穿琥宁对致热大鼠下丘脑组织中PGE_2和cAMP含量的影响
摘要
前言
穿琥宁注射液(Chuanhuning injection)为纯中药制剂，其主要成分为脱水穿心莲内酯琥珀酸半酯单钾盐(Potassium Dehydroandrographolide Succinate)。该药对细菌、内毒素引起的发热具有很好的解热作用。穿琥宁注射液是否通过影响中枢发热介质而实现其解热作用，PGE_2和cAMP是否参与了穿琥宁注射液的解热机制至今未见报道。本实验就是运用酵母混悬液复制大鼠发热模型，通过测定该药解热时大鼠下丘脑组织中PGE_2和cAMP含量的变化，从中枢发热介质的角度探讨穿琥宁注射液解热的化学信息传递机制，为穿琥宁注射液的临床应用提供实验依据和理论基础。
材料与方法
一、实验动物与体温测量
健康雄性Wistar系大鼠，实验前动物在实验环境中适应3d，实验室温度控制在20±1℃。实验时将大鼠放入特制的鼠笼中，数字温度计探头插入直肠6cm，以实验前测得的两次体温的平均值为大鼠的正常体温(两次温差＞0.3℃者剔除)。
二、实验分组及处理
1、对照组：无处理；2、发热组：测得正常体温后背部皮下注射20％酵母混悬液(10ml／kg)；3、药物1组：复制发热模型(同发热组)后240min腹腔注入穿琥宁注射液(125mg／kg)；4、药物2组：复制发热模型(同发热组)后240min腹腔注入穿琥宁注射液(250 mg／kg)。各组在测得正常体温180min后开始，每15min测体温一次直至360min(该时点为预实验发热组体温高峰期)后，立即断头取下丘脑组织速置液氮中，20min后存放于-70℃冰箱内保存备测。
三、PGE_2和cAMP样品的处理及测定
取一定量下丘脑组织按试剂盒说明书进行操作。
四、观察指标及统计学处理
观察指标：(1)360min平均体温变化曲线。(2)体温高峰期(注射酵母后360min时)的温差(ΔT(℃))。(3)下丘脑组织中PGE_2和cAMP含量。
统计学处理:所有数据均用均数土标准差表示，各组均数间显著性差异用
检验法。用相关性检验分析法作相关分析。
结果
一、穿唬宁注射液对酵母性发热大鼠体温的影响
对照组体温在实验过程中无明显变化;发热组大鼠体温明显升高，
360mhi时体温达高峰，明显高于对照组(p<0.01);药物1组大鼠在注射酵
母后360min即药后120min时△rr与发热组比较，差异非常显著(p<0·
01);药物2组大鼠在给药后120 min时△T与发热组比较，差异亦非常显著
(p<0.01);两用药组比较，温差亦有非常显著的差异(p<0.01)。表明穿
唬宁注射液具有明显抑制酵母性发热的作用，并与剂量相关。
二、穿唬宁注射液解热时下丘脑组织中PGE:和cAMP含量的变化
发热组下丘脑组织中PGE:和cAMP含量明显高于对照组，两组均数差
异非常显著(p<0.01);用药组可有效地抑制了下丘脑组织中PGE:和
cAMP含量的升高，与发热组比较，差异非常显著(p<0.01和p<0.01)，两
用药组比较，差异也十分显著(p<0.05)。将对照组、发热组和药物组的第
36Omin时么T与下丘脑组织中PGEZ和cAMP含量变化作相关分析，结果表
明△T与PGE:和cAMP之间均呈明显正相关(p<0.01和p<0.01)o
结论
一、穿唬宁注射液对酵母性发热大鼠有明显的解热作用，并具有量效关
系。
二、穿唬宁注射液可明显抑制酵母性发热时下丘脑组织中PGEZ含量的
升高，且PGEZ含量的变化与体温变化之间存在明显的正相关。
三、穿唬宁注射液可明显抑制酵母性发热时下丘脑组织中CAMP含量
的升高，且cAMP含量的变化与体温变化之间存在明显的正相关。
四、穿唬宁注射液可能部分通过抑制下丘脑组织中PGEZ和cAMP合
成、释放，和/或促进其分解，降低下丘脑组织中PGE:和cAMP的含量，使体
温调定点下移而呈现解热作用的。
Preface
The chuanhuning injection is a pure herb, preparation. Its main component is Potassium Dehydroandrographolide Succinate. This drug has effective antipyretic effects on fever induced by bacteria or endotoxin. Whether this herb exerts its antipyretic effects through changing the level of neural mediators of fever, and whether PGE2 and cAMP play a role in its .antipyretic effects has not yet reports up to present. In our study we used dry yeast to making fever model, through maturating the content of PGE2 and cAMP in hypothalamus of rats and from the point of view of neural mediators of fever we studied its antipyretic mechanism of chemical information transfer. This will introduce experimental thereunder and theoretic base for clinical use and further usage.
Materials and Methods
1. animals and temperature survey
Healthy male Wistar rats were kept in our experimental environment 3 days before the experiment. The temperature was kept at 20 ?1X.. In our work we put the rats into special cages and the probe of the thermometer was put into the rectum for 6 cm. We took the mean value of two temperature before experiment as the normal temperature (if the differences in temperature were more than 0. 3C then the rat was knock out).
2. experiment group and disposal
(1) control group: no treatment; ( 2 ) model group: After measuring the
normal temperature the suspension of yeast was injected hypodermically(10ml/ kg ). ( 3 ) herb group 1: 240min after making model the celiac injection of chua-nhuning was administered ( 125mg/kg ). (4) herb group 2: the method is the same as last group , the dosage is 250mg/kg. The rats' temperature of every group was noted every 15min from 180min after recording normal temperature until 360min(the point is peak period of the fever induced by yeast) , then rats were decapitated quickly and hypothalamus tissues were taken out and put into liquid nitrogen for fixation, 20min later the samples were transferred to refrigerator (-70C).
3. distillation and assay of the content of PGE2 and cAMP
Take some hypothalamus tissues, then we began to assay the content of cAMP and PGE2 according to specification of the Test Kit.
4. observation and statistical analysis
observation item: (1) the mean temperature curve (360min). (2) differences in temperature at peak time(360min after yeast injection). (3)the content of PGE2 and cAMP. Statistical analysis; all data were mean + SD. The differences between groups were assessed by student' s test, and the correlation was evaluated by correlation analysis.
Results
1. the effects of chuanhuning injection on fevered rats caused by yeast The temperature of control group did not change magnificently during the
experiment. The model group animals' temperature raised significantly which was more higher than control group ( P < 0. 01 ) ; and the peak time is at 360min. Compared with model group the difference in temperature at 120min of both herb group 1 and 2 had significant differentia( P <0.01) ; compare between these two herb groups also had significant differentia( P <0. 01). This data indicates that chuanhuning injection can magnificently inhibit the yeast inducing fever, and the effect is correlated with dosage.
2. changes of PGE2 and cAMP content in hypothalamus of fevered rats treated, with chuanhuning
Compared with control group the PGE2 and cAMP content in hypothalamus of model group is more higher, and difference of mean value has significant val-ue(P <0.01) ; The herb can efficiently inhibit the upraise of PGE2 and cAMP content in hypothalamus tissues, compared with model group (P<0.01,P<0. 01). Difference between these two herb groups also has statistical significance ( P < 0. 05 ). The content of PGE2 and cAMP in hypothalamus is positively correlative with the difference in temperature at 360min in all these groups (P <0. 01,P<0.01).
Conclusion
1. Our test has proved that chuanhuning has significant antipyretic effect on fevered rats induced by dry - yeast and this effect has dosage independence.
穿琥宁注射液(Chuanhuning injection)为纯中药制剂，其主要成分为脱水穿心莲内酯琥珀酸半酯单钾盐(Potassium Dehydroandrographolide Succinate)。该药对细菌、内毒素引起的发热具有很好的解热作用。穿琥宁注射液是否通过影响中枢发热介质而实现其解热作用，PGE_2和cAMP是否参与了穿琥宁注射液的解热机制至今未见报道。本实验就是运用酵母混悬液复制大鼠发热模型，通过测定该药解热时大鼠下丘脑组织中PGE_2和cAMP含量的变化，从中枢发热介质的角度探讨穿琥宁注射液解热的化学信息传递机制，为穿琥宁注射液的临床应用提供实验依据和理论基础。
材料与方法
一、实验动物与体温测量
健康雄性Wistar系大鼠，实验前动物在实验环境中适应3d，实验室温度控制在20±1℃。实验时将大鼠放入特制的鼠笼中，数字温度计探头插入直肠6cm，以实验前测得的两次体温的平均值为大鼠的正常体温(两次温差＞0.3℃者剔除)。
二、实验分组及处理
1、对照组：无处理；2、发热组：测得正常体温后背部皮下注射20％酵母混悬液(10ml／kg)；3、药物1组：复制发热模型(同发热组)后240min腹腔注入穿琥宁注射液(125mg／kg)；4、药物2组：复制发热模型(同发热组)后240min腹腔注入穿琥宁注射液(250 mg／kg)。各组在测得正常体温180min后开始，每15min测体温一次直至360min(该时点为预实验发热组体温高峰期)后，立即断头取下丘脑组织速置液氮中，20min后存放于-70℃冰箱内保存备测。
三、PGE_2和cAMP样品的处理及测定
取一定量下丘脑组织按试剂盒说明书进行操作。
四、观察指标及统计学处理
观察指标：(1)360min平均体温变化曲线。(2)体温高峰期(注射酵母后360min时)的温差(ΔT(℃))。(3)下丘脑组织中PGE_2和cAMP含量。
统计学处理:所有数据均用均数土标准差表示，各组均数间显著性差异用
检验法。用相关性检验分析法作相关分析。
结果
一、穿唬宁注射液对酵母性发热大鼠体温的影响
对照组体温在实验过程中无明显变化;发热组大鼠体温明显升高，
360mhi时体温达高峰，明显高于对照组(p<0.01);药物1组大鼠在注射酵
母后360min即药后120min时△rr与发热组比较，差异非常显著(p<0·
01);药物2组大鼠在给药后120 min时△T与发热组比较，差异亦非常显著
(p<0.01);两用药组比较，温差亦有非常显著的差异(p<0.01)。表明穿
唬宁注射液具有明显抑制酵母性发热的作用，并与剂量相关。
二、穿唬宁注射液解热时下丘脑组织中PGE:和cAMP含量的变化
发热组下丘脑组织中PGE:和cAMP含量明显高于对照组，两组均数差
异非常显著(p<0.01);用药组可有效地抑制了下丘脑组织中PGE:和
cAMP含量的升高，与发热组比较，差异非常显著(p<0.01和p<0.01)，两
用药组比较，差异也十分显著(p<0.05)。将对照组、发热组和药物组的第
36Omin时么T与下丘脑组织中PGEZ和cAMP含量变化作相关分析，结果表
明△T与PGE:和cAMP之间均呈明显正相关(p<0.01和p<0.01)o
结论
一、穿唬宁注射液对酵母性发热大鼠有明显的解热作用，并具有量效关
系。
二、穿唬宁注射液可明显抑制酵母性发热时下丘脑组织中PGEZ含量的
升高，且PGEZ含量的变化与体温变化之间存在明显的正相关。
三、穿唬宁注射液可明显抑制酵母性发热时下丘脑组织中CAMP含量
的升高，且cAMP含量的变化与体温变化之间存在明显的正相关。
四、穿唬宁注射液可能部分通过抑制下丘脑组织中PGEZ和cAMP合
成、释放，和/或促进其分解，降低下丘脑组织中PGE:和cAMP的含量，使体
温调定点下移而呈现解热作用的。
Preface
The chuanhuning injection is a pure herb, preparation. Its main component is Potassium Dehydroandrographolide Succinate. This drug has effective antipyretic effects on fever induced by bacteria or endotoxin. Whether this herb exerts its antipyretic effects through changing the level of neural mediators of fever, and whether PGE2 and cAMP play a role in its .antipyretic effects has not yet reports up to present. In our study we used dry yeast to making fever model, through maturating the content of PGE2 and cAMP in hypothalamus of rats and from the point of view of neural mediators of fever we studied its antipyretic mechanism of chemical information transfer. This will introduce experimental thereunder and theoretic base for clinical use and further usage.
Materials and Methods
1. animals and temperature survey
Healthy male Wistar rats were kept in our experimental environment 3 days before the experiment. The temperature was kept at 20 ?1X.. In our work we put the rats into special cages and the probe of the thermometer was put into the rectum for 6 cm. We took the mean value of two temperature before experiment as the normal temperature (if the differences in temperature were more than 0. 3C then the rat was knock out).
2. experiment group and disposal
(1) control group: no treatment; ( 2 ) model group: After measuring the
normal temperature the suspension of yeast was injected hypodermically(10ml/ kg ). ( 3 ) herb group 1: 240min after making model the celiac injection of chua-nhuning was administered ( 125mg/kg ). (4) herb group 2: the method is the same as last group , the dosage is 250mg/kg. The rats' temperature of every group was noted every 15min from 180min after recording normal temperature until 360min(the point is peak period of the fever induced by yeast) , then rats were decapitated quickly and hypothalamus tissues were taken out and put into liquid nitrogen for fixation, 20min later the samples were transferred to refrigerator (-70C).
3. distillation and assay of the content of PGE2 and cAMP
Take some hypothalamus tissues, then we began to assay the content of cAMP and PGE2 according to specification of the Test Kit.
4. observation and statistical analysis
observation item: (1) the mean temperature curve (360min). (2) differences in temperature at peak time(360min after yeast injection). (3)the content of PGE2 and cAMP. Statistical analysis; all data were mean + SD. The differences between groups were assessed by student' s test, and the correlation was evaluated by correlation analysis.
Results
1. the effects of chuanhuning injection on fevered rats caused by yeast The temperature of control group did not change magnificently during the
experiment. The model group animals' temperature raised significantly which was more higher than control group ( P < 0. 01 ) ; and the peak time is at 360min. Compared with model group the difference in temperature at 120min of both herb group 1 and 2 had significant differentia( P <0.01) ; compare between these two herb groups also had significant differentia( P <0. 01). This data indicates that chuanhuning injection can magnificently inhibit the yeast inducing fever, and the effect is correlated with dosage.
2. changes of PGE2 and cAMP content in hypothalamus of fevered rats treated, with chuanhuning
Compared with control group the PGE2 and cAMP content in hypothalamus of model group is more higher, and difference of mean value has significant val-ue(P <0.01) ; The herb can efficiently inhibit the upraise of PGE2 and cAMP content in hypothalamus tissues, compared with model group (P<0.01,P<0. 01). Difference between these two herb groups also has statistical significance ( P < 0. 05 ). The content of PGE2 and cAMP in hypothalamus is positively correlative with the difference in temperature at 360min in all these groups (P <0. 01,P<0.01).
Conclusion
1. Our test has proved that chuanhuning has significant antipyretic effect on fevered rats induced by dry - yeast and this effect has dosage independence.
引文
1．曹瑛侠，等。中西医结合治疗高热60例。陕西中医2002；23(9)：815
2. Dinaraello CA, et al. New concepts on the pathogenesis of fever. Rev Inf Dis 1998 ; 10 : 168
3. Kluger MJ, et al. Fever:Role of pyrogens and cryogens. Physiol Rev 1991;71:93
4．李楚杰．发热机制研究的新近展．中国病理生理杂志1990；6(5)：375
5. Clark M, et al. Neuromodulative action of cytokines. Yale J Biol Med 1990 ;63 : 133
6. Bligh J. Effects on temperature of monoamines injected into the lateral ventricles of sleep. J Physiol 1996;185:46
7. Myers RD et al. Fever: reciprocal shift in brain sodium to calcium ratio as the setpoint temperature rises. Science 1972 ; 178:765
8. Breckenridge BM, et al. Cyclic adenylate and hypothalamic regulatory function. Proc Soc Exp Biol Med 1969;131:934
9. Tsagakaris S, et al. Interleukin - 1 directly stimulates the release of corticotropin releasing factor from rat hypothalamus. Neuroendocrinology 1989 ;49 :98
10．王华东，等．中枢CRH在大鼠应激性体温升高和LPS性发热机制中的作用．中国病理生理杂志2001；17(2)：124
11. Blatteis CM, et al. Neuromodulation of fever; Apparent involvement of opioids. Brain Res Bull 1991 ;26:219
12. Milton AS,et al. Effects on body temperature of prostaglandins of the A. E and F series on injection into the third ventricle of unanaestheized cat and rabbits. J Physiol Lond 1971 ;218:325
13. Bernheim HA, et al. Prostaglandin E levels in third ventricalar cerebrospinal fluid of rabbits during fever and changes in body temperature. J Physiol 1980 ;301:69
14. Coceani F, et al. Further evidence implicationing prostaglandin E_2 in the genesis of pyrogen fever. Am J Physiol 1988 ;254:463
15．杨永景，等．AVP MSH和PGE在体温调节和发热中的作用及相互关系．解放军医学高等专科学校学报1997；25(4)：47
16. Stitt JT. Prostaglandin E as the neural mediator of the febrile response. Yale J Biol Med 1986 ;59 : 137
17．郭中钰，等．电针退热时家兔的血浆及脑脊液中前列腺素E_2含量的变化．中国病理生理杂志1991；7(1)：25
18．孙连坤，等．家兔内毒素性发热时脑区组织PGE_2含量的变化．中国病理生理杂志1991；7(6)：659
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23. Coceani F, Akarsu E. Prostaglandin E2 in the pathogenesis of fever an update. Ann NY Acad Sci, 1998 ;850:76
24．王琳，等．发热机制的研究进展．中医研究2000；13(2)：57
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27．胡景新，等。内毒素发热时家兔不同脑区组织、脑脊液及血浆中cAMP含量变化的比较．中国病理生理杂志1990；6(1)：9
28．徐方云，等．家兔EP性发热时血浆、脑脊液和不同脑区组织含量的变化．中国病理生理杂志1997；13(1)：52
29．刘基巍，等．内毒素性发热家兔血浆、不同脑区组织cAMP含量的变化及电针的影响．中国病理生理杂志1990；6(6)：487
30. Lisa RL. Cytokine regulation of fever:studies using gene knockout mice. J Appl Physio ,2002 ; 92 : 2648
31. Horai R, Asano M, Sudo K et al. Production of mice deficient in genes for in-
erleukinlL -1α, IL - 1β, IL - 1α/βand IL - 1 receptor antagonist shows that IL - 1βis crucial in turpentine - induced fever development and glucocorticoid secretion. Exp Med 1998 ; 187 : 1463
32．富杭育，等．桂枝汤对体温双向调节作用机理探讨-对下丘脑及血浆中环核苷酸的影响[J]．中药药理与临床1994；10(4)：1
2. Dinaraello CA, et al. New concepts on the pathogenesis of fever. Rev Inf Dis 1998 ; 10 : 168
3. Kluger MJ, et al. Fever:Role of pyrogens and cryogens. Physiol Rev 1991;71:93
4．李楚杰．发热机制研究的新近展．中国病理生理杂志1990；6(5)：375
5. Clark M, et al. Neuromodulative action of cytokines. Yale J Biol Med 1990 ;63 : 133
6. Bligh J. Effects on temperature of monoamines injected into the lateral ventricles of sleep. J Physiol 1996;185:46
7. Myers RD et al. Fever: reciprocal shift in brain sodium to calcium ratio as the setpoint temperature rises. Science 1972 ; 178:765
8. Breckenridge BM, et al. Cyclic adenylate and hypothalamic regulatory function. Proc Soc Exp Biol Med 1969;131:934
9. Tsagakaris S, et al. Interleukin - 1 directly stimulates the release of corticotropin releasing factor from rat hypothalamus. Neuroendocrinology 1989 ;49 :98
10．王华东，等．中枢CRH在大鼠应激性体温升高和LPS性发热机制中的作用．中国病理生理杂志2001；17(2)：124
11. Blatteis CM, et al. Neuromodulation of fever; Apparent involvement of opioids. Brain Res Bull 1991 ;26:219
12. Milton AS,et al. Effects on body temperature of prostaglandins of the A. E and F series on injection into the third ventricle of unanaestheized cat and rabbits. J Physiol Lond 1971 ;218:325
13. Bernheim HA, et al. Prostaglandin E levels in third ventricalar cerebrospinal fluid of rabbits during fever and changes in body temperature. J Physiol 1980 ;301:69
14. Coceani F, et al. Further evidence implicationing prostaglandin E_2 in the genesis of pyrogen fever. Am J Physiol 1988 ;254:463
15．杨永景，等．AVP MSH和PGE在体温调节和发热中的作用及相互关系．解放军医学高等专科学校学报1997；25(4)：47
16. Stitt JT. Prostaglandin E as the neural mediator of the febrile response. Yale J Biol Med 1986 ;59 : 137
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