外周丝裂原活化蛋白激酶在蜜蜂毒肽诱致病理性痛中的作用

发布时间：2018-08-07 20:19

【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A_2(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A_2、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。
[Abstract]:Over the past 10 years, this research group has conducted further studies on the basis of Lariviere and Melzack's proposed Bee venom (BV) model. The model is characterized by the ability to simulate three major manifestations of clinicopathological pain, including: 1) persistent self pain, 2) primary thermo pain and mechanical pain, 3) secondary pain sensitivity and "mirror pain". At the same time, the study group also used the body electrophysiological single cell extracellular recording technique to induce the spontaneous discharge response of the wide-dynamic-range (WDR) neurons in the wide dynamic region of the spinal dorsal horn and to enhance the response to the heat and mechanical stimulation of the injection site in the corresponding segment of the dorsal horn of the spinal cord. The results of the form are in accordance with the results of behavioral studies. Then, the research group also carried out a deep study on the components of the bee venom. The bee venom is a complex containing more than twenty components. The main components include: (1) Melittin, the most important substance in the crude venom of bees (about 50%) in the crude venom of the freeze-dried, and (2) Apamin, in the bee venom. Only 2% of the crude venom of freeze-dried; (3) other major damage components, such as phospholipase A_2 (12%), hyaluronidase (less than 3%), mast cell degranulation peptide (2%), histamine (1.5%), and Melittin F, etc. because there have been reports that subcutaneous injection of phospholipase A_2, hyaluronidase, mast cell degranulation peptide, histamine and 5-HT can not cause images. The long duration of bee venom and Faure Marin's long duration spontaneous spontaneous contraction reflex behavior, and not long duration activation of the peripheral primary afferent C fibers and spinal dorsal horn nociceptive neurons continued to increase, so we speculate that these substances are not the main cause of inflammation in the bee venom. We also use behavioral pharmacology, in vivo single cell outside the cell. The biological function and mechanism of honeybee venom peptide, such as single cell whole cell patch clamp recording in vitro and calcium imaging technique, were studied. It was found that Melittin is the main inflammatory chemical component in bee venom, and the main factor of honeybee venom induced persistent self pain, heat and mechanical pain sensitivity and inflammatory reaction. It can sensitize the peripheral primary sensory neurons directly, open the non selective cation channel (transient receptor potential vanilloid 1, TRPV1) on the capsaicin receptor on the open cell, and mediate the continuous spontaneous pain and thermo algesia induced by the bee venom peptide and the functional changes of the spinal dorsal horn nociceptive neurons, and can also activate phosphorus by activating phosphorus. Lipase A2- lipoxygenase metabolic pathway and regulating the opening of TRPV1 under the help of intracellular protein kinase A, C, resulting in persistent self pain and heat pain sensitivity after subcutaneous injection of honeybee venom, and human experiments have shown that the apaptide can cause spontaneous pain and primary mechanical pain sensitivity.
However, the peripheral afferent mechanism of a variety of pain related behaviors caused by honeybee venom peptide is not fully described. MITOGEN-ACTIVED protein kinases (MAPKs), as a class of highly conserved protein kinases in evolution, participates in the regulation of many sides of the cell, which is converted into intracellular by extracellular stimulation. Transcriptional and posttranslational reactions, linking the key regulatory targets within the cell surface of the recipient, play a key role in the cell signaling pathway.
In mammalian cells, three members of the MAPK family have been clearly described, namely, ERK1/2 (extracellular signal-regulated kinase 1 and 2), c-Jun N terminal kinase / stress activated protein kinase (JNK/SAPK, c-Jun N-terminal kinase/stress-actived) and kinase. Does the protein kinase play a role in the noxious stimulus response and the pain sensitization induced by melittin? Is the action of ERK, JNK, and P38 consistent? Can the current study help us further elaborate on the peripheral afferent mechanism of a variety of pain related behaviors induced by MELITIN? And we have developed a peripheral mitogen activated protein kinase in the bee The study of the role of toxic peptide induced pathological pain is mainly by injecting Extracellular signal-regulated kinase (ERK) inhibitor U0126, c-Jun amino terminal kinase (C-Jun N-terminal kinase,), respectively, after the local injection of honeybee venom in the subplantar subcutaneous part of the rat. JNK) the inhibitor SP600125 and the P38 mitogen activated protein kinase inhibitor SB239063 were used to observe their role in the pathological pain induced by bee venom peptide by behavioral pharmacology.
Results: (1) subcutaneous injection of ERK, JNK, and P38 inhibitors could not change the latent period of heat reaction and the threshold of response to mechanical stimulation in rats without any treatment, which suggests that the three main peripheral mitogen activated protein kinase subfamilies play a smaller role in normal pain sensation transmission in normal physiological state. (2) ERK, JNK and P38 inhibitors can obviously inhibit the production of persistent self pain induced by melittin, and it is dose-dependent; and the way of after administration can also significantly inhibit the maintenance of persistent self pain induced by melittin. (3) for the thermal pain sensitivity, the ERK, JNK, and P38 inhibitors in the pre administration group can inhibit the bee Toxic peptide induced primary thermal pain sensitization; and subcutaneous injection of ERK for 2 to 3 hours after subcutaneous injection of melittin in the drug group, JNK, P38 inhibitors also partially reverse thermal pain sensitivity, indicating that these three protein kinases play a role in the process of promeltin induced primary thermo algesia production and maintenance. (4) for mechanical properties. ERK and JNK inhibitors have no inhibitory effect on the primary mechanical pain induced by melittin, both through the pre administration and after the post administration, indicating that the two protein kinase does not participate in the production and maintenance of the primary mechanical pain sensitization induced by the bee venom peptide; and the P38 mitogen activated protein kinase inhibitor is used for the primary mechanical pain sensitivity. There is no inhibitory effect on the maintenance of primary mechanical pain sensitization. (5) local injection of these three kinase inhibitors on the contralateral foot has no effect on the sustained spontaneous pain of the injection of the one side of the foot of the bee venom, the production and maintenance of the primary heat and mechanical pain sensitivity, which eliminates the systematic action of the three inhibitors. Diameter.
Conclusion: the activation of peripheral mitogen activated protein kinase may be involved in the spontaneous pain induced by melittin and the production and maintenance of primary thermo pain sensitization, but it has no effect on the primary mechanical pain induced by melittin, which suggests the separation of the transmission of the peripheral mechanical and thermo algesia.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R363

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