内吗啡肽2参与镇痛效应的形态学和机能学研究
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摘要
疼痛是长期困扰人类的顽疾，严重影响患者的生活质量。在疼痛治疗中，最常用的镇痛药包括吗啡，可待因，度冷丁，美沙酮，曲马多等，其来源于植物或人工合成，主要作用于体内的μ型阿片受体（mu opioidreceptor; MOR）产生镇痛作用。但是这些外源性镇痛药都有副作用和成瘾性，长期使用无异于饮鸩止渴，而寻求一种疗效好，副作用小的镇痛新药迫在眉睫。
Zadina研究组经过20年的努力终于找到了两种MOR的内源性配体，分别被称为内吗啡肽1(Tyr-Pro-Trp-Phe-NH2, endomorphin-1, EM1)和内吗啡肽2（Tyr-Pro-Phe-Phe-NH2, endomorphin-2, EM2）。EM对MOR的选择性和亲和力是δ，κ阿片受体的1000倍以上，而且具有明确的、比吗啡更强的镇痛作用。而作为一种内源性物质，EM的副作用非常小。因此，在临床应用中就完全有可能使用较小剂量的EM产生有效的镇痛作用，而避免常见的副作用，成瘾性和耐受性。
然而EM的镇痛机理仍然不很清楚，只有搞清了EM镇痛作用的神经机制，才能开发出镇痛作用强和成瘾性及不良反应弱或无的止痛药物，有益于临床工作中对疼痛的治疗，提高患者的生活质量。经典的疼痛传递通路为：背根神经节初级传入纤维在脊髓背角释放致痛物质作用于脊髓背角二级投射神经元，二级投射神经元将痛信息传递到高位脑中枢。当然，结状结和孤束核也参与了内脏痛信息的调控。脊髓背角或孤束核作为痛觉传递的闸门，在痛觉调控中起着至为关键的作用。脊髓背角主要含有EM2，其主要来源于含EM2的背根节神经元发出的初级传入纤维；专职内脏信息传递的结状结中是否含有EM2至今无人报道。EM2在初级传入以及脊髓背角或孤束核的镇痛效应并不明确，其作用机理尚处于空白，亟需深入开展研究。本论文综合利用形态学、分子生物学、行为学、药理学以及电生理学等实验方法，在初级传入以及脊髓背角或孤束核水平深入探索EM2镇痛的具体分子机理。本研究具体分为三部分。
一、内吗啡肽2在结状结参与内脏痛调控的机制
以往的研究表明，内吗啡肽2(EM2)与μ型阿片受体(MOR)及其它一些神经递质共存于背根节神经元和初级传入纤维。然而，专职内脏信息传递的结状结中是否含有EM2至今尚无报道。在本实验中：
1.免疫荧光组织化学方法显示有43.5%的结状结神经元呈EM2阳性，这些阳性神经元以中小型细胞为主。
2.免疫荧光双标染色显示在这些EM2免疫阳性的神经元里，有15.2%,27.8%,74.4%以及25.2%的神经元同时表达有P物质(SP)，降钙素基因相关肽(CGRP),一氧化氮合酶(NOS)以及血管活性肠肽(VIP)。另外，EM2免疫阳性的神经元里几乎有90.8%的神经元包含有MOR，并且在迷走神经干上面我们发现了EM2/MOR双标的结状结外周突神经纤维。
3.利用生物素化葡聚糖胺注入结状结顺行追踪外加免疫荧光三标的方法，我们在孤束核(NTS)里发现了大量的EM2/MOR双标的结状结中枢突神经纤维。
4.进一步的免疫荧光三标显示一些EM2/MOR双标的结状结神经元和轴突同时包含有SP或者CGRP。
5.在超微结构水平，包埋后免疫电镜双标显示在NTS的突触前终末里，EM2免疫阳性金颗粒和SP免疫阳性金颗粒共存于相同的突触囊泡里面，并且MOR免疫阳性金颗粒同时存在于突触前膜和后膜之上。
6.此外，在将福尔马林灌入胃内造成急性内脏伤害性刺激的大鼠模型中，我们发现结状结里Fos阳性的神经元大部分是EM2阳性的，并且与对照组相比EM2阳性神经元的数量有明显的增加。
以上这些结果表明EM2与MOR及其它神经递质共存于大鼠结状结的神经元和轴突内，EM2有可能在结状结神经元的轴突终末释放出以后，通过作用于突触前的MOR去调控其它神经递质的释放或者作用于突触后的MOR去调控其它神经递质的突触后效应，进而调节内脏痛信息的传递和内脏反射的形成。
二、内吗啡肽2在背根节参与躯体痛调控的机制
最近的一些形态学实验证实SP与EM2和MOR共存于背根节神经元的胞体和初级传入纤维，这强烈预示着EM2极有可能通过作用于脊髓背角突触前的MOR进而抑制SP的释放，最终调控伤害性信息的传递。但是，至今并未有实验报道EM2能抑制SP的释放，在本实验中：
1.将CFA注入大鼠足底，建立炎性痛模型。
2.我们以镇痛药的有效镇痛剂量除以呼吸抑制剂量或循环抑制剂量的比值作为评判药物安全性的指标。EM2的该比值远远低于吗啡等阿片类镇痛药的该比值。另外，我们还发现连续使用EM2，其镇痛作用风采依旧，而吗啡等则产生了明显的耐受。
3.来自于DRG的中枢突在脊髓背角突触前释放致痛物质：SP， SP作用于突触后相应受体产生突触后效应，是痛觉传递的核心环节。在形态学实验中发现，EM2与SP和MOR共存于DRG神经元的胞体、中枢突和脊髓背角的终末内。
4.为了更直观地显示脊髓背角阳性终末的微观结构。我们用免疫电镜包埋后染色显示，EM2, SP分别或同时存在于突触前囊泡，而MOR同时存在于突触前膜和突触后膜。
基于上述形态学研究结果，我们提出假设：当痛觉信息从外周传递到脊髓背角时，EM2与SP共同释放，EM2结合于突触前膜的MOR，抑制SP的释放，实现镇痛效应。
5.为了证实以上假设，首先，在行为学实验中，我们发现SP的拮抗剂与EM2之间具有协同镇痛效应。
6.其次，在脊髓背角神经元膜片钳全细胞记录中，预先阻断电压门控钠通道和突触后受体，EM2仍然可以作用于突触前膜的MOR抑制谷氨酸的释放。
7.最后，由于膜片钳无法精确定量SP释放量的变化，我们利用新购置的微透析装置证明：EM2确实能作用于突触前膜的MOR，从而有效地抑制突触前SP的释放。
上述研究结果证实了我们的假设，即EM2会结合于突触前膜的MOR有效抑制致痛物质的释放，最终起到镇痛作用。
三、内吗啡肽2与μ型阿片受体在脊髓背角参与糖尿病性痛调控的机制
随着生活水平的提高，糖尿病的发病率一再上升，目前我国有糖尿病患者五千万，每年新增两百万。糖尿病性痛是糖尿病最严重的并发症之一，表现为下肢远侧端的痛觉过敏和异常疼痛，严重地影响着患者的生活质量。以往对糖尿病性痛的研究都集中在外周神经末梢变性和炎性脱髓鞘的基础之上，而本项目另辟蹊径，从糖尿病性痛状态下脊髓背角内吗啡肽2（EM2）与μ型阿片受体（MOR）减少的角度出发，综合利用多种实验方法对糖尿病性痛进行诊断和治疗方面的深入研究，以期能够在脊髓水平对糖尿病性痛进行有效的干预和治疗，为临床实践工作提供有力的理论依据。
我们综合利用各种实验技术，得出以下实验结果：
1.糖尿病组小鼠血糖迅速升高并且维持在高水平状态，出现了明显的机械性痛觉过敏。
2.在糖尿病性痛形成时，一些经典的兴奋性或抑制性的神经递质和调质（如SP、CGRP、NO、Glu、GABA等）没有参与糖尿病性痛的形成。
3.与对照组相比，在糖尿病性痛形成时糖尿病组脊髓背角EM2或MOR免疫组化阳性产物的密度显著减少，且表达的减少与痛觉阈值的降低密切相关。与此同时，EM2和MOR免疫荧光双标产物也出现了显著减少。
4.在糖尿病性痛行为学研究中观察到，虽然EM2与各种阿片类镇痛药都能剂量依赖地产生镇痛作用，但EM2的镇痛作用更明显；与吗啡相比，相当剂量的EM2具有更好的镇痛效果。
5.我们对背根神经节神经元进行了NeuN和EM2的双重染色，NeuN和MOR的双重染色以及TUNEL染色。结果显示，与对照组相比，糖尿病组的背根神经节中神经元的总数量并没有减少，且没有出现凋亡。但是，与对照组相比，糖尿病组的背根神经节中EM2或MOR免疫阳性神经元的数量出现了显著减少。
6. Western blot检测显示，与对照组相比，糖尿病组脊髓背角MOR的表达在突触前和突触后两个水平都出现了显著减少。
7.脊髓背角神经元细胞外电生理检测结果提示在糖尿病性痛状态下，痛觉信息在脊髓水平的传递出现了中枢敏化。
8.利用膜片钳全细胞记录的方法我们发现EM2作用于MOR以后主要通过G蛋白偶联受体途径发挥胞内信号转导效应。
9.在糖尿病组，系统给予胰岛素样生长因子(insulin-like growth factor；IGF)，醛糖还原酶抑制剂fidarestat或者蛋白质糖基化抑制剂alagebrium，都能不同程度地阻止痛觉过敏的产生。而在对照组，上述药物对于痛觉阈值并没有影响。
10.利用病毒载体感染的方法在糖尿病性痛状态下上调脊髓背角内EM2和MOR的表达，产生了持续的镇痛作用。
以上结果我们提出如下结论：慢性糖尿病状态下，由于某个或某些致病因素的影响，导致了背根节神经元中EM2和MOR以及背角神经元中MOR的合成减少，进而使脊髓背角的EM2和MOR显著减少，内源性镇痛作用的减低导致了痛觉过敏的产生，即糖尿病性痛。
揭示出一种新型镇痛剂的镇痛机理，对于开发临床镇痛新药至关重要。综上所有结果，本论文综合利用现代生命科学研究的先进技术，在初级传入（结状结和背根节）和中枢水平（孤束核和脊髓背角）深入研究内吗啡肽2(EM2)参与调控内脏痛和躯体痛的具体分子机制。本论文的结果不仅对于阐明疼痛的中枢机制、理解内源性阿片肽与阿片受体在病理状态下的功能、推动学科发展具有重要的理论意义，而且对于指导临床用药、开发新一代临床镇痛药物、造福疼痛患者具有重要的实际意义。
Pain is a common and severely disabling state that affects millions of peopleworldwide. Pain could be experienced after nerve injury, inflammation or as partof diseases that affect nervous system. The μ-opioid receptor (MOR), aG-protein coupled receptor expressed in the central and peripheral nervoussystems, is activated by opioid compounds such as morphine, codeine, pethidine,methadone and tramadol. Activated MOR initiates signaling cascades whichresult in potent analgesia. Opioids have been used for centuries to provideeffective pain relief and continue to be essential tools in modern clinical painmanagement. However, the clinical utility of opioid drugs is limited byside-effects that include gastrointestinal complications, respiratory depression,tolerance and dependence with long-term use. These limitations have stimulatedthe search for more effective analgesics that possess fewer side effects, and donot lead to tolerance or dependence.
The endomorphins EM-1(Tyr-Pro-Trp-Phe-NH2) and EM-2(Tyr-Pro-Phe-Phe-NH2) were identified from brain extracts as highly selective MOR agonists and represent the endogenous ligands of MOR. Moreover, thesepeptides are the first endogenous agonists for the MOR and, like morphine, theyare able to regulate neuroendocrine and cardiovascular functions, motivation,feeding, sexual behavior, and pain processing, indicating their significantclinical potential.
An important role of endomorphins in pain modulation is indicated by theirpresence in well-characterized nociceptive pathways. Spinal dorsal horn playsthe most important role in the nociceptive pathways, and is known to beinvolved in the transmission of the nociceptive information by direct input fromthe primary afferents and/or as relay nuclei to other higher pain-processingregion.
While EM-1-like immunoreactivity is primarily restricted to brain, EM-2-likeimmunoreactivity is found primarily in the spinal cord and peripheral nervoussystem and may modulate spinal level pain signaling. MOR have highconcentration in the superficial laminae (laminae I and II) of the spinal dorsalhorn, distributing on both primary afferent terminals and neuronal elements. Thesuperficial laminae of the spinal dorsal horn are also occupied by endogenousEM-2synthesized in neurons of the dorsal root ganglia.
It has been concluded that the two peptides have similar potencies in somebehavioral studies, including the tail-flick test, paw pressure test andcarrageenan-induced inflammation pain test. In contrast, Tseng et al.(2000) haveobserved that EM-2administered intrathecally is more potent than morphine ininhibiting pain responses in the tail-flick test. EM-2was also shown to be more effective than morphine in paw pressure and formalin-induced inflammationpain tests.
However, the precise pathophysiological mechanisms of EM2mediatedanalgesia remain unclear. The molecular mechanism of EM2mediated analgesianeeds to be further elucidated. In the present study, we try our best to exploreEM2mediated analgesic effects by using the methods of morphology, molecularbiology, ethology, pharmacology and pharmacology. There are three parts in mystudy:
Ⅰ. Endomorphin-2is involved in vagally mediated visceral sensationnociception.
It has been revealed that sensory neurons in somatic ganglia (e.g., DRG andtrigeminal ganglion) contain EM2. However, it is still unclear whether or notneurons in the visceral sensory ganglia (e.g., nodose ganglion, NG) also expressEM2. The pseudo-unipolar neurons in the NG play important roles in visceralinformation transmission. In the present study, morphological methods wereused to identify the distribution of EM2and its coexistence with variousneurotransmitters and MOR in both NG neurons and vagal axon terminals. Also,immunofluorescent double-labeling was used to identify whether activated NGneurons were EM2-immunopositive by employing a visceral noxiousstimulation rat model.
1. Immunofluorescent histochemistry showed that43.5%of nodose neuronalprofiles contained EM2and these neurons were small to medium in size.
2. Immunofluorescent double-labeling demonstrated that about15.2%,27.8%,74.4%and25.2%of EM2-like immunoreactive (-LI) neurons expressedsubstance P (SP), calcitonin gene-related peptide (CGRP), nitric oxide synthaseand vasoactive intestinal peptide, respectively. In addition, almost90.8%ofEM2-LI neurons also contained mu-opioid receptor (MOR; endogenous receptorfor endomorphin); and EM2/MOR double-labeled peripheral axons wereobserved on the vagal trunk.
3. Biotinylated dextran amine (BDA) anterograde tracing combined withtriple-labeling showed EM2/MOR double-labeled vagal afferents in the nucleustractus solitarii (NTS).
4. Further immunofluorescent triple-labeling showed EM2/MOR/SP orEM2/MOR/CGRP triple-labeled nodose neurons and axons.
5. Importantly, at the ultrastructrual level, post-embedding electronmicroscopy revealed that EM2-LI and SP-LI gold particles coexisted in thesame large dense-cored synaptic vesicles (LDSV) in the pre-synaptic button,while MOR-LI gold particles existed on both pre-and post-synaptic membranesin the NTS.
6. Moreover, in a formalin visceral nociceptive stimuli rat model, most Fos-LInodose neurons were EM2-LI and the number of EM2-LI neurons wassignificantly increased after stimulus.
The present study of this part provides morphological evidence for theco-localizations of EM2with MOR and other neurotransmitters in the vagalnodose neuron, and EM2with SP in the same LDSV or EM2and MOR in thesame pre-synaptic terminal in the NTS were also identified. These observations,together with the fact that most of the Fos-immunopositive NG neurons showedEM2-immunopositive staining and the number of EM2-LI neurons increasedafter visceral nociceptive stimulation, seem to suggest that EM2mightfunctionally affect visceral reflex by modulating other neurotransmitter releasefrom vagal afferent terminals through binding to pre-synaptically localizedmu-opioid autoreceptor and through post-synaptic mechanisms in the NTS.
Ⅱ. Endomorphin-2mediated inhibition of substance P releasing fromthe primary afferents in the spinal dorsal horn exert antinociception in aninflammatory pain rat model
The endogenous ligand for neurokinin-1receptor (NK1R), substance P(SP), is thought to be involved in pain transmission. Many SP containingprimary afferents terminate in the spinal dorsal horn, from which SP could bereleased from primary afferent terminals and binds to postsynaptic NK1R afternoxious stimulation. While endomorphin-2(EM2) exerts an analgesic role,substance P (SP), commonly coexists with EM2in the same neurons in thedorsal root ganglion (DRG), plays an algesic role during nociception. However,the interaction between these two neuropeptides is still unknown. Further, therewas still no report on the EM2mediated regulation of SP release at presynapticlevel. Whether or not EM2could surely regulate SP release needs to beelucidated.
1. In this study, CFA (complete Freund’s adjuvant) induced inflammatory painmodel was employed to investigate the interaction between EM2and SP atspinal level.
2. Immunofluorescent staining showed that EM2/MOR/SP triple-labeledimmunoreactivity was located in neurons of the DRG, primary afferents andspinal dorsal horn.
3. Post-embedding electron microscopy revealed that EM2-immunoreactiveproducts and SP-immunoreactive gold particles coexisted in the samepre-synaptic large dense-cored vesicles, while MOR-immunoreactive goldparticles existed on both pre-and post-synaptic membranes in spinal dorsalhorn.
4. Intrathecal injection of L-732138[antagonist for (neurokinin-1receptor;NK1R)] and EM2dose-dependently reversed the mechanical allodynia. On theother hand, intrathecal injection of SP and CTAP [antagonist for (mu-opioidreceptor; MOR)] each could facilitate the mechanical allodynia. Mostimportantly, EM2and L-732138had synergic effect for antinociception but SPcould antagonize the analgesic effect of EM2.
5. Subsequently, immunofluorescent staining showed that MOR and NK1Rcoexisted in spinal dorsal horn. Whole cell patch-clamp recordings from spinaldorsal horn neurons showed that AMPA perfusion induced postsynaptic currentwas inhibited by EM2and facilitated by SP. The facilitory effect of SP could be reversed by EM2.
6. Intrathecal microdialysis analysis showed that spinal SP release wassignificantly increased after CFA injection, and SP release could be facilitatedby intrathecal injection of CTAP and antagonized by injection of EM2.
The present study of this part provides evidence for the regulation of EM2on SP mediated algesic effect at the spinal level. These results seem to suggestthat EM2might functionally affect nociception through modulating SP releasefrom primary afferent terminals by binding to pre-synaptically localizedmu-opioid autoreceptor in the spinal dorsal horn.
Ⅲ Endomorphin-2and mu opioid receptor are involved in themediation of diabetic neuropathic pain in spinal dorsal horn
Although activation of the polyol pathway, immune mechanisms,peripheral nerve ischemia and/or loss of insulin-like growth factors have beenhypothesized, the precise underlying pathophysiological mechanisms of painfuldiabetic neuropathy remain unclear. In the present study, we comparativelytested the effects of endomorphins and morphin on neuropathic pain in diabeticmice. Furthermore, to take insight into the role of endogenous spinal EM2andMOR in the development of diabetic mechanical allodynia, doubleimmunofluorecent histochemistry was used to observe the variance ofendogenous EM2and MOR in the diabetic spinal cord compared to control rats.Accordingly, we also tested whether intrathecal administration ofβ-funaltrexamine (MOR-selective antagonist) could facilitate diabeticmechanical allodynia.
1. Diabetic mice developed mechanical allodynia as soon as2weeks afterstreptozotocin (STZ) injection. Thermal nociceptive threshold was not alteredup to8weeks after STZ injection.
2. More importantly, double immunofluorecent histochemical staining of EM2and MOR in the spinal dorsal horn of diabetic rats showed decreased EM2immunoreactivity, MOR immunoreactivity and EM2/MOR double-labeledimmunoreactivity in comparison to normal controls. Interestingly, pearsoncorrelation analysis showed that EM2-LI in the dorsal horn was significantlycorrelated with diabetic mechanical allodynia, which indicated that decreasedEM2in the dorsal horn may be involved in diabetic mechanical allodynia.
3. Intrathecal injections of EM1, EM2or morphine each dose-dependentlyreversed4week-diabetes-induced mechanical allodynia. Although EM-1andEM-2showed a shorter duration than morphine, they were more effectivecompared with corresponding doses of morphine. On the other hand, intrathecalinjections of β-funaltrexamine could facilitate mechanical allodynia in thediabetic rats but had no effect on the control rats.
4. Double immunofluorescence histochemical staining showed that thedouble-labeling of EM2/MOR in the dorsal horn was also significantlydecreased in the diabetic rats. Further colocalization rate analysis showed thatthe neuronal number of DRG was unchanged during diabetes butEM2-immunopositive neurons was significantly decreased in the diabetes group.Next, TUNEL staining was negative in spinal cord of diabetic group. Also,TUNEL staining was also negative in DRG of diabetic group.
5. Further Western blott for MOR in intact and dorsal rhizotomized spinal cord and DRG showed that MOR was decreased in both presynaptic and postsynapticlevel in the spinal cord during diabetes.At this stage of the present study, it can be supposed that normally, EM2issynthesized in DRG neurons, and transported to dosal horn. But during diabetes,some etiological factors contribute to the decreased EM2expression in DRGneurons and dosal horn. Finally, diabetic mechanical allodynia occurs.
6. It was showed that after chronic diabetic stimulation, WDR neurons inlamina1and2has significantly increased firing response to degradedmechanical stimuli, and the increased firing response could be reversed byintrathecal injection of EM2and could be facilitated by intrathecal injection ofnalaxone or β-FNA.
7. Patch clamp whole cell recoding showed that EM2mediated analgesia wasthough bing to MOR to activate intracellular G-protein pathway.
8. In the diabetic group, to seek the specific pathogeny of the decrease of EM2and MOR, systematic administration of insulin-like growth factor, fidarestat andalagebrium each could to some extent alleviate diabetic neuropathic pain andelevate the spinal level of EM2and MOR.
9. Finally, viral vector transfection could elevate the expression level of EM2and MOR in spinal dorsal horn, and exert analgesic effect.
Thus, the present study of this part reveals the spinal antinociceptive action ofendomorphins in diabetic rats and suggest that reduced endogeneous EM2andMOR in the spinal dorsal horn might induce ongoing diabetic mechanical allodynia. Our rearch group strives to provide original theories for the diagnosisand therapy of DNP though the above work.
All the above results indicated that EM2may exert analgesic effect throughbinding to pre-synaptic MOR to inhibit neurotransmitter release and alsothrough binding to post-synaptic MOR to antagonize neurotransmitter mediatedpost-synaptic effect in NTS or spinal dorsal horn. These findings suggest thedetailed pathophysiological mechanisms underlying EM2mediated analgesia,and provide original theories for the diagnosis and treatment of visceral orsomatic nociception.
Zadina研究组经过20年的努力终于找到了两种MOR的内源性配体，分别被称为内吗啡肽1(Tyr-Pro-Trp-Phe-NH2, endomorphin-1, EM1)和内吗啡肽2（Tyr-Pro-Phe-Phe-NH2, endomorphin-2, EM2）。EM对MOR的选择性和亲和力是δ，κ阿片受体的1000倍以上，而且具有明确的、比吗啡更强的镇痛作用。而作为一种内源性物质，EM的副作用非常小。因此，在临床应用中就完全有可能使用较小剂量的EM产生有效的镇痛作用，而避免常见的副作用，成瘾性和耐受性。
然而EM的镇痛机理仍然不很清楚，只有搞清了EM镇痛作用的神经机制，才能开发出镇痛作用强和成瘾性及不良反应弱或无的止痛药物，有益于临床工作中对疼痛的治疗，提高患者的生活质量。经典的疼痛传递通路为：背根神经节初级传入纤维在脊髓背角释放致痛物质作用于脊髓背角二级投射神经元，二级投射神经元将痛信息传递到高位脑中枢。当然，结状结和孤束核也参与了内脏痛信息的调控。脊髓背角或孤束核作为痛觉传递的闸门，在痛觉调控中起着至为关键的作用。脊髓背角主要含有EM2，其主要来源于含EM2的背根节神经元发出的初级传入纤维；专职内脏信息传递的结状结中是否含有EM2至今无人报道。EM2在初级传入以及脊髓背角或孤束核的镇痛效应并不明确，其作用机理尚处于空白，亟需深入开展研究。本论文综合利用形态学、分子生物学、行为学、药理学以及电生理学等实验方法，在初级传入以及脊髓背角或孤束核水平深入探索EM2镇痛的具体分子机理。本研究具体分为三部分。
一、内吗啡肽2在结状结参与内脏痛调控的机制
以往的研究表明，内吗啡肽2(EM2)与μ型阿片受体(MOR)及其它一些神经递质共存于背根节神经元和初级传入纤维。然而，专职内脏信息传递的结状结中是否含有EM2至今尚无报道。在本实验中：
1.免疫荧光组织化学方法显示有43.5%的结状结神经元呈EM2阳性，这些阳性神经元以中小型细胞为主。
2.免疫荧光双标染色显示在这些EM2免疫阳性的神经元里，有15.2%,27.8%,74.4%以及25.2%的神经元同时表达有P物质(SP)，降钙素基因相关肽(CGRP),一氧化氮合酶(NOS)以及血管活性肠肽(VIP)。另外，EM2免疫阳性的神经元里几乎有90.8%的神经元包含有MOR，并且在迷走神经干上面我们发现了EM2/MOR双标的结状结外周突神经纤维。
3.利用生物素化葡聚糖胺注入结状结顺行追踪外加免疫荧光三标的方法，我们在孤束核(NTS)里发现了大量的EM2/MOR双标的结状结中枢突神经纤维。
4.进一步的免疫荧光三标显示一些EM2/MOR双标的结状结神经元和轴突同时包含有SP或者CGRP。
5.在超微结构水平，包埋后免疫电镜双标显示在NTS的突触前终末里，EM2免疫阳性金颗粒和SP免疫阳性金颗粒共存于相同的突触囊泡里面，并且MOR免疫阳性金颗粒同时存在于突触前膜和后膜之上。
6.此外，在将福尔马林灌入胃内造成急性内脏伤害性刺激的大鼠模型中，我们发现结状结里Fos阳性的神经元大部分是EM2阳性的，并且与对照组相比EM2阳性神经元的数量有明显的增加。
以上这些结果表明EM2与MOR及其它神经递质共存于大鼠结状结的神经元和轴突内，EM2有可能在结状结神经元的轴突终末释放出以后，通过作用于突触前的MOR去调控其它神经递质的释放或者作用于突触后的MOR去调控其它神经递质的突触后效应，进而调节内脏痛信息的传递和内脏反射的形成。
二、内吗啡肽2在背根节参与躯体痛调控的机制
最近的一些形态学实验证实SP与EM2和MOR共存于背根节神经元的胞体和初级传入纤维，这强烈预示着EM2极有可能通过作用于脊髓背角突触前的MOR进而抑制SP的释放，最终调控伤害性信息的传递。但是，至今并未有实验报道EM2能抑制SP的释放，在本实验中：
1.将CFA注入大鼠足底，建立炎性痛模型。
2.我们以镇痛药的有效镇痛剂量除以呼吸抑制剂量或循环抑制剂量的比值作为评判药物安全性的指标。EM2的该比值远远低于吗啡等阿片类镇痛药的该比值。另外，我们还发现连续使用EM2，其镇痛作用风采依旧，而吗啡等则产生了明显的耐受。
3.来自于DRG的中枢突在脊髓背角突触前释放致痛物质：SP， SP作用于突触后相应受体产生突触后效应，是痛觉传递的核心环节。在形态学实验中发现，EM2与SP和MOR共存于DRG神经元的胞体、中枢突和脊髓背角的终末内。
4.为了更直观地显示脊髓背角阳性终末的微观结构。我们用免疫电镜包埋后染色显示，EM2, SP分别或同时存在于突触前囊泡，而MOR同时存在于突触前膜和突触后膜。
基于上述形态学研究结果，我们提出假设：当痛觉信息从外周传递到脊髓背角时，EM2与SP共同释放，EM2结合于突触前膜的MOR，抑制SP的释放，实现镇痛效应。
5.为了证实以上假设，首先，在行为学实验中，我们发现SP的拮抗剂与EM2之间具有协同镇痛效应。
6.其次，在脊髓背角神经元膜片钳全细胞记录中，预先阻断电压门控钠通道和突触后受体，EM2仍然可以作用于突触前膜的MOR抑制谷氨酸的释放。
7.最后，由于膜片钳无法精确定量SP释放量的变化，我们利用新购置的微透析装置证明：EM2确实能作用于突触前膜的MOR，从而有效地抑制突触前SP的释放。
上述研究结果证实了我们的假设，即EM2会结合于突触前膜的MOR有效抑制致痛物质的释放，最终起到镇痛作用。
三、内吗啡肽2与μ型阿片受体在脊髓背角参与糖尿病性痛调控的机制
随着生活水平的提高，糖尿病的发病率一再上升，目前我国有糖尿病患者五千万，每年新增两百万。糖尿病性痛是糖尿病最严重的并发症之一，表现为下肢远侧端的痛觉过敏和异常疼痛，严重地影响着患者的生活质量。以往对糖尿病性痛的研究都集中在外周神经末梢变性和炎性脱髓鞘的基础之上，而本项目另辟蹊径，从糖尿病性痛状态下脊髓背角内吗啡肽2（EM2）与μ型阿片受体（MOR）减少的角度出发，综合利用多种实验方法对糖尿病性痛进行诊断和治疗方面的深入研究，以期能够在脊髓水平对糖尿病性痛进行有效的干预和治疗，为临床实践工作提供有力的理论依据。
我们综合利用各种实验技术，得出以下实验结果：
1.糖尿病组小鼠血糖迅速升高并且维持在高水平状态，出现了明显的机械性痛觉过敏。
2.在糖尿病性痛形成时，一些经典的兴奋性或抑制性的神经递质和调质（如SP、CGRP、NO、Glu、GABA等）没有参与糖尿病性痛的形成。
3.与对照组相比，在糖尿病性痛形成时糖尿病组脊髓背角EM2或MOR免疫组化阳性产物的密度显著减少，且表达的减少与痛觉阈值的降低密切相关。与此同时，EM2和MOR免疫荧光双标产物也出现了显著减少。
4.在糖尿病性痛行为学研究中观察到，虽然EM2与各种阿片类镇痛药都能剂量依赖地产生镇痛作用，但EM2的镇痛作用更明显；与吗啡相比，相当剂量的EM2具有更好的镇痛效果。
5.我们对背根神经节神经元进行了NeuN和EM2的双重染色，NeuN和MOR的双重染色以及TUNEL染色。结果显示，与对照组相比，糖尿病组的背根神经节中神经元的总数量并没有减少，且没有出现凋亡。但是，与对照组相比，糖尿病组的背根神经节中EM2或MOR免疫阳性神经元的数量出现了显著减少。
6. Western blot检测显示，与对照组相比，糖尿病组脊髓背角MOR的表达在突触前和突触后两个水平都出现了显著减少。
7.脊髓背角神经元细胞外电生理检测结果提示在糖尿病性痛状态下，痛觉信息在脊髓水平的传递出现了中枢敏化。
8.利用膜片钳全细胞记录的方法我们发现EM2作用于MOR以后主要通过G蛋白偶联受体途径发挥胞内信号转导效应。
9.在糖尿病组，系统给予胰岛素样生长因子(insulin-like growth factor；IGF)，醛糖还原酶抑制剂fidarestat或者蛋白质糖基化抑制剂alagebrium，都能不同程度地阻止痛觉过敏的产生。而在对照组，上述药物对于痛觉阈值并没有影响。
10.利用病毒载体感染的方法在糖尿病性痛状态下上调脊髓背角内EM2和MOR的表达，产生了持续的镇痛作用。
以上结果我们提出如下结论：慢性糖尿病状态下，由于某个或某些致病因素的影响，导致了背根节神经元中EM2和MOR以及背角神经元中MOR的合成减少，进而使脊髓背角的EM2和MOR显著减少，内源性镇痛作用的减低导致了痛觉过敏的产生，即糖尿病性痛。
揭示出一种新型镇痛剂的镇痛机理，对于开发临床镇痛新药至关重要。综上所有结果，本论文综合利用现代生命科学研究的先进技术，在初级传入（结状结和背根节）和中枢水平（孤束核和脊髓背角）深入研究内吗啡肽2(EM2)参与调控内脏痛和躯体痛的具体分子机制。本论文的结果不仅对于阐明疼痛的中枢机制、理解内源性阿片肽与阿片受体在病理状态下的功能、推动学科发展具有重要的理论意义，而且对于指导临床用药、开发新一代临床镇痛药物、造福疼痛患者具有重要的实际意义。
Pain is a common and severely disabling state that affects millions of peopleworldwide. Pain could be experienced after nerve injury, inflammation or as partof diseases that affect nervous system. The μ-opioid receptor (MOR), aG-protein coupled receptor expressed in the central and peripheral nervoussystems, is activated by opioid compounds such as morphine, codeine, pethidine,methadone and tramadol. Activated MOR initiates signaling cascades whichresult in potent analgesia. Opioids have been used for centuries to provideeffective pain relief and continue to be essential tools in modern clinical painmanagement. However, the clinical utility of opioid drugs is limited byside-effects that include gastrointestinal complications, respiratory depression,tolerance and dependence with long-term use. These limitations have stimulatedthe search for more effective analgesics that possess fewer side effects, and donot lead to tolerance or dependence.
The endomorphins EM-1(Tyr-Pro-Trp-Phe-NH2) and EM-2(Tyr-Pro-Phe-Phe-NH2) were identified from brain extracts as highly selective MOR agonists and represent the endogenous ligands of MOR. Moreover, thesepeptides are the first endogenous agonists for the MOR and, like morphine, theyare able to regulate neuroendocrine and cardiovascular functions, motivation,feeding, sexual behavior, and pain processing, indicating their significantclinical potential.
An important role of endomorphins in pain modulation is indicated by theirpresence in well-characterized nociceptive pathways. Spinal dorsal horn playsthe most important role in the nociceptive pathways, and is known to beinvolved in the transmission of the nociceptive information by direct input fromthe primary afferents and/or as relay nuclei to other higher pain-processingregion.
While EM-1-like immunoreactivity is primarily restricted to brain, EM-2-likeimmunoreactivity is found primarily in the spinal cord and peripheral nervoussystem and may modulate spinal level pain signaling. MOR have highconcentration in the superficial laminae (laminae I and II) of the spinal dorsalhorn, distributing on both primary afferent terminals and neuronal elements. Thesuperficial laminae of the spinal dorsal horn are also occupied by endogenousEM-2synthesized in neurons of the dorsal root ganglia.
It has been concluded that the two peptides have similar potencies in somebehavioral studies, including the tail-flick test, paw pressure test andcarrageenan-induced inflammation pain test. In contrast, Tseng et al.(2000) haveobserved that EM-2administered intrathecally is more potent than morphine ininhibiting pain responses in the tail-flick test. EM-2was also shown to be more effective than morphine in paw pressure and formalin-induced inflammationpain tests.
However, the precise pathophysiological mechanisms of EM2mediatedanalgesia remain unclear. The molecular mechanism of EM2mediated analgesianeeds to be further elucidated. In the present study, we try our best to exploreEM2mediated analgesic effects by using the methods of morphology, molecularbiology, ethology, pharmacology and pharmacology. There are three parts in mystudy:
Ⅰ. Endomorphin-2is involved in vagally mediated visceral sensationnociception.
It has been revealed that sensory neurons in somatic ganglia (e.g., DRG andtrigeminal ganglion) contain EM2. However, it is still unclear whether or notneurons in the visceral sensory ganglia (e.g., nodose ganglion, NG) also expressEM2. The pseudo-unipolar neurons in the NG play important roles in visceralinformation transmission. In the present study, morphological methods wereused to identify the distribution of EM2and its coexistence with variousneurotransmitters and MOR in both NG neurons and vagal axon terminals. Also,immunofluorescent double-labeling was used to identify whether activated NGneurons were EM2-immunopositive by employing a visceral noxiousstimulation rat model.
1. Immunofluorescent histochemistry showed that43.5%of nodose neuronalprofiles contained EM2and these neurons were small to medium in size.
2. Immunofluorescent double-labeling demonstrated that about15.2%,27.8%,74.4%and25.2%of EM2-like immunoreactive (-LI) neurons expressedsubstance P (SP), calcitonin gene-related peptide (CGRP), nitric oxide synthaseand vasoactive intestinal peptide, respectively. In addition, almost90.8%ofEM2-LI neurons also contained mu-opioid receptor (MOR; endogenous receptorfor endomorphin); and EM2/MOR double-labeled peripheral axons wereobserved on the vagal trunk.
3. Biotinylated dextran amine (BDA) anterograde tracing combined withtriple-labeling showed EM2/MOR double-labeled vagal afferents in the nucleustractus solitarii (NTS).
4. Further immunofluorescent triple-labeling showed EM2/MOR/SP orEM2/MOR/CGRP triple-labeled nodose neurons and axons.
5. Importantly, at the ultrastructrual level, post-embedding electronmicroscopy revealed that EM2-LI and SP-LI gold particles coexisted in thesame large dense-cored synaptic vesicles (LDSV) in the pre-synaptic button,while MOR-LI gold particles existed on both pre-and post-synaptic membranesin the NTS.
6. Moreover, in a formalin visceral nociceptive stimuli rat model, most Fos-LInodose neurons were EM2-LI and the number of EM2-LI neurons wassignificantly increased after stimulus.
The present study of this part provides morphological evidence for theco-localizations of EM2with MOR and other neurotransmitters in the vagalnodose neuron, and EM2with SP in the same LDSV or EM2and MOR in thesame pre-synaptic terminal in the NTS were also identified. These observations,together with the fact that most of the Fos-immunopositive NG neurons showedEM2-immunopositive staining and the number of EM2-LI neurons increasedafter visceral nociceptive stimulation, seem to suggest that EM2mightfunctionally affect visceral reflex by modulating other neurotransmitter releasefrom vagal afferent terminals through binding to pre-synaptically localizedmu-opioid autoreceptor and through post-synaptic mechanisms in the NTS.
Ⅱ. Endomorphin-2mediated inhibition of substance P releasing fromthe primary afferents in the spinal dorsal horn exert antinociception in aninflammatory pain rat model
The endogenous ligand for neurokinin-1receptor (NK1R), substance P(SP), is thought to be involved in pain transmission. Many SP containingprimary afferents terminate in the spinal dorsal horn, from which SP could bereleased from primary afferent terminals and binds to postsynaptic NK1R afternoxious stimulation. While endomorphin-2(EM2) exerts an analgesic role,substance P (SP), commonly coexists with EM2in the same neurons in thedorsal root ganglion (DRG), plays an algesic role during nociception. However,the interaction between these two neuropeptides is still unknown. Further, therewas still no report on the EM2mediated regulation of SP release at presynapticlevel. Whether or not EM2could surely regulate SP release needs to beelucidated.
1. In this study, CFA (complete Freund’s adjuvant) induced inflammatory painmodel was employed to investigate the interaction between EM2and SP atspinal level.
2. Immunofluorescent staining showed that EM2/MOR/SP triple-labeledimmunoreactivity was located in neurons of the DRG, primary afferents andspinal dorsal horn.
3. Post-embedding electron microscopy revealed that EM2-immunoreactiveproducts and SP-immunoreactive gold particles coexisted in the samepre-synaptic large dense-cored vesicles, while MOR-immunoreactive goldparticles existed on both pre-and post-synaptic membranes in spinal dorsalhorn.
4. Intrathecal injection of L-732138[antagonist for (neurokinin-1receptor;NK1R)] and EM2dose-dependently reversed the mechanical allodynia. On theother hand, intrathecal injection of SP and CTAP [antagonist for (mu-opioidreceptor; MOR)] each could facilitate the mechanical allodynia. Mostimportantly, EM2and L-732138had synergic effect for antinociception but SPcould antagonize the analgesic effect of EM2.
5. Subsequently, immunofluorescent staining showed that MOR and NK1Rcoexisted in spinal dorsal horn. Whole cell patch-clamp recordings from spinaldorsal horn neurons showed that AMPA perfusion induced postsynaptic currentwas inhibited by EM2and facilitated by SP. The facilitory effect of SP could be reversed by EM2.
6. Intrathecal microdialysis analysis showed that spinal SP release wassignificantly increased after CFA injection, and SP release could be facilitatedby intrathecal injection of CTAP and antagonized by injection of EM2.
The present study of this part provides evidence for the regulation of EM2on SP mediated algesic effect at the spinal level. These results seem to suggestthat EM2might functionally affect nociception through modulating SP releasefrom primary afferent terminals by binding to pre-synaptically localizedmu-opioid autoreceptor in the spinal dorsal horn.
Ⅲ Endomorphin-2and mu opioid receptor are involved in themediation of diabetic neuropathic pain in spinal dorsal horn
Although activation of the polyol pathway, immune mechanisms,peripheral nerve ischemia and/or loss of insulin-like growth factors have beenhypothesized, the precise underlying pathophysiological mechanisms of painfuldiabetic neuropathy remain unclear. In the present study, we comparativelytested the effects of endomorphins and morphin on neuropathic pain in diabeticmice. Furthermore, to take insight into the role of endogenous spinal EM2andMOR in the development of diabetic mechanical allodynia, doubleimmunofluorecent histochemistry was used to observe the variance ofendogenous EM2and MOR in the diabetic spinal cord compared to control rats.Accordingly, we also tested whether intrathecal administration ofβ-funaltrexamine (MOR-selective antagonist) could facilitate diabeticmechanical allodynia.
1. Diabetic mice developed mechanical allodynia as soon as2weeks afterstreptozotocin (STZ) injection. Thermal nociceptive threshold was not alteredup to8weeks after STZ injection.
2. More importantly, double immunofluorecent histochemical staining of EM2and MOR in the spinal dorsal horn of diabetic rats showed decreased EM2immunoreactivity, MOR immunoreactivity and EM2/MOR double-labeledimmunoreactivity in comparison to normal controls. Interestingly, pearsoncorrelation analysis showed that EM2-LI in the dorsal horn was significantlycorrelated with diabetic mechanical allodynia, which indicated that decreasedEM2in the dorsal horn may be involved in diabetic mechanical allodynia.
3. Intrathecal injections of EM1, EM2or morphine each dose-dependentlyreversed4week-diabetes-induced mechanical allodynia. Although EM-1andEM-2showed a shorter duration than morphine, they were more effectivecompared with corresponding doses of morphine. On the other hand, intrathecalinjections of β-funaltrexamine could facilitate mechanical allodynia in thediabetic rats but had no effect on the control rats.
4. Double immunofluorescence histochemical staining showed that thedouble-labeling of EM2/MOR in the dorsal horn was also significantlydecreased in the diabetic rats. Further colocalization rate analysis showed thatthe neuronal number of DRG was unchanged during diabetes butEM2-immunopositive neurons was significantly decreased in the diabetes group.Next, TUNEL staining was negative in spinal cord of diabetic group. Also,TUNEL staining was also negative in DRG of diabetic group.
5. Further Western blott for MOR in intact and dorsal rhizotomized spinal cord and DRG showed that MOR was decreased in both presynaptic and postsynapticlevel in the spinal cord during diabetes.At this stage of the present study, it can be supposed that normally, EM2issynthesized in DRG neurons, and transported to dosal horn. But during diabetes,some etiological factors contribute to the decreased EM2expression in DRGneurons and dosal horn. Finally, diabetic mechanical allodynia occurs.
6. It was showed that after chronic diabetic stimulation, WDR neurons inlamina1and2has significantly increased firing response to degradedmechanical stimuli, and the increased firing response could be reversed byintrathecal injection of EM2and could be facilitated by intrathecal injection ofnalaxone or β-FNA.
7. Patch clamp whole cell recoding showed that EM2mediated analgesia wasthough bing to MOR to activate intracellular G-protein pathway.
8. In the diabetic group, to seek the specific pathogeny of the decrease of EM2and MOR, systematic administration of insulin-like growth factor, fidarestat andalagebrium each could to some extent alleviate diabetic neuropathic pain andelevate the spinal level of EM2and MOR.
9. Finally, viral vector transfection could elevate the expression level of EM2and MOR in spinal dorsal horn, and exert analgesic effect.
Thus, the present study of this part reveals the spinal antinociceptive action ofendomorphins in diabetic rats and suggest that reduced endogeneous EM2andMOR in the spinal dorsal horn might induce ongoing diabetic mechanical allodynia. Our rearch group strives to provide original theories for the diagnosisand therapy of DNP though the above work.
All the above results indicated that EM2may exert analgesic effect throughbinding to pre-synaptic MOR to inhibit neurotransmitter release and alsothrough binding to post-synaptic MOR to antagonize neurotransmitter mediatedpost-synaptic effect in NTS or spinal dorsal horn. These findings suggest thedetailed pathophysiological mechanisms underlying EM2mediated analgesia,and provide original theories for the diagnosis and treatment of visceral orsomatic nociception.
引文
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