筛选镇痛抗炎中药循证实验医学方法的建立及应用探讨
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摘要
中医学对痛证的认识及其理、法、方、药的形成，有着悠久的历史。但长期以来，一方面因中医证和方剂等多因素复杂性，临床观察和临床实验仅限于低水平重复，缺乏设计良好的大的多中心、随机分组、双盲、安慰剂对照实验的系统评价的客观证据，而主要靠“经验医学”方法，因此造成中药镇痛疗效一直难以得到国际上公认。另一方面，在镇痛中药的基础研究中，
由于知识老化和对国际上关于临床病理性痛发生机制研究的跟踪不及时，在
筛选镇痛新药时，还不能认识到“生理性痛”与“病理性痛”发生机制的差
异性，以及病理性痛发生和持续慢性化过程中机制的多样性，所以常困动物
模型的选择不当，导致药效无法正确评价，也使药物的作用部位和机制研究
难以深入下去，这严重阻碍了镇痛中药的研究和开发。要克服和解决上述问
题的关键是在临床和基础双轨研究中建立一整套“循证医学”的实验方法，
对中药疗效进行客现的而非主观的临床前和临床期评价。为了探讨此问题，
本实验拟探讨建立一个“循证医学”的动物实验方法，即为了筛选和评价有
效镇痛抗炎中药而建立了一套动物实验模型方法。
外周组织或神经损伤可引起持续性自发痛、原发性热和机械性痛敏以及
损伤部位红、肿、热等炎症反应。而建立与临床病理性炎性痛相似的动物模
型对全面评价药物的镇痛抗炎作用意义重大。成年清醒大鼠一侧后肢足底皮
下注射蜜蜂毒（BV，0．2％，50…）可立即诱发注射侧长达1小时以上的单相
自发痛行为（如注射侧持续自发缩足反射行为），并伴有持续3－4天的原发性
热和机械性痛敏现象以及注射肢体红肿等炎症反应。皮下注射福尔马林（F，
2．5％，50 pl）也可诱导出一双相的持续性自发痛过程，但无长时程的痛超敏
现象．因为经过大量实验研究，这两个动物痛模型能够反映临床病理性痛特
点，而且发生机制较清楚，所以较适合筛选镇痛中药。
根据药性，中药可大致分为平性、温热和寒凉三大类，在筛选有效镇痛
杭炎中药时应该考虑这三类药性的天然植物药在作用效果和机制上是否一致
的问题．困此，本实验采用蜜蜂毒模型和福尔马林模型，选用已在其他模型
上证实有镇痛杭炎作用的平性类、温热类和寒凉类的三类中药的镇痛抗炎效
果做比较评价，以期为今后大规模筛选和评价镇痛抗炎中药的有效成分，开
展有效成分血液动力学及其神经药理学机制研究提供模型系统。
此外，为了进一步了解这三类中药在预防和治疗两方面的作用效果是否
一致，本研究在给药时间上设计分为两个给药组，即致痛前（致痛剂注射前
10－30Inin）和或致痛后（在持续性自发痛完全建立之后smin，或在痛敏完
全建立并达到稳定状态的 2－4小时期间）给药。
一、4夯匆搅衅亿翔织拨涝诊早种燃牲g发疗封懈疲嘉
1．乎性类中来对夕人寄亿学纽织握涝诊早种样尊牲8发瘀时件刀痰果
为了研究平性类中药的抗外周化学组织损伤诱导的持续性自发痛的
作用效果，本部分实验选择两种市售中药镇痛制剂一鸡矢藤和野木瓜注
射液，评价了这两种注射液静脉给药在BV模型和F模型上的抗伤害作
用。在 BV模型上，致痛前分别注射 0．32 mwg、1石 mwg和 9刀 mwg
三个剂量的市售浓度为 500％门 支）鸡矢藤注射液或市售浓度为
250o门．og／Zml／支）野木瓜注射液，对 BV诱致的 lh自发缩足脓次数
具有剂量依赖性抑制作用；致痛 5 min后分别给予鸡矢藤或野木瓜注射液
对BV诱发的自发痛反应也产生显著的抑制作用。在F模型上，致痛前
或致痛后分别给予单一有效剂量门石 mwg）的鸡矢藤或野木瓜注射液
对F诱发的自发痛反应也产生显著的抑制作用。实验结果提示，对外周
化学组织损伤诱致的持续性自发痛反应，鸡矢藤或野木瓜注射液有显著
的抑制作用，而且没有模型间差异。
2．水喇和挟于捞乃种中穷勿斡对今尸lithe织握涝劳早勿燃性力发瘀
Mk＄ ＃lbf1Hg
内服药物是中医临床治疗的最主要的方法。汤剂（即水煎剂）和粉
－3－
第 四 军 医 大 学 硕 士 学 位 论 文
剂是最为常用的制备简便、便于服用和保存的口服中药剂型。由于制备
工艺不同，水煎剂和冻干粉的对持续性自发痛的抗伤害是否有差异？困
此，本部分实验选择了被认为有镇痛抗炎作用的白芜，比较观察了水煎
剂和冻干粉两种剂型对化学组织损伤诱致的持续性自发痛反应的抗伤害
作用。致痛前分别给予 0．lgiml、0．4 g／ml和 2．0 g／il三个剂量的白茧水
煎剂和冻干粉门 ml／kg）对 BV诱致的 lh自发缩足反射次数具有剂量
相关性抑制作用；0．4g／inl的白芜水煎剂和冻干粉对F诱致的双相性自发
缩足反射也有显著的抑制作用，表明白茧对外周化学组织损伤诱导的持
续性自发痛的抑制作用，没有制剂间和模型间差异。
3．温娇姜4符对外尸化翔织损涝诊早的燃姓g发虏伽发果的地致研
S
实验1、2的结果显示中药（天然植
The Traditional Chinese Medical (TCM) herbs have been long used as analgesic and anti-inflammatory drugs in clinic. However, due to severe lack of Good-Clinical-Practice (GCP) trials that should be in accord with randomly-divided, double-blind and placebo-controlled standard (RDP standard), the efficacy of the TCM analgesics and anti-inflammatory drugs has not been world-widely accepted. On the other hand, the knowledge about pain and its underlying mechanisms has been greatly changed due to the findings that
-10-
functional and/or structural plasticity or change can be induced in the central somatosensory system following peripheral tissue and nerve injury (pathological pain state). Therefore pain can be generally divided into physiological and pathological types. The former is transient in duration and plays an alarm function to tell where, what and how pain is produced (good and wanted pain). Under physiological state, there is no spontaneous pain and hyperalgesia (allodynia), nor is there inflammation and obvious tissue or nerve injury. The animal models which can be used to test normal nocifensive threshold are tail flick test and hot plate test. While the latter is persistent or chronic in duration and often occurs spontaneously and shows abnormally sensitive (hypersensitivity and hyperalgesia or allodynia) to natural stimuli such as thermal and mechanical stimuli and is not wanted by human life (bad and unwanted pain). Under pathological state, tissue or nerve injury is often the origin of pain and some types of pain are insensitive to conventional pain-killers (analgesics). The animal models which can mimic pathological pain are of particular importance and useful in study of pathological pain mechanisms and in screening novel effective endogenous or exogenous analgesics.
The present study is trying to establish a new method for screening TCM herbs based on experimental evidence-based knowledge by using two persistent pathological pain models: the bee venom test and the formalin test.
Subcutaneous (s.c.) injection of bee venom (BV) solution into one hindpaw of rats has been demonstrated to be able to produce a prolonged time course of persistent spontaneous pain-related behaviors such as flinching reflex in a monophasic manner for 1 h, and accompanied by a subsequent period of profound, persistent primary thermal and mechanical hypersensitivity (hyperalgesia) in the
-11-
injected hind paw for 3-4 days. Meanwhile, s.c. BV injection also produces a striking edema and redness of the plantar surface for nearly the same period as the development of hyperalgesia. Injection s.c. of formalin solution can produce two phases of persistent pain-related behavior but without obvious hyperalgesia and allodynia. Both of the two animal models are suitable to study the effects of TCM herbs on tissue-injury induced pain and/or hyperalgesia and inflammation.
According to the natures of TCM, Chinese herbs have been divided into three classes: Mild class, Warm-Heat class and Cool - Cold class. In the present study, effects of the three classes of TCM herbs were tested to see whether they could produce the same effects on persistent spontaneous pain, heat and mechanical hyperalgesia (or allodynia) and inflammatory responses (such as edema and plasma extravasation).
Moreover, to see whether the three classes of TCM drugs are effective in both prevention and relief of the peripheral chemical tissue-injury induced pain, hyperalgesia and inflammation, the drugs were administered: (1) 10-30 min prior to chemical pain-producing agents; (2) 5-10 min after good establishment of persistent pain; (3) 2-4 h after good establishment of the thermal and mechanical hyperalgesia as well as inflammation. The major results are as follows:
Anti-nociceptive effects of TCM herbs
1. Effects of TCM herbs on the chemical tissue injury-induced persistent spontaneous pain-related responses
-12-
(1) Effects o/the Mild class of TCM herbs
To study anti-nociceptive effects of the Mild class of TCM herbs
由于知识老化和对国际上关于临床病理性痛发生机制研究的跟踪不及时，在
筛选镇痛新药时，还不能认识到“生理性痛”与“病理性痛”发生机制的差
异性，以及病理性痛发生和持续慢性化过程中机制的多样性，所以常困动物
模型的选择不当，导致药效无法正确评价，也使药物的作用部位和机制研究
难以深入下去，这严重阻碍了镇痛中药的研究和开发。要克服和解决上述问
题的关键是在临床和基础双轨研究中建立一整套“循证医学”的实验方法，
对中药疗效进行客现的而非主观的临床前和临床期评价。为了探讨此问题，
本实验拟探讨建立一个“循证医学”的动物实验方法，即为了筛选和评价有
效镇痛抗炎中药而建立了一套动物实验模型方法。
外周组织或神经损伤可引起持续性自发痛、原发性热和机械性痛敏以及
损伤部位红、肿、热等炎症反应。而建立与临床病理性炎性痛相似的动物模
型对全面评价药物的镇痛抗炎作用意义重大。成年清醒大鼠一侧后肢足底皮
下注射蜜蜂毒（BV，0．2％，50…）可立即诱发注射侧长达1小时以上的单相
自发痛行为（如注射侧持续自发缩足反射行为），并伴有持续3－4天的原发性
热和机械性痛敏现象以及注射肢体红肿等炎症反应。皮下注射福尔马林（F，
2．5％，50 pl）也可诱导出一双相的持续性自发痛过程，但无长时程的痛超敏
现象．因为经过大量实验研究，这两个动物痛模型能够反映临床病理性痛特
点，而且发生机制较清楚，所以较适合筛选镇痛中药。
根据药性，中药可大致分为平性、温热和寒凉三大类，在筛选有效镇痛
杭炎中药时应该考虑这三类药性的天然植物药在作用效果和机制上是否一致
的问题．困此，本实验采用蜜蜂毒模型和福尔马林模型，选用已在其他模型
上证实有镇痛杭炎作用的平性类、温热类和寒凉类的三类中药的镇痛抗炎效
果做比较评价，以期为今后大规模筛选和评价镇痛抗炎中药的有效成分，开
展有效成分血液动力学及其神经药理学机制研究提供模型系统。
此外，为了进一步了解这三类中药在预防和治疗两方面的作用效果是否
一致，本研究在给药时间上设计分为两个给药组，即致痛前（致痛剂注射前
10－30Inin）和或致痛后（在持续性自发痛完全建立之后smin，或在痛敏完
全建立并达到稳定状态的 2－4小时期间）给药。
一、4夯匆搅衅亿翔织拨涝诊早种燃牲g发疗封懈疲嘉
1．乎性类中来对夕人寄亿学纽织握涝诊早种样尊牲8发瘀时件刀痰果
为了研究平性类中药的抗外周化学组织损伤诱导的持续性自发痛的
作用效果，本部分实验选择两种市售中药镇痛制剂一鸡矢藤和野木瓜注
射液，评价了这两种注射液静脉给药在BV模型和F模型上的抗伤害作
用。在 BV模型上，致痛前分别注射 0．32 mwg、1石 mwg和 9刀 mwg
三个剂量的市售浓度为 500％门 支）鸡矢藤注射液或市售浓度为
250o门．og／Zml／支）野木瓜注射液，对 BV诱致的 lh自发缩足脓次数
具有剂量依赖性抑制作用；致痛 5 min后分别给予鸡矢藤或野木瓜注射液
对BV诱发的自发痛反应也产生显著的抑制作用。在F模型上，致痛前
或致痛后分别给予单一有效剂量门石 mwg）的鸡矢藤或野木瓜注射液
对F诱发的自发痛反应也产生显著的抑制作用。实验结果提示，对外周
化学组织损伤诱致的持续性自发痛反应，鸡矢藤或野木瓜注射液有显著
的抑制作用，而且没有模型间差异。
2．水喇和挟于捞乃种中穷勿斡对今尸lithe织握涝劳早勿燃性力发瘀
Mk＄ ＃lbf1Hg
内服药物是中医临床治疗的最主要的方法。汤剂（即水煎剂）和粉
－3－
第 四 军 医 大 学 硕 士 学 位 论 文
剂是最为常用的制备简便、便于服用和保存的口服中药剂型。由于制备
工艺不同，水煎剂和冻干粉的对持续性自发痛的抗伤害是否有差异？困
此，本部分实验选择了被认为有镇痛抗炎作用的白芜，比较观察了水煎
剂和冻干粉两种剂型对化学组织损伤诱致的持续性自发痛反应的抗伤害
作用。致痛前分别给予 0．lgiml、0．4 g／ml和 2．0 g／il三个剂量的白茧水
煎剂和冻干粉门 ml／kg）对 BV诱致的 lh自发缩足反射次数具有剂量
相关性抑制作用；0．4g／inl的白芜水煎剂和冻干粉对F诱致的双相性自发
缩足反射也有显著的抑制作用，表明白茧对外周化学组织损伤诱导的持
续性自发痛的抑制作用，没有制剂间和模型间差异。
3．温娇姜4符对外尸化翔织损涝诊早的燃姓g发虏伽发果的地致研
S
实验1、2的结果显示中药（天然植
The Traditional Chinese Medical (TCM) herbs have been long used as analgesic and anti-inflammatory drugs in clinic. However, due to severe lack of Good-Clinical-Practice (GCP) trials that should be in accord with randomly-divided, double-blind and placebo-controlled standard (RDP standard), the efficacy of the TCM analgesics and anti-inflammatory drugs has not been world-widely accepted. On the other hand, the knowledge about pain and its underlying mechanisms has been greatly changed due to the findings that
-10-
functional and/or structural plasticity or change can be induced in the central somatosensory system following peripheral tissue and nerve injury (pathological pain state). Therefore pain can be generally divided into physiological and pathological types. The former is transient in duration and plays an alarm function to tell where, what and how pain is produced (good and wanted pain). Under physiological state, there is no spontaneous pain and hyperalgesia (allodynia), nor is there inflammation and obvious tissue or nerve injury. The animal models which can be used to test normal nocifensive threshold are tail flick test and hot plate test. While the latter is persistent or chronic in duration and often occurs spontaneously and shows abnormally sensitive (hypersensitivity and hyperalgesia or allodynia) to natural stimuli such as thermal and mechanical stimuli and is not wanted by human life (bad and unwanted pain). Under pathological state, tissue or nerve injury is often the origin of pain and some types of pain are insensitive to conventional pain-killers (analgesics). The animal models which can mimic pathological pain are of particular importance and useful in study of pathological pain mechanisms and in screening novel effective endogenous or exogenous analgesics.
The present study is trying to establish a new method for screening TCM herbs based on experimental evidence-based knowledge by using two persistent pathological pain models: the bee venom test and the formalin test.
Subcutaneous (s.c.) injection of bee venom (BV) solution into one hindpaw of rats has been demonstrated to be able to produce a prolonged time course of persistent spontaneous pain-related behaviors such as flinching reflex in a monophasic manner for 1 h, and accompanied by a subsequent period of profound, persistent primary thermal and mechanical hypersensitivity (hyperalgesia) in the
-11-
injected hind paw for 3-4 days. Meanwhile, s.c. BV injection also produces a striking edema and redness of the plantar surface for nearly the same period as the development of hyperalgesia. Injection s.c. of formalin solution can produce two phases of persistent pain-related behavior but without obvious hyperalgesia and allodynia. Both of the two animal models are suitable to study the effects of TCM herbs on tissue-injury induced pain and/or hyperalgesia and inflammation.
According to the natures of TCM, Chinese herbs have been divided into three classes: Mild class, Warm-Heat class and Cool - Cold class. In the present study, effects of the three classes of TCM herbs were tested to see whether they could produce the same effects on persistent spontaneous pain, heat and mechanical hyperalgesia (or allodynia) and inflammatory responses (such as edema and plasma extravasation).
Moreover, to see whether the three classes of TCM drugs are effective in both prevention and relief of the peripheral chemical tissue-injury induced pain, hyperalgesia and inflammation, the drugs were administered: (1) 10-30 min prior to chemical pain-producing agents; (2) 5-10 min after good establishment of persistent pain; (3) 2-4 h after good establishment of the thermal and mechanical hyperalgesia as well as inflammation. The major results are as follows:
Anti-nociceptive effects of TCM herbs
1. Effects of TCM herbs on the chemical tissue injury-induced persistent spontaneous pain-related responses
-12-
(1) Effects o/the Mild class of TCM herbs
To study anti-nociceptive effects of the Mild class of TCM herbs
引文
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2. Ali-BH. Some effects of Cassia italica on the central nervous system in mice. J-Pharm-Pharmacol, 1997; 49(5): 500-504.
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9. Chen HS, Chen J, Chen J, Guo WG, Zheng MH. Establishment of bee venom-induced contralateral heat hyperalgesia in the rat is dependent upon central temporal summation of afferent input from the site of injury. Neurosci Lett, 2001; 298:57-60.
10. Chen HS, Chen J. Secondary heat, but not mechanical hyperalgesia induced by subcutaneous injection of bee venom in the conscious rat: effect of systemic MK-801, a non-competitive NMDA receptor antagonist. Eur. J Pain, 2000; 4:389-401.
11. Chen HS, Chen J, Sun YY. Contralateral heat hyperalgesia induced by unilaterally intra-plantar bee venom injection is produced by central changes: a
behavioral study in the conscious rat. Neurosci. Lett, 2000; 284: 45-48.
12. Chen J, Koyama N. Differential activation of spinal dorsal horn units by subcutaneous formalin injection in the cat: An electrophysiological study. Exp Brain Res., 1998; 118: 14-18.
13. Chen J, Chen HS. Pivotal role of capsaicin-sensitive primary afferents in development of both heat and mechanical hyperalgesia induced by intraplantar bee venom injection. Pain, 2001; 91: 367-376.
14. Chen J. Diversity of pathological pain: new evidence from the bee venom model. Chin J of Neuroanat, 2001; 17(Suppl): 28-34.
15. Chen J, Koyama N. Effects of subcutaneous formalin on responses of identified primary afferent units in the cat: An electrophysiological study. Chin J Neuroanat, 1996; 12:381-393.
16. Chen J, Koyama N, Kaneko T, Mizuno N. Distribution of μ-opioid receptor-like immunoreactivity in the brain of the japanese monkey (macaca fuscata):Ⅱ. Rostrocaudal root gangila. Chin J Neuroanat, 1998a; 14: 321-327.
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