| 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 modelThe 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 hornAlthough 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. |
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