摘要
研究背景与目的
神经病理性疼痛(neuropathic pain)是外周或中枢神经系统的结构受到损伤或功能发生紊乱而引起的疼痛。可表现为自发性疼痛(spontaneous pain)、痛觉过敏(hyperalgesia)和触诱发痛(allodynia)等多种临床症状。这些疾病的共同特点是临床常规的镇痛治疗效果不佳。由此引发了人们对神经病理性疼痛产生机制及药物镇痛机制的广泛研究。
脊髓神经元敏感化(spinal cord central sensitization)是病理性疼痛的产生、发展和维持的重要的细胞学基础之一。越来越多的证据表明多肽在神经病理性疼痛的调控中发挥重要作用。近来,一种较新的内源性阿片肽类受体-递质系统—孤啡肽和其受体在痛信号的传递和调制过程中的作用已引起了人们的高度重视。在脊髓中,孤啡肽富集于脊髓背角。脊髓背角尤其胶质区是伤害性信息传入和调制的第一站,在此部位抑制痛觉信息是最经济而有效的。因此,在脊髓水平研究孤啡肽与痛觉的感受和调控有着重要的理论和实践意义。
敏感化的产生有赖于神经元细胞膜上受体增多或离子通道功能的增强,而上述改变又依赖于细胞内信号转导系统特别是激酶系统的激活,激活后的激酶可通过磷酸化神经元膜受体或离子通道,改变神经元的兴奋性。蛋白激酶C(proteinkinase C,PKC)是神经系统中最重要的蛋白激酶之一。已有大量的证据表明PKC的活化参与伤害性刺激引起的脊髓神经元敏感化的形成、发展和维持。NMDA受体NR1亚单位是NMDA受体复合物的主要功能亚单位,丝氨酸896位点(serine896)是NR1亚单位发生磷酸化的主要位点之一。激活的PKC可磷酸化NR1亚单位serine 896位点,从而增强NMDA受体功能。NR1亚单位异常激活是脊髓敏感化乃至痛觉过敏产生的重要机制。由于NMDA受体及PKC的活化都受孤啡肽信号传导的调控。因此推测,脊髓PKC依赖性NMDA受体NR1亚单位磷酸化可能参与孤啡肽脊髓水平的痛觉调节作用。本研究的目的在于探讨脊髓NMDA受体上PKC磷酸化位点NR1亚单位serine 896的磷酸化水平与孤啡肽痛觉调节作用的关系。
论文一鞘内注射孤啡肽对大鼠脊髓NMDA受体NR1亚单位磷酸化的影响
实验材料和方法
1、雄性SD大鼠16只,随机分为生理盐水(normal saline,NS)对照组和孤啡肽(orphanin FQ,OFQ)组(n=8)。以动物疼痛相关反应的时间为指标,在OFQ鞘内注射后20分钟内每间隔5分钟对动物自发性疼痛表现作综合评分。
2、以甩尾潜伏期作为痛阈指标,用热水甩尾法观察OFQ鞘内注射后20分钟甩尾潜伏期的变化。
3、观察鞘内预先注射NMDA受体拮抗剂MK-801,PKC抑制剂chelerythrine或OFQ拮抗剂痛稳素(nocistatin,NST)对OFQ诱发的自发性疼痛表现及热痛敏形成的影响。
4、利用western blot方法观察OFQ鞘内注射后5,30,60,120分钟后大鼠脊髓NMDA受体PKC磷酸化位点NR1 serine-896的磷酸化水平及磷酸化PKCα(pPKCα)表达的变化。
实验结果
1、鞘内注射OFQ 50 pg后10～15分钟诱发了特征性自发性疼痛反应,表现为:过度舔爪、搔抓、嘶叫以及间隙性后肢的跛行。与NS组比较,OFQ组大鼠自发性疼痛表现的总时间明显增多(40.9±3.4 vs.5.2±0.9),差异有显著性(P＜0.01)。疼痛反应在OFQ注射后20分钟消失。
2、和基础值比,鞘内注射NS对大鼠的甩尾潜伏期无明显影响。而鞘内注射OFQ后10～15分钟,甩尾潜伏期较基础值和NS组缩短(P＜0.05),注射后15分钟OFQ的作用达顶峰。
3、鞘内单独注射MK-801(0.5μg),chelerythrine(20μg)和NST(10μg)对动物行为活动及甩尾潜伏期无明显影响,但明显减少OFQ鞘内注射引发的疼痛相关行为反应的时间并取消OFQ缩短大鼠甩尾潜伏期的效应。
4、OFQ注射后30分钟磷酸化NMDA受体NR1亚单位serine-896(pNR1serine-896)与磷酸化PKCα(pPKCα)水平开始上升,60分钟达高峰,维持120分钟。与NS对照组比较,OFQ注射后30～120分钟间pNR1 serine-896及pPKCα表达明显增加,差异有显著性(P＜0.05)。
论文二鞘内注射痛稳素对NMDA诱发的疼痛及大鼠脊髓一氧化氮合酶的影响
实验材料和方法
1、参照文献报道,鞘内注射NMDA建立疼痛模型。SD大鼠,随机分为二组(n=8):NS+NMDA组和痛稳素(nocistatin,NST)+NMDA组。在NST(10μg)鞘内注射后20分钟,鞘内注射NMDA 1μg,在NMDA注射后20分钟内对动物自发性疼痛表现作综合评分;并用热水甩尾法观察NMDA鞘内注射后60分钟内动物甩尾潜伏期的变化。旨在观察鞘内预先注射OFQ受体拮抗剂NST对NMDA诱发的自发性疼痛和热痛觉异常的影响。
2、NMDA受体被激活后,引发一系列下游反应,包括激活一氧化氮合酶(NOS)。本文运用依赖还原型辅酶Ⅱ的黄递酶(NADPH-d)组织化学法,通过观察鞘内注射NST对大鼠NMDA鞘内注射诱发痛过敏过程中脊髓背角NADPH-d阳性神经元数量(NADPH-d反映NOS的活性)的影响,进一步佐证脊髓水平孤啡肽诱发的自发性疼痛及热痛敏现象与NMDA受体的关系。
实验结果
1、鞘内注射NMDA(1μg)诱发了自发性疼痛反应,表现为:过度舔爪、嘶叫以及间隙性后肢的跛行以及抓挠。NMDA注射10～60分钟内大鼠的甩尾潜伏期较基础值和NS组缩短,差异显著(P＜0.05)。提示,鞘内注射NMDA后动物出现热痛敏。
2、与NS+NMDA组比,NST+NMDA组大鼠的自发性疼痛反应的总时间减少。与之相对应,NST+NMDA组大鼠和甩尾潜伏期较NS+NMDA组延长,差异显著(P＜0.05)。提示,NST对NMDA鞘内注射引起的自发性疼痛及热痛觉过敏有显著的抑制作用。
3、鞘内注射NMDA后1小时大鼠脊髓NADPH-d阳性神经元、纤维和终末表达明显增加。与NS+NMDA组比,NST+NMDA组大鼠脊髓NADPH-d阳性神经元明显减少,差异显著(P＜0.05)。提示,NST可抑制NMDA受体被激活后其下游产物NOS的活化。
论文三鞘内注射痛稳素对甲醛致痛大鼠脊髓NMDA受体NR1亚单位磷酸化的影响
实验材料和方法
1、大鼠于右后足掌部皮下注射4%甲醛100μl建立甲醛炎性痛疼痛模型。NST和NS组分别在甲醛前30分钟鞘内注射。以1h内动物舔咬后爪的时间为指标来评价疼痛反应。
2、在甲醛注射后1小时将所有动物处死取脊髓腰膨大处,采用western blot观察脊髓磷酸化PKCα及磷酸化NMDA受体NR1亚单位serine-896表达的变化。
3、采用Fos免疫组织化学、依赖还原型辅酶Ⅱ的黄递酶(NADPH-d)组织化学及Fos/NADPH-d双标技术,观察甲醛诱发疼痛后大鼠脊髓NADPH-d阳性神经元、Fos样免疫反应蛋白(Fos-LI)和Fos-LI/NADPH-d双标阳性神经元数量的变化。
实验结果
1、大鼠右后肢足底皮下注射甲醛后立即出现缩腿舔爪反应,并呈明显的两个阶段,第一阶段为注射后立即开始,持续约5min,经过10min的间歇期,开始第二阶段,持续约40min。非注射足活动自如,无缩腿舔爪反应。NST组疼痛反应持续时间较NS组短,差异显著(P＜0.05)。与这种行为学的变化相对应,NST可抑制甲醛增加大鼠脊髓pPKCα及pNR1 serine 896的表达的作用,与NS组比较,差异显著(P＜0.05,P＜0.01)。
2、甲醛刺激后,大鼠脊髓Fos LI、NADPH d阳性神经元、纤维和终末表达明显增加,且出现Fos LI/NADPH d双标神经元表达。甲醛刺激前给予NST,脊髓背角各层Fos LI神经元和Fos LI/NADPH d双标神经元的数量与NS对照组比均减少(P＜0.05或P＜0.01)。
小结
本研究发现:①大鼠鞘内注射OFQ可诱发自发性疼痛反应和热痛觉过敏;OFQ的这种作用可分别被鞘内预先注射NMDA受体拮抗剂MK-801,PKC抑制剂chelerythrine,OFQ受体拮抗剂NST明显抑制。与行为学研究相一致,鞘内注射OFQ明显增加大鼠脊髓PKCα和NR1 serine-896磷酸化水平;②大鼠鞘内注射OFQ受体拮抗剂NST抑制NMDA诱发的疼痛以及NMDA受体下游产物NOS的活化;③在甲醛致痛模型上,鞘内注射NST抑制甲醛致痛大鼠的疼痛反应及甲醛所致脊髓PKCα和NR1-serine 896的磷酸化。在此基础上,我们得出以下结论。
结论
1、脊髓水平微量注射孤啡肽(50 pg)可诱发自发性疼痛反应及热痛敏现象。
2、PKCα以及NMDA受体PKC磷酸化位点NR1亚单位serine-896的磷酸化参与孤啡肽在脊髓水平的痛觉调节作用。
3、痛稳素有效对抗甲醛诱发的痛觉过敏,该作用可能与痛稳素抑制甲醛致痛大鼠脊髓PKCα、NMDA受体NR1亚单位serine-896的磷酸化以及NOS的活性有关。
Objective
Neuropathic pain arising from peripheral nerve injury is a clinical disorder characterized by a combination of spontaneous pain,hyperalgesia and allodynia,and remains a significant clinical problem as it is often poorly relieved by conventional analgesics.Although central sensitization resulting from neuronal plasticity in the spinal cord has been considered to play a crucial role,the mechanisms of neuropathic pain are not fully understood.
Orphanin FQ was discovered as the endogenous ligand for the opioid receptor-like-1(ORL-1) receptor.ORL-1 receptor and its mRNA are densely distributed in the spinal dorsal horn.In addition,nociceptin transcripts and the precursor protein for nociceptin are highly expressed in the dorsal horn of the spinal cord and medulla,particularly in the superficial superficial dorsal horn of the spinal cord.In accordance with these localizations,OFQ is shown to be involved in nociception at the spinal level,but the results obtained by intrathecal administration of N/OFQ are contradictory,depending on the testing paradigms,animal species,and doses.OFQ at low doses(picogram range) induced allodynia and thermal hyperalgesia in conscious mice and facilitated the flexor reflex.By contrast,N/OFQ at high doses, produced analgesia,antihyperalgesic and antiallodynic effects.Fast excitatory and inhibitory neurotransmission in the spinal cord,as well as in other regions of the brain, is mediated by the amino acids L-glutamate and glycine and.Similar to behavioral studies,electrophysiological studies demonstrated that N/OFQ inhibited excitatory glutamatergic neurotransmission,without affecting glycine or GABA receptormediated synaptic responses,by decreasing the synaptic release of glutamate from synaptic terminals.However,the mechanisms of low dose of OFQ pain facilitation are not understood.The cellular mechanisms responsible for the spinal actions of N/OFQ largely remain to be clarified.
Considerable evidence has demonstrated that activation of the N-methyl-D-aspartate(NMDA) subtype of glutamate receptors and subsequent nitric oxide(NO) production are key events in pain perception and central hyperexcitability in the spinal cord.In fact,a previous study demonstrated that the analgesic effect of an ORL receptor antagonist on neuropathic pain was mediated by inhibition of neuronal NO synthase(nNOS),the enzyme required for NO production.Study also showed that the NMDA receptor antagonist D-AP5 blocked the nociceptin-induced allodynia. Collectively,these results support the current interpretation that OFQ ligands may directly or indirectly modulate NMDA receptor complex functions.
The function and localization of NMDA receptors is also modulated by post-translational modifications including phosphorylation.Phosphorylation of the NR1 subunit(pNR1) at protein kinase C(PKC)-dependent sites(serine 896) has been demonstrated to play a key role in enhancement of NMDA receptor activity related to pain transmission in the spinal cord.
The present study was designed to characterize nociceptive response induced by OFQ and to investigate effects of pretreatment with intrathecal MK-801,a NMDA receptor antagonist chelerythrine(Chel),an inhibitor of PKC,and nocistatin,a functional antagonist of OFQ on OFQ-induced spontaneous pain and hyperalgesia were investigated.In addition,we examined whether thisOFQ mediated PKC dependent phosphorylation of NR1at serine 896 sites plays an important role in sensory function using formaldehyde -induced pain model.
Part One Effect of intrathecal administration of orphanin FQ on protein kinase C dependent phosphorylation of the spinal cord NMDA receptor subunit NR1 in rats
Materials and Methods
All animal procedures were conducted after approval of protocol by the Chinese Medical University Animal Care Committee.Male SD rats were kept under standard conditions with unrestricted access to food and water.
1.Studies on Spontaneous Pain.Immediately following the intrathecal injection of orphanin FQ(OFQ),each rat was placed into the transparent cage,and the time spent for induced spontaneous behavioral pain responses was measured at 5-min intervals for 20 min.
2.Studies on Hyperalgesia.Warm water tail-flick test was used to assess the nociceptive response.The thermal nociceptive stimulus was 50℃water,with the latency to tail-flick or withdrawal taken as the endpoint.The response time of the rats to the heat was measured at 15 min,the point of the maximal hyperalgesic effect obtained with 50 pg of OFQ.
3.The effects of pretreatment with intrathecal MK-801,a NMDA receptor antagonist chelerythrine,an inhibitor of PKC,and nocistatin,a functional antagonist of orphanin FQ on OFQ-induced spontaneous pain and hyperalgesia were investigated.
4.The effects of intrathecal administration of OFQ on protein kinase C alpha(PKCα) dependent phosphorylation of the NMDA receptor subunit NR1(phosphorylated at serine 896) as a marker of NMDA receptor sensitization was analyzed by western blot methods.
Results
1.Orphanin FQ induced nociceptive behavior.The intrathecal administration of OFQ(50 pg) resulted in a characteristic behavioral response consisting of vigorous biting and licking with occasional crutching,which peaked at 10-15 min and had disappeared by 20 min post injection.
2.There was no significant difference in the tail flick latency period between rat that had not received an intrathecal injection and those treated with intrathecal saline(at 15 min).Intrathecal administration of OFQ(50 pg) shortened the response latency at 15 min after intrathecal injection,demonstrating that the experimental rats developed increased sensitivity to the thermal stimuli by intrathecal OFQ.
3.Intrathecal treatment with MK-801(0.5μg),chelerythrine(20μg) or nocistatin (10μg) alone did not produce any noticeable pain behaviour for 20 min after injection, but significantly inhibited spontaneous pain and hyperalgesia induced by intrathecal injection of OFQ.
4.Expression of phosphorylation of NR1(at serine896) and PKCαin the spinal cord was found to increase as early as 30 min after intrathecal injection of either OFQ ),and reached the peak effect 60 min after injection.Collectively,these findings demonstrate that PKC dependent phosphorylation of the NMDA receptor subunit NR1 (phosphorylated at Ser896) is involved in the facilitation of nociception associated with OFQ receptor activation.
Part Two Effects of intrathecal administration of orphanin FQ receptor antagonist nocistatin on intrathecal NMDA-induced pain and spinal NOS activity
Materials and Methods
The present study was aimed at determining whether an intrathecal injected OFQ receptor antagonist,NST,can modulate NMDA-induced behavioral responses and neuronal activity.
1.NMDA at a dose of(μg) was administered intrathecally 10 min after treatment with the NST or saline.Immediately following the NMDA injection,cumulative time spent scratching,biting and lifting the paw was measured.Warm water tail flick test was used to assess thermal hyperalgesia.Rats received intrathecal administration of NST or saline followed by intrathecal NMDA 10 min later.The effects of pretreatment with NST on NMDA-induced pain were also observed.
2.As the subsequent activation of NMDA receptors promotes an increase in intracellular calcium resulting in the production of nitric oxide(NO) in the spinal cord. Nicotinamide adenine dinucleotide phosphatediaphorase(NADPH-d) histochemistry method was used to investigat possible role for nocistatin in the effects of NMDA-induced NOS activitation.
Results
1.Intrathecal administration of NMDA(1μg) induced nociceptive behaviors, including scratching,biting and lifting and significantly decreased tail-flick latency responses to thermal stimulation.Pretreatment with intrathecal NST significanty decreased NMDA-induced spontaneous pain and NMDA-induced decrease in tail tick latency.
2.Intrathecal injection of NST significantly inhibited NMDA-evoked increase in NOS expression,compared with intrathecal injection of NS.
Part Three Modulatory role of intrathecal administration of nocistatin on protein kinase C dependent phosphorylation of the spinal cord NMDA receptor subunit NR1 induced by formaldehyde
Materials and Methods
1.The formaldehyde test was carried out.100μl of 4%formaldehyde in PBS was injected subcutaneously into the dorsal surface of the right hind paw.Immediately after the injection,each rat was returned to the observation chamber.The amount of time that animals spent licking the injected paw was monitored.
2.Immunocytochemistry and histochemistry were performed to determine the effects of pretreatment with NST on formaldehyde -induced changes of Fos-like immunoreactive(Fos-LI) and nicotinamide adenine dinucleotide phosphatediaphorase (NADPH-d) positive neurons,respectively.
3,Western blot was used to determine the effects of the intrathecal injection of NST on formaldehyde-induced phosphorylation of NMDA NR1 subunit at serine 896 site(pNR1) and phosphorylated PKCα.
Results
1.Intrathecal pretreatment with the selective OFQ receptor antagonist,NST significantly reduced formaldehyde-induced pain behaviors.
2.Formaldehyde-induced pain behaviors were associated with the expression of Fos-like immunoreactivity(Fos LI) throughout the spinal dorsal horn with highest effect seen in laminaeⅠ-Ⅱ.Formaldehyde also induced an increase in nitric oxide synthase(NOS) activity in superficial layers of the dorsal horn as revealed by NADPH diaphorase histochemistry.Pretreatment with the NST significantly inhibited formaldehyde-evoked Fos-LI,NADPH-d-positive and Fos-LI/NADPH-d double-labeled neurons in the spinal dorsal horn.
Intrathecal injection of NST also reduced formaldehyde-evoked pNR1 expression at the protein kinase C-dependent site,serine-896 in spinal cord.
Summary
Intrathecal injection of OFQ elicited spontaneous pain and hyperalgesia.On the other hand,the nociceptive response was inhibited by pretreatment with intrathecal MK-801,a NMDA receptor antagonist chelerythrine,an inhibitor of PKC,or nocistatin, a functional antagonist of orphanin FQ.Western blot assays of pNR1 in the spinal cord indicated that intrathecal injection of the OFQ significantly enhanced pNR1 at the protein kinase C-dependent site,serine-896 expression in the spinal cord.In another set of experiments,NST reduced NMDA-induced pain behaviour and the nitric-oxide synthase(NOS) immunoreactivity.Finally,pretreatment with the NST significantly inhibited formaldehyde evoked pain and formaldehyde-evoked pNR1 expression at the protein kinase C-dependent site,serine-896 in spinal cord.
Conclusions
1.Activation of the ORL-1 receptor by orphanin FQ produces spontaneous pain and thermal hyperalgesia in conscious rats.
2.PKCαand PKC dependent phosphorylation of the NMDA receptor subunit NR1(phosphorylated at serine896) is involved in the facilitation of orphanin FQ associated with OFQ receptor activation.
3.Inhibition of of spinal orphanin FQ inhibites formaldehyde-induced pain via PKC dependent phosphorylation of the NMDA receptor NR 1 subunit and subsequent activation of NOS.
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