Roles Of Peripheral Mitogen-activated Protein Kinases In Melittin-induced Nociception And Hyperalgesia

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.