两面针中木脂素化合物结晶-8的镇痛作用及机制的研究
摘要
研究目的:
探讨两面针木脂素化合物结晶-8的镇痛作用;观察侧脑室注射结晶-8对大鼠痛阀的影响,同时了解结晶-8的镇痛作用与阿片受体及其单胺类递质的关系,并观察氟哌啶醇、东莨菪碱等对结晶-8镇痛作用的影响;建立大鼠福尔马林疼痛模型,研究结晶-8的镇痛作用机制。
研究方法:
1.结晶-8的镇痛作用采用小鼠醋酸扭体法、热板法,观察用药后扭体反应数,舔足后潜伏期;采用福尔马林法观察其Ⅰ相和Ⅱ相的疼痛反应;通过小鼠辐射热刺激法观察用药后痛阀变化。
2.结晶-8的镇痛机制的初步研究采用辐射热刺激法,结晶-8侧脑室注射后观察大鼠痛阀变化;同时考察纳络酮、利血平、氟哌啶醇、东莨菪碱等对结晶-8镇痛作用的影响。
3.结晶-8的镇痛机制的研究
3.1福尔马林疼痛模型的建立实验组动物在右后肢趾部皮下注射5%的福尔马林100μl,参考Mestre的分级评分法,进行模型的评估,合格动物进入下一步实验。HE染色和尼氏染色观察模型组动物病理改变,同时透射电镜观察其超微结构。
3.2结晶-8的镇痛作用与中枢NO、PGE2、MDA关系疼痛大鼠脑组织中的NO、PGE2、MDA含量测定采用比色法。
3.3结晶-8镇痛作用与中枢神经递质的关系造模成功后的大鼠,ICV结晶-8后,采用酶联免疫法测定了脑组织匀浆中的NE、5-TH、DA的含量。
3.4结晶-8对中枢组织FOS的表达影响实验免疫组化观察疼痛大鼠中枢FOS的表达情况。
3.5结晶-8对致痛大鼠脑内β-内啡肽的影响海马、下丘脑连续切片,免疫组化染色,对照图谱在显微镜下观察β-内啡肽在海马、下丘脑的阳性细胞数。
实验结果:
1.结晶-8(15、30、60mg/kg)均可明显减少小鼠的扭体反应次数,延长小鼠舔足后的潜伏期,对福尔马林致痛动物的Ⅰ和Ⅱ相疼痛反应均有抑制作用,并延长小鼠甩尾潜伏期。
2.结晶-8侧脑室注射能提高大鼠痛阀;利血平能拮抗结晶-8镇痛的作用;纳络酮、氟哌啶醇、东莨宕碱等未影响结晶-8的镇痛作用。
3.动物注射福尔马林以后,出现明显的完整的两期伤害性反应;HE染色和尼氏染色未见其明显的病理改变;超微电镜显示模型动物海马、下丘脑可见核周,胞浆线粒体肿胀等情况。
4.ICV结晶-8可以降低疼痛大鼠脑组织NO、PGE2、MDA含量。
5.结晶-8在中枢中能够显著升高疼痛大鼠的多巴胺,5-羟色胺的含量,降低去甲肾上腺素的含量。
6.结晶-8抑制疼痛大鼠中枢FOS的表达,与模型组相比具有显著的差异(p<0.01)。
7.结晶-8能提高海马、下丘脑的β-内啡肽阳性细胞的表达数。
实验结论:
1.结晶-8有较显著的镇痛作用。
2.结晶-8镇痛作用可能有中枢机制的参与,与单胺类递质有关;无阿片系统、M胆碱系统的介导参与,与抑制中枢DA系统无关。
3.结晶-8有中枢镇痛机制,其机制可能与抑制脑组织中的PGE2合成,脂质过氧化及NO的释放有关。
4.结晶-8调整体内单胺类神经递质含量是其镇痛的机理之一。
5.结晶-8的镇痛机制可能与中枢FOS的表达有关。
6.炎症致痛大鼠的中枢系统对β-内啡肽存在影响,提示结晶-8的镇痛作用有一定的中枢机制的参与。
5Objective: To study the analgesic effect of crystal-8 and observe the effect of intracrwbroventricular injection of crystal-8 on pain threshold; also we explored the relathionship between analgesic effect of crystal-8 and opiun acceptor, monoamine neural transmitter, haloperidol, scopolamine. Using the formalin painful model rats, we studied the mechanism of analagesic effect of crystal-8.
Methods:
1.analgesic effect of the crystal-8 Adopting acetic acid writhing and hot plate test in mice,the changes of the number of writhing,the latencies of paw licking after adiminstration were recorded. TheⅠandⅡphase pain were observed with the pain modle caused by formalin test;tail flick test was used to determinate the pain thresholds of mice.
2.Initial studay of analgesic mechanism of the crystal-8 The tail-flick test was used to measure the change of pain threshold fowlling intracrwbroventricular (ICV) injection of crystal-8,also the influences of naloxone and reserpine, haloperidol, scopolamine to crystal-8 were evaluated by the tail-flick test.
3.Study of analgesic mechanism of the crystal-8
3.1 Building the formalin pain rat model We would adopt injecting the 5% formalin solution 100μl into the right paw of rats to made the pain model and reference Mestre grading score ways to assessment the model. The qualified experimental rats would enter the next step. We explored the pathological changes of the model rats by HE staining and Nissl staining, and the ultrastructural changes would be observed by the electron microscpy.
3.2Relations between analgesic effect of the crystal-8 and NO, PGE2, MDA The contents of nitric oxide (NO), the malondialdehyde(MDA), and prostaglandin E2 ( PGE2) in the brain tissue were recorded by colorimetry.
3.3Relations between analgesic effect of the crystal-8 and central neurotransmitter. We detected the content of monamine neural transmitter including NE, 5-TH,DA in the brain tissue with the pain model rats induced by formalin by Elisa.
3.4 Effect of the crystal-8 on the expression of Fos in the brain of painful rats. Expriment detected the expression of c-fos gene in central tissue by using the method of immunohistochmistry.
3.5 Effect of the crystal-8 on the expression ofβ-endorphine in the brain of painful rats. Hippocampus and thalamus were cut down and made into serial sections. The positive cells ofβ-endorphine expression were calculated in hippocampus and thalamus increased in high and low dose group,when compared with mode group.
Results:
1.Crystal-8 (15, 30, 60 mg·kg-1 ) could remarkly reduce the numbers of wrinting as wells as prolong the latencies of paw licking. crystal-8 had inhibitory effect onⅠandⅡphase pain in formalin test, also the fail-flick latencies were prolonged.
2. It was found that pain threshold was increased significantly after injecting crystal-8 into the lateral cerebral ventricle of rats. Naloxone, haloperidol, and scopolamine had no effect on analagesic effect of crystal-8, but reserpin could reverese analgsic effect of crystal-8.
3.There were obvious two phase nociceptive response after injecting formalin solution into rats. It showed that the rats had no pathological changes by HE staining and nissl staining,yet there was swelling in mitochondria of prerinuclear and cytoplasm in hippocampus and thalamus.
4.Cystal-8 could obviously lowered contents of NO, MDA, PGE2 in the brain tissue.
5.Cystal-8 could increase DA, 5-TH, yet decreased NE in the brain tissue significantly.
6.Cystal-8 could restrain the c-fos gene expression of central tissue.
7.After that cystal-8 was given to the rats,the expression ofβ-endorphine in hippocampus and thalamus increased in high and low dose group when compared with model group.
Conclusions:
1. The crystal-8 is shown to have excellent analgesic effect.
2. The analgesic effect of crystal-8 may be related to the central mechanism; and the analgesic effect of crystal-8 to the thermal stimulation has effect on monoamine neural transmitter,while the analgesic effect has little effect on opium acceptor, DA acceptor, M acceptor.
3. The site of analagesic action of crystal-8 may be in the central nervous system. It ,s mechanism of analagesic action may be related to reducing the NO, lipid peroxidtion,as well as inhibiting PGE2 production.
4. Crystal-8 adjusts pain through regulating the content of monamine neural transmitter of pain model rats.
5.The analagesic effect of crystal-8 is probably associated with it,s effect on restraining the Fos expression of central tissue.
6.The analgesic effect of crystal-8 has effect on the expression ofβ-endorphine, it may be related to the central mechanism.
探讨两面针木脂素化合物结晶-8的镇痛作用;观察侧脑室注射结晶-8对大鼠痛阀的影响,同时了解结晶-8的镇痛作用与阿片受体及其单胺类递质的关系,并观察氟哌啶醇、东莨菪碱等对结晶-8镇痛作用的影响;建立大鼠福尔马林疼痛模型,研究结晶-8的镇痛作用机制。
研究方法:
1.结晶-8的镇痛作用采用小鼠醋酸扭体法、热板法,观察用药后扭体反应数,舔足后潜伏期;采用福尔马林法观察其Ⅰ相和Ⅱ相的疼痛反应;通过小鼠辐射热刺激法观察用药后痛阀变化。
2.结晶-8的镇痛机制的初步研究采用辐射热刺激法,结晶-8侧脑室注射后观察大鼠痛阀变化;同时考察纳络酮、利血平、氟哌啶醇、东莨菪碱等对结晶-8镇痛作用的影响。
3.结晶-8的镇痛机制的研究
3.1福尔马林疼痛模型的建立实验组动物在右后肢趾部皮下注射5%的福尔马林100μl,参考Mestre的分级评分法,进行模型的评估,合格动物进入下一步实验。HE染色和尼氏染色观察模型组动物病理改变,同时透射电镜观察其超微结构。
3.2结晶-8的镇痛作用与中枢NO、PGE2、MDA关系疼痛大鼠脑组织中的NO、PGE2、MDA含量测定采用比色法。
3.3结晶-8镇痛作用与中枢神经递质的关系造模成功后的大鼠,ICV结晶-8后,采用酶联免疫法测定了脑组织匀浆中的NE、5-TH、DA的含量。
3.4结晶-8对中枢组织FOS的表达影响实验免疫组化观察疼痛大鼠中枢FOS的表达情况。
3.5结晶-8对致痛大鼠脑内β-内啡肽的影响海马、下丘脑连续切片,免疫组化染色,对照图谱在显微镜下观察β-内啡肽在海马、下丘脑的阳性细胞数。
实验结果:
1.结晶-8(15、30、60mg/kg)均可明显减少小鼠的扭体反应次数,延长小鼠舔足后的潜伏期,对福尔马林致痛动物的Ⅰ和Ⅱ相疼痛反应均有抑制作用,并延长小鼠甩尾潜伏期。
2.结晶-8侧脑室注射能提高大鼠痛阀;利血平能拮抗结晶-8镇痛的作用;纳络酮、氟哌啶醇、东莨宕碱等未影响结晶-8的镇痛作用。
3.动物注射福尔马林以后,出现明显的完整的两期伤害性反应;HE染色和尼氏染色未见其明显的病理改变;超微电镜显示模型动物海马、下丘脑可见核周,胞浆线粒体肿胀等情况。
4.ICV结晶-8可以降低疼痛大鼠脑组织NO、PGE2、MDA含量。
5.结晶-8在中枢中能够显著升高疼痛大鼠的多巴胺,5-羟色胺的含量,降低去甲肾上腺素的含量。
6.结晶-8抑制疼痛大鼠中枢FOS的表达,与模型组相比具有显著的差异(p<0.01)。
7.结晶-8能提高海马、下丘脑的β-内啡肽阳性细胞的表达数。
实验结论:
1.结晶-8有较显著的镇痛作用。
2.结晶-8镇痛作用可能有中枢机制的参与,与单胺类递质有关;无阿片系统、M胆碱系统的介导参与,与抑制中枢DA系统无关。
3.结晶-8有中枢镇痛机制,其机制可能与抑制脑组织中的PGE2合成,脂质过氧化及NO的释放有关。
4.结晶-8调整体内单胺类神经递质含量是其镇痛的机理之一。
5.结晶-8的镇痛机制可能与中枢FOS的表达有关。
6.炎症致痛大鼠的中枢系统对β-内啡肽存在影响,提示结晶-8的镇痛作用有一定的中枢机制的参与。
5Objective: To study the analgesic effect of crystal-8 and observe the effect of intracrwbroventricular injection of crystal-8 on pain threshold; also we explored the relathionship between analgesic effect of crystal-8 and opiun acceptor, monoamine neural transmitter, haloperidol, scopolamine. Using the formalin painful model rats, we studied the mechanism of analagesic effect of crystal-8.
Methods:
1.analgesic effect of the crystal-8 Adopting acetic acid writhing and hot plate test in mice,the changes of the number of writhing,the latencies of paw licking after adiminstration were recorded. TheⅠandⅡphase pain were observed with the pain modle caused by formalin test;tail flick test was used to determinate the pain thresholds of mice.
2.Initial studay of analgesic mechanism of the crystal-8 The tail-flick test was used to measure the change of pain threshold fowlling intracrwbroventricular (ICV) injection of crystal-8,also the influences of naloxone and reserpine, haloperidol, scopolamine to crystal-8 were evaluated by the tail-flick test.
3.Study of analgesic mechanism of the crystal-8
3.1 Building the formalin pain rat model We would adopt injecting the 5% formalin solution 100μl into the right paw of rats to made the pain model and reference Mestre grading score ways to assessment the model. The qualified experimental rats would enter the next step. We explored the pathological changes of the model rats by HE staining and Nissl staining, and the ultrastructural changes would be observed by the electron microscpy.
3.2Relations between analgesic effect of the crystal-8 and NO, PGE2, MDA The contents of nitric oxide (NO), the malondialdehyde(MDA), and prostaglandin E2 ( PGE2) in the brain tissue were recorded by colorimetry.
3.3Relations between analgesic effect of the crystal-8 and central neurotransmitter. We detected the content of monamine neural transmitter including NE, 5-TH,DA in the brain tissue with the pain model rats induced by formalin by Elisa.
3.4 Effect of the crystal-8 on the expression of Fos in the brain of painful rats. Expriment detected the expression of c-fos gene in central tissue by using the method of immunohistochmistry.
3.5 Effect of the crystal-8 on the expression ofβ-endorphine in the brain of painful rats. Hippocampus and thalamus were cut down and made into serial sections. The positive cells ofβ-endorphine expression were calculated in hippocampus and thalamus increased in high and low dose group,when compared with mode group.
Results:
1.Crystal-8 (15, 30, 60 mg·kg-1 ) could remarkly reduce the numbers of wrinting as wells as prolong the latencies of paw licking. crystal-8 had inhibitory effect onⅠandⅡphase pain in formalin test, also the fail-flick latencies were prolonged.
2. It was found that pain threshold was increased significantly after injecting crystal-8 into the lateral cerebral ventricle of rats. Naloxone, haloperidol, and scopolamine had no effect on analagesic effect of crystal-8, but reserpin could reverese analgsic effect of crystal-8.
3.There were obvious two phase nociceptive response after injecting formalin solution into rats. It showed that the rats had no pathological changes by HE staining and nissl staining,yet there was swelling in mitochondria of prerinuclear and cytoplasm in hippocampus and thalamus.
4.Cystal-8 could obviously lowered contents of NO, MDA, PGE2 in the brain tissue.
5.Cystal-8 could increase DA, 5-TH, yet decreased NE in the brain tissue significantly.
6.Cystal-8 could restrain the c-fos gene expression of central tissue.
7.After that cystal-8 was given to the rats,the expression ofβ-endorphine in hippocampus and thalamus increased in high and low dose group when compared with model group.
Conclusions:
1. The crystal-8 is shown to have excellent analgesic effect.
2. The analgesic effect of crystal-8 may be related to the central mechanism; and the analgesic effect of crystal-8 to the thermal stimulation has effect on monoamine neural transmitter,while the analgesic effect has little effect on opium acceptor, DA acceptor, M acceptor.
3. The site of analagesic action of crystal-8 may be in the central nervous system. It ,s mechanism of analagesic action may be related to reducing the NO, lipid peroxidtion,as well as inhibiting PGE2 production.
4. Crystal-8 adjusts pain through regulating the content of monamine neural transmitter of pain model rats.
5.The analagesic effect of crystal-8 is probably associated with it,s effect on restraining the Fos expression of central tissue.
6.The analgesic effect of crystal-8 has effect on the expression ofβ-endorphine, it may be related to the central mechanism.
引文
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[1]宋小平,陈志武,张建华,等.杜鹃花总黄酮的镇痛作用的研究[J].山东医药,2007,47(14):14-16
[2]韦国锋,韦启后,黄祖良,等.仙人掌总黄酮的提取及镇痛作用研究[J].右江民族医学院学报,2004,26(6):779-781
[3]陈志武,方明,马传庚.银杏叶总黄酮的镇痛作用[J].安徽医科大学学报,1997,32(1):15-16.
[4]张黎,赵春晖,陈志武,等.银杏叶总黄铜的镇痛作用及机制的探讨[J].安徽医科大学学报,2001,36(4):263-266.
[5]赵铁华,杨鹤松,邓淑华,等.黄芩茎叶总黄铜的镇痛作用的实验[J].中国中医药科技,2001,8(3):116-117.
[6]聂犇,余陈欢,王芳芳,等.石荠苧总黄酮镇痛作用及作用机制研究[J].中国实用医药,2007,2(21):116-117.
[7]罗学娅,张学梅,高卫,等.苦参碱的镇痛作用部位及机制研究[J].中草药,2001,32(1):42-43.
[8]杨宇杰,王春民,袁亚非,等.野罂粟总生物镇痛部位的研究[J].中草药,2005,36(4):5554-557.
[9]李小芳,罗庆洪,任文.延胡索炮制后生物碱含量测定及镇痛作用的对比研究[J].湖南中医药导报,2001,7(5):253-255.
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[12]YANG Q, LU JT .Antiociceptive effect of astragalosides and its mechanism of action .Acta phamacol sin ,2001,22(9):809-812.
[13]徐仙祥,夏伦祝,戴敏,等.威灵仙总皂苷抗炎镇痛作用的研究[J].中药药理与临床,2005,21(4):34-35.
[14]高崇凯,吴雁,王勇,等.白芍总苷粉针的抗炎,镇痛的研究[J].中药新药与临床药理,2002,13(3):163-165.
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[19]王德才,马健,孔志峰,等.白花前胡总香豆素解热镇痛抗炎作用的研究[J].中国中医信息杂志,2004,11(8):668-670.
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[1]宋小平,陈志武,张建华,等.杜鹃花总黄酮的镇痛作用的研究[J].山东医药,2007,47(14):14-16
[2]韦国锋,韦启后,黄祖良,等.仙人掌总黄酮的提取及镇痛作用研究[J].右江民族医学院学报,2004,26(6):779-781
[3]陈志武,方明,马传庚.银杏叶总黄酮的镇痛作用[J].安徽医科大学学报,1997,32(1):15-16.
[4]张黎,赵春晖,陈志武,等.银杏叶总黄铜的镇痛作用及机制的探讨[J].安徽医科大学学报,2001,36(4):263-266.
[5]赵铁华,杨鹤松,邓淑华,等.黄芩茎叶总黄铜的镇痛作用的实验[J].中国中医药科技,2001,8(3):116-117.
[6]聂犇,余陈欢,王芳芳,等.石荠苧总黄酮镇痛作用及作用机制研究[J].中国实用医药,2007,2(21):116-117.
[7]罗学娅,张学梅,高卫,等.苦参碱的镇痛作用部位及机制研究[J].中草药,2001,32(1):42-43.
[8]杨宇杰,王春民,袁亚非,等.野罂粟总生物镇痛部位的研究[J].中草药,2005,36(4):5554-557.
[9]李小芳,罗庆洪,任文.延胡索炮制后生物碱含量测定及镇痛作用的对比研究[J].湖南中医药导报,2001,7(5):253-255.
[10]HayesAG ,Skingle M ,TyersMB ,Effects of single doses of capsaisin on nociceptive tkhresholds in the rotent .Neuropharmology ,1981.20(4):505.
[11]杨卫东,郝银菊,李红兵,等.老头瓜总碱镇痛,抗炎作用的实验研究[J].宁夏医学院院报,2005.27(30):191-193.
[12]YANG Q, LU JT .Antiociceptive effect of astragalosides and its mechanism of action .Acta phamacol sin ,2001,22(9):809-812.
[13]徐仙祥,夏伦祝,戴敏,等.威灵仙总皂苷抗炎镇痛作用的研究[J].中药药理与临床,2005,21(4):34-35.
[14]高崇凯,吴雁,王勇,等.白芍总苷粉针的抗炎,镇痛的研究[J].中药新药与临床药理,2002,13(3):163-165.
[15]李小川,郭胜民,孙海燕,等.怀牛膝总皂苷镇痛作用的研究[J].陕西医学杂志,1999,28(12):735-736.
[16]乐江,彭红琇,孔锐,等.当归多糖镇痛作用的实验研究[J].中国药学杂志,2002,37(10):746-748.
[17]张玉英,龚珊,张慧琴.云芝多糖肽镇痛抗炎作用的实验研究[J].苏州大学学报(医学版),2004,24(5):652-653.
[18]Gongshang ,Yinqizhan,et.Involvement of interleukin-2 in analgesia produced corilus versicolor poly saccharide peptides[J].zhong guo yao li xue bao ,1998,01 .
[19]王德才,马健,孔志峰,等.白花前胡总香豆素解热镇痛抗炎作用的研究[J].中国中医信息杂志,2004,11(8):668-670.
[20]上馆珠,连其深,曾靖,等.花淑毒酚的药效学研究[J].赣南医学院报,1997,17(3):201-203。
[21]王德才,李珂,徐晓燕,等.杭白芷香豆素组份解热镇痛抗炎作用的实验研究[J].2005,12(11):36-38.
[22]相应证,雷汉民,姜孝文,等.老鹤草鞣质类化合物的抗炎,免疫,和镇痛作用[J].西北国防医学杂志,1998,29(5):325-327.
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[24]殷银霞,蔺兴遥.生姜挥发油的镇痛作用及其对胃溃疡大鼠血清中引哚类神经递质的影响[J].卫生职业教育,2007年,25(11):142-143.
[25]吴小松,肖飞,张志东,等.砂仁挥发油中乙酸龙脑酯的镇痛作用及机制研究[J].中药材,2005,28(6):505-506.
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[27]邓毅,张艳萍,王淳,等.油松叶挥发油镇痛抗炎作用的实验研究[J].中国中医药科技.2004,11(6):357-358.
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