180－2209，由中国医科大
学实验动物中心提供。室温（2252）℃，湿度O0f2）％，光照6：
皿J：00，单笼饲养，自由进食、水。数字温度计探头插入大鼠直
肠6cm，待数值稳定后读数。实验前给大鼠测温，使其服习。实验
中每隔30ndn测一次温。以给药前一小时测得的三次温的平均值
为大鼠给药前温度基线。所有实验均在7：见一4：00进行。
z侧脑室插管及脑室给药
大鼠在盐酸氯氨酮和盐酸甲苯嚷嚷联合麻醉下，立体定位后
按ouls J．Peleghae著图谱向一侧侧脑室置人一外径为 Inun的
插管。用牙科水泥固定，封闭插管上端。常规、单笼饲养一周后实
验。实验结束后，检查插管位置，会掉插管位置不正确的数据。经
侧脑室插管给药，要注意速度一致性。
3．AVP含量测定
实验结束后师 人立即断头处死大鼠，迅速取出鼠脑
放人沸生理盐水中，煮沸sndn。称取下丘脑30mg、腹中隔区
10mg用放射免疫法测定AVP含量。－，
实验结果
1．侧脑室注射不同剂量的BNJ－IFN对大鼠直肠温的影响
侧脑室注射a－IFN引起剂量依赖性升温反应k＜o．防人注
药后150分时升温幅度最大；而侧脑室注射BN引起剂盖依赖性
降温反应订利．01＊注药后的分时降温鹏最大。
2．侧脑室注射BN对VSA和下丘脑中AVP含量的影响
侧脑室注射 BN 0．5 gO gi）后 60Illlll迅速断头测 AyP含量，
＊犯中＊w含量明显高于对照组u＜o．洒人下丘脑中AyP含
量无明显变化河＞o．防人
3．侧脑室注射BN对a－IFN性发热反应的影响
·2·
Iry－NS组大鼠直肠温，与NS－NS组比较，显著升高瞻＜0．
05h IFN－BN组在注射 BN后大鼠直肠温与 IFN－NS组比较，显
．
著降低（P＜o．05）；与＊S一＊S组比较滁150Wu（P＞o．05）外，其
它均有显著差异瞩＜o．05人
4．侧脑室注射BN对a－IFN性发热大鼠腹中隔区、下丘脑中
AVP含量的影响
IFN－NS组VSA中AVP含量明显升高，与对照组相比，差异
显著（P＜O．05）；＊S－＊N组＊sA中＊＊P含量也明显升高，与对照
组相比，差颗著（P＜0．05）；而＊N－BN组 VSA中 AVP含量接
近对照组水平tP＞0．05），而明显低于IFN－NS组（P＜0．05）。
下丘脑中 AVP含量在四组中无显著差异（p＞Q．05人
讨 论
本实验中室温下侧脑室注射BN引起剂量依赖性降温反应；
侧脑室注射IFN引起剂量依赖性升温反应。这与以前报道结果一
致。
本实验中观察到，侧脑室注射BN引起降温的同时，VSA中
AVP含量升高。结果表明在N降温作用可能与AVP有关。Pain
等学者曾发现，侧脑室注射 BNN．25刀．5 ug）可使下丘脑室旁核
（PVN）及正中隆起弓状核MdM）中AVP含量增高。而隔区
内的AVP纤维主要来自于室旁核、视上核瞩ON）和终纹床核。故
、推测，注射BN后隔区AVP含量的升高，可能由于PyN等下丘脑
核团AW合成增加所致。Pain等还测得，BN可使PyN及M分批
。中AVP含量增高的同时还使下丘脑腹内侧核（VMH）中AVP含量
跳，而下丘脑前区（AH人下丘脑外侧区（LH）及下丘脑背内侧
核（DMN）中 AyP含量无明显变化。Pain等没有检测整个下丘脑
中AyP含量。本实验中，注射BN后测得整个下丘脑中AyP含量
·3·
无明显变化，可能由于下丘脑各核团中AVP含量增、减幅度相差
不大，而无明显变化。
本实验观察到，侧脑室注?
Introduction
Bombesin (BN ) is a tetradecapeptide which was isolated from an-uran skin in 1970s. Bombesin - like peptides which were similar to BN in many respects ,such as,structure,biological characteristic, physiological role,etc. have been reported to be found in mammalian intestine and brain. They have many physiological role. In this study ,the mechanism underlying thermoregulatory action of bombesin is to be further elucidated.
When given intracerebrally bombesin produces hypothermia in many animals, such as, rats, mice, rabbits, etc. Moreover,bombesin can prevent the hyperthermia produced by TRF and PGE2 in rats kept at room temperature. Interferon - a, one of endogenous pyrogens, given intracerebrally, caused a dose - dependent fever. It has been reported that two processes may mediate the pyrogenic effects of a -IFN, viz, an endogenous opioid - and a PGE - dependent mechanism. It is possible that bombesin blocks the changes induced by a -IFN.
Now, there is evidence that arginine vasopressin( AVP) acts as a neurotransmitter in the ventral septal area(VSA) where is the most susceptible and first site of action. Intracerebroventricular injection of
BN increased AVP level in the PVN, and neurofibrae of AVP in VSA primarily come from paraventricular nucleus, superaoptic nucleus and bed nucleus of stria terminalis. It is possible that central thermoregu-latory action of BN involves the increase of AVP in the VSA. The aim of the present study was to test that hypothesis by investigating the effects of centrally administered BN on IFN - induced febrile response and AVP level in the brain.
Materials and Methods
1. Animals and body temperature response measurement
Adult male Wistar rats (180 -220g, obtained from the Experimental Animal Center, Chinese Medical University. ) were used in this study . Animals were caged individually and had free access to food and water. The ambient temperature of the animal room was (22 + 2)C; light and darkness were alternated; with light on from 6:00 to 18:00. During experiments each rat was placed individually in a plastic cage without restraint. Rectal temperature was monitored by insertion of a thermister probe 6cm in the rectum, the thermistor was fed into digital thermometer ( SN2202 Type, Beijing Sinan Instruments Factory). Temperature records were taken at 30 min intervals until the experiment was over. All experimental trials were carried out between 7:00 and 14;00.
2. Surgical Procedures and administration of test substances Under Ketamin hydrochloride and acepromazine meleate injection
anesthesia ,a guide cannula was inserted into one of the lateral ventricles as described in atlas writen by Louis J. Pellegrine. The animals were allowed to recover for at least 6 days before expermentation. Po-
sitioning of cannula was verified histologically at the end of the experiment . Recombinant human interferon a2b ( obtained from ANHUI ANKE Biotechnology co. Ltd) , BN (purchased from Sigma Chemicals Ltd) were injected with the similar velocity.
3. Determination of AVP content
The rats was decapitated at the end of the experiments, then the brain was removed quickly and boiled in normal saline for 5 min. The VSA and hypothalamus were dissected for AVP assay. The concentration of AVP in the VSA or hypothalamus was determined by radioim-munoassay as described earilier using AVP assay kits ( obtained from the Second Military University,Shanghai, China).
Results
1. The rectal temperature response to intracerebroventricular injection of BN or a - IFN of different doses.
Injection (i. c. v. ) of a - IFN caused a dose - dependent fever in rats( P < 0.05); Injection (i. c. v. ) of BN induced a dose - dependent hypothermia( P < 0.01).
2. Effect of injection (i. c. v. ) of BN on AVP content hi the VSA and hypothlamus.
Introcerebroventricular administration of BN ( 0. 5ug , 5ul ) caused a significant increase in AVP levels of the VSA ( P < 0.05 ) and had no significant changes in AVP levels of the hypothalamus compared with control group ( P > 0.
蛙皮素(bombesin，BN)是20世纪70年代初从欧洲铃蟾皮肤中提取出来的一种14肽。此后陆续发现许多与BN结构相似、生物学特性、生理作用相似的物质，统称为蛙皮素样肽。哺乳动物脑和胃肠道内存在大量的蛙皮素样肽，它们有很多的生理作用。本文旨在探讨蛙皮素的降体温及抗热作用机制。
脑内注射蛙皮素可使大鼠、小鼠、兔等多种动物体温降低，还可拮抗PGE2、TRH所致大鼠的发热反应。干扰素-α(α-IFN)是一种内生性致热原，侧脑室注射可引起剂量依赖性体温升高。研究表明，α-IFN可能通过作用于阿片受体，继之引起前列腺素释放，而引起发热的。故推测，蛙皮素也能拮抗α-IFN的致热作用。
精氨酸加压素(AVP)是一种内生解热物质，AVP在体温调节和退热中的最敏感和最先作用点位于脑内的腹中隔区(VSA)。脑室注射BN能引起室旁核(PVN)中AVP含量升高。而隔区AVP纤维主要来自于室旁核、视上核和终纹床核。故推测BN可引起VSA中AVP含量升高。关于BN能否引起VSA中AVP增加，及BN降温及解热作用是否与AVP相关，还未见报道。本研究观察侧脑室注射BN对大鼠α-IFN性发热反应及下丘脑、VSA中AVP含量的影响。
材料与方法
1．动物和体温测量
健康WSthe大鼠，72只，雄性，体重180－2209，由中国医科大
学实验动物中心提供。室温（2252）℃，湿度O0f2）％，光照6：
皿J：00，单笼饲养，自由进食、水。数字温度计探头插入大鼠直
肠6cm，待数值稳定后读数。实验前给大鼠测温，使其服习。实验
中每隔30ndn测一次温。以给药前一小时测得的三次温的平均值
为大鼠给药前温度基线。所有实验均在7：见一4：00进行。
z侧脑室插管及脑室给药
大鼠在盐酸氯氨酮和盐酸甲苯嚷嚷联合麻醉下，立体定位后
按ouls J．Peleghae著图谱向一侧侧脑室置人一外径为 Inun的
插管。用牙科水泥固定，封闭插管上端。常规、单笼饲养一周后实
验。实验结束后，检查插管位置，会掉插管位置不正确的数据。经
侧脑室插管给药，要注意速度一致性。
3．AVP含量测定
实验结束后师 人立即断头处死大鼠，迅速取出鼠脑
放人沸生理盐水中，煮沸sndn。称取下丘脑30mg、腹中隔区
10mg用放射免疫法测定AVP含量。－，
实验结果
1．侧脑室注射不同剂量的BNJ－IFN对大鼠直肠温的影响
侧脑室注射a－IFN引起剂量依赖性升温反应k＜o．防人注
药后150分时升温幅度最大；而侧脑室注射BN引起剂盖依赖性
降温反应订利．01＊注药后的分时降温鹏最大。
2．侧脑室注射BN对VSA和下丘脑中AVP含量的影响
侧脑室注射 BN 0．5 gO gi）后 60Illlll迅速断头测 AyP含量，
＊犯中＊w含量明显高于对照组u＜o．洒人下丘脑中AyP含
量无明显变化河＞o．防人
3．侧脑室注射BN对a－IFN性发热反应的影响
·2·
Iry－NS组大鼠直肠温，与NS－NS组比较，显著升高瞻＜0．
05h IFN－BN组在注射 BN后大鼠直肠温与 IFN－NS组比较，显
．
著降低（P＜o．05）；与＊S一＊S组比较滁150Wu（P＞o．05）外，其
它均有显著差异瞩＜o．05人
4．侧脑室注射BN对a－IFN性发热大鼠腹中隔区、下丘脑中
AVP含量的影响
IFN－NS组VSA中AVP含量明显升高，与对照组相比，差异
显著（P＜O．05）；＊S－＊N组＊sA中＊＊P含量也明显升高，与对照
组相比，差颗著（P＜0．05）；而＊N－BN组 VSA中 AVP含量接
近对照组水平tP＞0．05），而明显低于IFN－NS组（P＜0．05）。
下丘脑中 AVP含量在四组中无显著差异（p＞Q．05人
讨 论
本实验中室温下侧脑室注射BN引起剂量依赖性降温反应；
侧脑室注射IFN引起剂量依赖性升温反应。这与以前报道结果一
致。
本实验中观察到，侧脑室注射BN引起降温的同时，VSA中
AVP含量升高。结果表明在N降温作用可能与AVP有关。Pain
等学者曾发现，侧脑室注射 BNN．25刀．5 ug）可使下丘脑室旁核
（PVN）及正中隆起弓状核MdM）中AVP含量增高。而隔区
内的AVP纤维主要来自于室旁核、视上核瞩ON）和终纹床核。故
、推测，注射BN后隔区AVP含量的升高，可能由于PyN等下丘脑
核团AW合成增加所致。Pain等还测得，BN可使PyN及M分批
。中AVP含量增高的同时还使下丘脑腹内侧核（VMH）中AVP含量
跳，而下丘脑前区（AH人下丘脑外侧区（LH）及下丘脑背内侧
核（DMN）中 AyP含量无明显变化。Pain等没有检测整个下丘脑
中AyP含量。本实验中，注射BN后测得整个下丘脑中AyP含量
·3·
无明显变化，可能由于下丘脑各核团中AVP含量增、减幅度相差
不大，而无明显变化。
本实验观察到，侧脑室注?
Introduction
Bombesin (BN ) is a tetradecapeptide which was isolated from an-uran skin in 1970s. Bombesin - like peptides which were similar to BN in many respects ,such as,structure,biological characteristic, physiological role,etc. have been reported to be found in mammalian intestine and brain. They have many physiological role. In this study ,the mechanism underlying thermoregulatory action of bombesin is to be further elucidated.
When given intracerebrally bombesin produces hypothermia in many animals, such as, rats, mice, rabbits, etc. Moreover,bombesin can prevent the hyperthermia produced by TRF and PGE2 in rats kept at room temperature. Interferon - a, one of endogenous pyrogens, given intracerebrally, caused a dose - dependent fever. It has been reported that two processes may mediate the pyrogenic effects of a -IFN, viz, an endogenous opioid - and a PGE - dependent mechanism. It is possible that bombesin blocks the changes induced by a -IFN.
Now, there is evidence that arginine vasopressin( AVP) acts as a neurotransmitter in the ventral septal area(VSA) where is the most susceptible and first site of action. Intracerebroventricular injection of
BN increased AVP level in the PVN, and neurofibrae of AVP in VSA primarily come from paraventricular nucleus, superaoptic nucleus and bed nucleus of stria terminalis. It is possible that central thermoregu-latory action of BN involves the increase of AVP in the VSA. The aim of the present study was to test that hypothesis by investigating the effects of centrally administered BN on IFN - induced febrile response and AVP level in the brain.
Materials and Methods
1. Animals and body temperature response measurement
Adult male Wistar rats (180 -220g, obtained from the Experimental Animal Center, Chinese Medical University. ) were used in this study . Animals were caged individually and had free access to food and water. The ambient temperature of the animal room was (22 + 2)C; light and darkness were alternated; with light on from 6:00 to 18:00. During experiments each rat was placed individually in a plastic cage without restraint. Rectal temperature was monitored by insertion of a thermister probe 6cm in the rectum, the thermistor was fed into digital thermometer ( SN2202 Type, Beijing Sinan Instruments Factory). Temperature records were taken at 30 min intervals until the experiment was over. All experimental trials were carried out between 7:00 and 14;00.
2. Surgical Procedures and administration of test substances Under Ketamin hydrochloride and acepromazine meleate injection
anesthesia ,a guide cannula was inserted into one of the lateral ventricles as described in atlas writen by Louis J. Pellegrine. The animals were allowed to recover for at least 6 days before expermentation. Po-
sitioning of cannula was verified histologically at the end of the experiment . Recombinant human interferon a2b ( obtained from ANHUI ANKE Biotechnology co. Ltd) , BN (purchased from Sigma Chemicals Ltd) were injected with the similar velocity.
3. Determination of AVP content
The rats was decapitated at the end of the experiments, then the brain was removed quickly and boiled in normal saline for 5 min. The VSA and hypothalamus were dissected for AVP assay. The concentration of AVP in the VSA or hypothalamus was determined by radioim-munoassay as described earilier using AVP assay kits ( obtained from the Second Military University,Shanghai, China).
Results
1. The rectal temperature response to intracerebroventricular injection of BN or a - IFN of different doses.
Injection (i. c. v. ) of a - IFN caused a dose - dependent fever in rats( P < 0.05); Injection (i. c. v. ) of BN induced a dose - dependent hypothermia( P < 0.01).
2. Effect of injection (i. c. v. ) of BN on AVP content hi the VSA and hypothlamus.
Introcerebroventricular administration of BN ( 0. 5ug , 5ul ) caused a significant increase in AVP levels of the VSA ( P < 0.05 ) and had no significant changes in AVP levels of the hypothalamus compared with control group ( P > 0.
引文
1．王仁鹏．蛙皮素样肽及其作用．生理科学进展，1982；13(1)：54-58．
2. Marvin Brown, Jean Rivier, Wylie Vale. Action of bomhesin, thyrotropin releasing factor, prostaglandin E2 and naloxone on thermoregulation in the rat. Life Sciences, 1977;20:1681-1688.
3. Shibata M, Blatteis CM. Differential effects of cytokines on thermosensitive neurons in guinea pig preoptic area slices. Am J Physiol, 1992 ;261: R1096-R1103.
4. Clark M Blatteis, Li Xin, Ning Quan. Neuromodulation of fever:apparent involvement of opioids. Brain Research Bulletin, 1991;26:219-223.
5. T Nakashima, T Hori, K Kuriyama, T Kiyohara. Naloxone blocks the interferon- a induced changes in hypothnlnmlc neuronal activity. Neuroscience Letter, 1987;82:332-336.
6．李楚杰．发热时体温的正调节和负调节．中国病理生理杂志1994；10(5)：553-557．
7. Para Kent, Hymie Anisman, Zul Merali. Central bombesin actives the Hypothalamic-pituitary-adrenalaxis. Neuroendocrinology, 2001;73:203-214.
8．杨永录，杨文辉．精氨酸加压素在体温调节与抗热中的作用．解放军医学情报，1995；9(6)：302-304．
9. WDRuwe, WL Veale, K E Cooper. Peptide neurohormones: their role in thermoregulation and fever. Can. J. Biochem. Cell biol, 1983 ;61:579-593.
10．杨永录，孟涛，王绪明．家兔发热时脑脊液和血浆中精氨酸加
压素含量的变化及禁水的抗热作用．中国病理生理杂志，1991；7：134-138．
11. Marvin Brown, Jean Vale. Bombesin: potent effects on thermoregulation in the rat. Science: 1977; 196:998-1000.
12. S.J. Won, M. T. Lin. Pyrogenicity of interferon and its inducer in rabbits. Am J Physiol, 1988 ;254: R499-R507.
13．徐刚，张冰，叶武军．清热中药对大鼠下丘脑组织AVP含量的影响．中国现代应用药学杂志，1999；16(1)：21-23．
2. Marvin Brown, Jean Rivier, Wylie Vale. Action of bomhesin, thyrotropin releasing factor, prostaglandin E2 and naloxone on thermoregulation in the rat. Life Sciences, 1977;20:1681-1688.
3. Shibata M, Blatteis CM. Differential effects of cytokines on thermosensitive neurons in guinea pig preoptic area slices. Am J Physiol, 1992 ;261: R1096-R1103.
4. Clark M Blatteis, Li Xin, Ning Quan. Neuromodulation of fever:apparent involvement of opioids. Brain Research Bulletin, 1991;26:219-223.
5. T Nakashima, T Hori, K Kuriyama, T Kiyohara. Naloxone blocks the interferon- a induced changes in hypothnlnmlc neuronal activity. Neuroscience Letter, 1987;82:332-336.
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