【作者】 郝键；

【导师】 陈军；

【作者基本信息】 第四军医大学 ， 神经生物学， 2008， 硕士

【摘要】 在过去的10年中,本研究组在Lariviere and Melzack提出蜜蜂毒（Bee venom,BV）模型的基础上进行了更进一步的研究。该模型的特点是可以模拟临床病理性痛的三大表现,包括:1)持续性自发痛,2)原发性热痛敏和机械痛敏,3)继发性痛敏和“镜像痛敏”。同时本研究组还应用在体电生理单细胞胞外记录技术,在蜜蜂毒足底注射侧脊髓背角相应节段诱导出脊髓背角广动力域（wide-dynamic-range,WDR）神经元自发放电反应增强及对注射部位热和机械刺激反应增强现象,其持续时程和反应形式与行为学的结果一致。随后本研究组还对蜜蜂毒成分进行了深入研究,蜜蜂毒是含有二十余种成分的复合物,其主要成分包括:（1）蜜蜂毒肽（Melittin）,是蜜蜂粗毒中最主要（在冻干的粗毒中占50%左右）的物质成分;（2）蜂毒明肽（Apamin）,在冻干的粗毒中仅占2%;（3）其他主要致伤害成分,如磷脂酶A₂（12%）、透明脂酸酶（小于3%）、肥大细胞脱颗粒肽（2%）、组胺（1.5%）、Melittin F等。因为已有报道称皮下分别注射磷脂酶A₂、透明脂酸酶、肥大细胞脱颗粒肽、组胺和5-HT等化学物质不能引起像蜜蜂毒和福尔马林那样的长时程持续自发缩足反射行为,而且也不能长时程激活外周初级传入C纤维和脊髓背角伤害性神经元持续放电增强,所以推测这些物质不是蜜蜂毒中主要致炎致痛成分。我们还应用行为药理学、在体单细胞胞外记录、离体单细胞全细胞膜片钳记录以及钙成像等技术对蜜蜂毒肽的生物学作用及机制进行深入研究,发现蜜蜂毒肽（Melittin）是蜜蜂毒中主要的致炎致痛化学成分,是蜜蜂毒诱发持续性自发痛、热和机械痛敏以及炎症反应的主要因素。它可以直接敏化外周初级感觉神经元,开放细胞上辣椒素受体的非选择性阳离子通道（transient receptor potential vanilloid 1,TRPV1）,从而介导蜜蜂毒肽诱致的持续性自发痛和热痛敏以及脊髓背角伤害性神经元的功能改变,还可以通过激活磷脂酶A2-脂氧合酶代谢途径并在胞内蛋白激酶A、C的协助下调节TRPV1的开放从而引起皮下注射蜜蜂毒肽后行为学上表现的持续性自发痛和热痛敏等,并且已有人体实验证明蜜蜂毒肽可以引起自发痛和原发性机械痛敏。然而蜜蜂毒肽引起的多种痛相关行为的外周传入机制还未完全清楚地描述。丝裂原活化蛋白激酶（mitogen-actived protein kinases, MAPKs）作为一类在进化中高度保守的蛋白激酶,参与了细胞内部许多方面的调节,它通过将胞外刺激转化为胞内的转录和翻译后反应,从而将细胞表面的受体同胞内关键的调节目标联系起来,在细胞信号传导通路中发挥关键的作用。在哺乳动物细胞中,有三个MAPK家族成员已经得到比较清楚的描述,即ERK1/2（extracellular signal-regulated kinase 1 and 2）,c-Jun N末端激酶/应激激活的蛋白激酶（JNK/SAPK,c-Jun N-terminal kinase/stress-actived protein kinase）和p38激酶。其中ERK、JNK以及P38丝裂原活化蛋白激酶在蜂毒肽引起的伤害性刺激反应以及痛敏中是否发挥了作用?其ERK、JNK、P38的作用是否一致?当前的研究能否帮我们进一步阐述蜂毒肽引起的多种痛相关行为的外周传入机制?于是我们展开了外周丝裂原活化蛋白激酶在蜜蜂毒肽诱致的病理性痛中作用的相关研究。实验方法主要是在大鼠后足底皮下局部注射蜜蜂毒肽引起的炎症反应部位先后分别注射细胞外信号调节激酶（Extracellular signal-regulated kinase,ERK）抑制剂U0126、c-Jun氨基末端激酶（C-Jun N-terminal kinase,JNK）抑制剂SP600125以及P38丝裂原活化蛋白激酶抑制剂SB239063,通过行为药理学的方法观察它们在蜜蜂毒肽诱致的病理性痛中的作用。结果:（1）在未进行任何处理的大鼠后足皮下注射ERK、JNK、P38抑制剂不能改变大鼠对热反应的潜伏期和对机械刺激的反应阈值,这提示在正常生理状态下,这三个主要外周丝裂原活化蛋白激酶亚家族成员在正常痛感觉传递过程中作用较小。（2）通过前给药方式ERK、JNK及P38抑制剂能明显的抑制蜂毒肽诱发的持续性自发痛的产生,并呈剂量依赖性;通过后给药的方式也能明显的抑制蜂毒肽诱发的持续性自发痛的维持。（3）对于热痛敏,在前给药组中,ERK、JNK、P38抑制剂均能抑制蜂毒肽诱发原发性热痛敏的产生;且在后给药组中于蜂毒肽皮下注射2到3小时后分别在同侧皮下注射ERK、JNK、P38抑制剂也能部分逆转热痛敏,说明这三种蛋白激酶在蜂毒肽诱发原发性热痛敏产生和维持的过程中发挥了作用。（4）对于机械性痛敏,无论是通过前给药方式还是通过后给药方式, ERK与JNK抑制剂对蜂毒肽诱发原发性机械痛敏无抑制作用,说明这两种蛋白激酶并未参与蜂毒肽诱发原发性机械痛敏的产生与维持;而P38丝裂原活化蛋白激酶抑制剂对原发性机械痛敏的产生无抑制作用,对原发性机械痛敏的维持有部分抑制作用。（5）在对侧足底局部注射这三种激酶抑制剂,对注射蜂毒肽一侧足底的持续性自发痛、原发性热和机械痛敏的产生和维持也无影响,这样就排除了这三种抑制剂的系统作用途径。结论:外周丝裂原活化蛋白激酶的活化可能参与蜂毒肽诱发的自发痛以及原发性热痛敏的产生和维持,但是对蜂毒肽诱发的原发性机械痛敏无作用,提示我们在外周机械和热痛敏传递的分离。更多还原

【Abstract】 In the past 10 years, we have been working on the bee venom （BV） test, a novel inflammatory pain model produced by the experimentally-produced honeybee’s sting, which was followed by a series of Chen’s group research work describing the model with both a feature of tonic nociception and a multifaceted feature of inflammatory pain hypersensitivity, including primary heat and mechanical hyperalgesia, secondary heat hyperalgesia and mirror-image heat hyperalgesia in behaviorally awake rodents following subcutaneous （s.c.） injection of a solution containing the whole bee-venom. In the in vivo electrophysiological recordings, the unique expressions of tonic or persistent spontaneous nociception （PSN） and inflammatory pain hypersensitivity have been demonstrated to be mediated by plastic changes in functions of spinal dorsal horn wide-dynamic-range （WDR） neurons, which are believed to play pivotal roles in mediation of spinally-organized nociception flexion reflex. Under pathological pain state of peripheral bee venom insult, spinal dorsal horn WDR neurons became hyperexcitable and hypersensitive with initial long-term ongoing spike discharges lasting for 1-2 h followed by a even much longer time of an enhanced responsiveness to both heat and mechanical stimuli applied to the cutaneous receptive field.Although a central change has been primarily believed to play an important role in the maintenance of the BV-induced abnormal pain behaviors, peripheral mechanisms are likely to be more crucial because some results of our previous studies suggest that establishment of the long-term central changes is peripherally dependent and requires a time window for temporal summation of the primary afferent input. These data imply that some specific biotoxins of the whole BV are potential candidates to be involved in production of the BV-induced abnormal pain behaviors and central changes according to the unique characteristics of the BV test. Bee venom is a compound with more than twenty components, and the major constitutents are claimed to be: （1） Melittin （the main chemical component of bee venom, accounting for 50% of the freeze dried preparation）, （2） Apamin （accounting for 2% of the freeze dried preparation）, （3） other nociceptive components, such as PLA₂（Phospholipase A₂, 12%）, hyalase （﹤3%）, MCDP （Mast cell degranulating peptide, 2%）, histamine （1.5%）,Melittin F etc.. It has been reported that subcutaneous injection of PLA2, hyalase, MCD, histamine and 5-HT （5-hydroxytrypatamine（serotonin）） et al. couldn’t induce a long-term expression of persistent spontaneous paw flinches as that of bee venom or formalin insult, nor was it able to elicit a long-term persistent spontaneous discharge of peripheral primary afferent C fiber and dorsal horn nociceptive neurons, indicating they are not the major components accounting for the hyperalgesia and inflammation of bee venom injection. Hower, over 50% of the component of the BV is melittin, a 26 amino acid amphipathic peptide. The toxic peptide has been assumed to produce pain by directly acting on peripheral nerve terminals and/or by releasing potassium ions through cell lysis. So we propose that melittin is one of the major algogenic components of the BV-produced long-term changes in peripheral and central neural plasticity as well as abnormal pain behaviors.By the behavioral surveys, extracellular electrophysiological recording, patch-clamp recording and calcium imaging studies in our lab, melittin, the major toxin of the whole BV, is likely to be responsible for production of the long-term spinal neuronal changes as well as persistent spontaneous nociception, heat and mechanical hypersensitivity and inflammatory responses that are produced by BV. And melittin can act directly on DRG cells and activate the non-selective cation channel- TRPV1 which might contribute to the increased tonic spontaneous neuronal activity and heat hypersensitivity, but not to mechanical hypersensitivity. Phospholipase A2-lipoxygenase pathway might be the major candidate in activation of TRPV1 on DRG cells following melittin administration and this might also be a molecular basis of melittin-induced ongoing pain and heat hypersensitivity. More recently, some researchers demonstrated that intradermal injection of melittin （5μg in 50μl saline） could produce spontaneous pain, mechanical hyperalgesia and neurogenic inflammation in human subjects that are similar to the animal responses to suncutaneous injection of the BV solution.However, the exact peripheral mechanisms underlying melittin-induced multiple pain-related behaviors are still less characterized. In the present study, we sought to investigate the potential roles of peripheral mitogen-activated protein kinases （MAPKs） in melittin-induced nociception and hyperalgesia by pre- and post-administration of three MAPK inhibitors, namely U0126 （1 mg, 10 mg） for extracellular signal-regulated kinase （ERK）, SP600125 （10 mg, 100 mg） for c-Jun N-terminal kinase （JNK） and SB239063 （10 mg, 100 mg） for p38 MAPK, into the local inflamed area of one hind paw of rats.Result:1. Prevention and reversal of PSN by inhibiting local activation of MAPKsPre-treatment with subcutaneous injection of three kinds of MAPK inhibitors prevented the occurrence of persistent flinching reflex in a dose-related manner when compared with DMSO-treated vehicle control group over the 1 h period of observation. And pre-treatment with the ERK and JNK inhibitors had a temporally limited effect on spontaneous nociception within the first 20 min after melittin injection. In contrast, the p38 inhibitor had a smaller inhibitory effect （compared with the other two compounds） but with a longer duration. Although it was observed that the time course of inhibitory actions of the three drugs differed from each other, all of them profoundly reduced the mean total number of paw flinches averaged from 1 h. The two doses of U0126 produced 23.72% and 33.57% , while the value of SB239063 was 29.48% and 29.78%. For the JNK inhibitor, the lower dose of SP600125 caused a 39.60% reduction of the averaged total number of paw flinches, but the higher dose failed to exert any significant influence upon the spontaneous nociception.In the post-treatment paradigm （5 min after s.c. melittin injection）, all agents remarkably suppressed the maintenance of melittin-induced persistent nociceptive behavioral responses. Furthermore, the duration of this inhibition was much longer than that of the pre-treatment paradigm for each drug. As for the mean total number of flinches, the inhibitory rate of U0126, SP600125 and SB239063 at 10μg was 40.05%, 34.55% and 46.04%, respectively. Taken together, the above results indicate that pharmacological blockade of peripheral MAPKs activation could both prevent and reverse the melittin-induced PSN, but differences might also exist between specific inhibitors according to different drug administration protocols.2. Prevention and reversal of primary heat hyperalgesia by inhibiting local activation of MAPKsPre-treatment with two doses of U0126 significantly prevented the development of thermal hyperalgesia identified in primary injury site with inhibitory rates of 48.89% and 44.47%, respectively. In comparison with the DMSO group, pre-treatment with SP600125 and SB239063 also partially blocked the development of primary heat hyperalgesia. The thermal latency in the injection site was increased by 33.81% and 40.21% for the former and 22.93% and 40.26% for the latter. As noted, all drugs reversed the established heat hyperalgesia tested at 2-3h after intraplantar melittin injection. The inhibitory rate at 10μg was 62.65% for U0126 , 21.49% for SP600125 and 57.10% for SB239063, respectively.3. Inhibition of peripheral MAPKs activation exerted no influence upon melittin-induced mechanical hyperalgesia.Neither pre- nor post-treatment with U0126 （1μg and 10μg） and SP600125 （10μg and 100μg） produced any effect on the PWMT values measured in melittin-treated rat hindpaw. With regard to the p38 inhibitor, s.c. pre-administration of SB239063 did not prevent the generation of mechanical hyperalgesia, but post-injection of the drug partially reversed its maintenance.4. Additional control studiesIn order to exclude systemic effects of the three drugs and to confirm their peripheral antinociceptive actions, all three compounds at 10μg, which was shown to be effective in antinociception, were also subcutaneously injected into a region on the contralateral hindpaw symmetrical to the melittin injection site. It was found that contralateral pre-administration of these three inhibitors did not affect the development of melittin-evoked PSN or thermal/mechanical hyperalgesia in the injury side. This result suggested that s.c. administration of MAPK inhibitors exerted only a local effect and that all those above-described findings were not due to systemic actions of the three drugs. In another series of experiments, the thermal and mechanical sensitivity were examined on the contralateral hindpaw following ipsilateral injection of the three MAPK inhibitors （or vehicle） and the melittin solution. And in the vehicle-treated group, s.c. injection of melittin did not cause any significant changes in the PWTL values when compared with the baseline measurements, implicating no generation of contralateral heat hyperalgesia, which was somewhat different from the BV model. Unilateral injection of melittin also failed to produce any alterations in the PWMT values measured in the non-injected paw, indicating no occurrence of contralateral mechanical hyperalgesia, which was consistent with the observations in the BV test. Moreover, local application of the three MAPK inhibitors did not result in any marked changes in the basal PWTL or PWMT values of the contralateral hindpaw at either dose, further confirming the local effects of s.c. injection of three inhibitors. To further exclude any local pharmacological effects of the three compounds on the basal pain sensitivity, we examined their effects in na?ve rats without receiving any nociceptive treatment. In comparison with the baseline control, no siginificant changes were observed for either basal thermal or mechanical pain sensitivity following local administration of 10μg of the three compounds. Conclusion:we conclude that activation of peripheral MAPKs, including ERK, JNK and p38, might contribute to the induction and maintenance of persistent ongoing pain and primary heat hyperalgesia in the melittin test. However, they are not likely to be involved in the processing of melittin-induced primary mechanical hyperalgesia, implicating a mechanistic separation between mechanical and thermal hyperalgesia in the periphery.更多还原