Study On The Molecular Mechanisms Of Spinal Macrophage Migration Inhibitory Factor (MIF) In The Pathogenesis Of Pain

The occurrence and development of diseases always evoke the generation of pain. Many drugs and methods so far have been being developed for pain treatment, whereas they are limited in therapeutic effectiveness. It has, thus, vital clinical implications to seek novel mediators of pain and tap corresponding interventions. Noxious stimulation or nervous injury-induced inflammatory immunity is strongly associated with the occurrence and development of pain. MIF is a pro-inflammatory cytokine that involves in almost all types of inflammation-related disorders reported, and published reports indicate that MIF is an essential modulator of nerve regeneration and function recovery after nervous injury. Given the causative relationship between nervous injury and pain, a role for MIF in pain was proposed that MIF decreases the threshold of pain by sensitizing nervous system after injury.By modeling acute inflammatory and chronic neuropathic pain in rodents, the correlation was assessed between the changes of pain threshold and spinal cord MIF levels through intrathecal administration of ISO-1, a low molecular weight inhibitor of MIF tautomerase. Further, the underlying signaling mechanisms of MIF-mediated pathogenesis of pain were investigated. This provides link for therapeutic target of pain, and theoretic basis for MIF-associated drug discovery in pain treatment. The entire study includes following three parts:Part I. MIF involves in the regulation of pain threshold in formalin-induced acute inflammatory pain and underlying mechanismsDifferent doses of MIF tautomerase inhibitor ISO-1 were administered intrathecally in formalin-induced inflammatory rats and found ISO-1 could improve the changes in formalin-induced two-phase behaviors in a dose-dependent manner. Along with the changes in pain behaviors, the expression of MIF and its receptor CD74 were upregulated in the spinal cord dorsal horn in a time-dependent manner after formalin injection; meanwhile, the levels of CSF MIF displayed an increase at the later period of the second phase of formalin-induced behaviors. These findings indicate that changes in spinal MIF involves in the occurrence and development of formalin-induced inflammatory pain. Based on these, the expression of ERK, p-p38 MAPK and NR2B were detected with immunoblotting and found they were blocked markedly by ISO-1; moreover, the inhibitors of ERK and p38 MAPK resulted in a significant downregulation of NR2B. These indicate the activation of ERK-p38 MAPK-NR2B signaling pathway mediates the pathogenesis of formalin-induced inflammatory pain. Furthermore, the expression of MIF, CD11b and CD3 in the spinal cord dorsal horn was measured using immunohistochemistry, and found MIF and CDllb are colocalized. This show the upregulated MIF after formalin stimulation is originated from spinal microglial cells. In order to confirm the equal effectiveness between MIF tautomerase inhibition and inhibition of MIF biological activities, i.e. whether the intrathecal ISO-1 could effectively inhibit MIF biological activities, this study used dopachrome methyl ester as the detecting tool of tautomerase activity, and tested the association between MIF tautomerase and MIF production. The results showed it is consistent between the inhibition of MIF tautomerase activity and MIF production after ISO-1 administration. So it confirms that MIF tautomerase inhibitors can be developed for MIF-associated pain therapy and drug discovery.Part II. MIF involves in the regulation of pain threshold in CCI-induced neuropathic pain and underlying mechanismsFollowing the upregulation of MIF expression in the ipsilateral spinal cord dorsal horn in CCI-induced neuropathic mice models, the decrease in mechanical threshold and shortening in thermal latency displayed a time-dependent manner, and the spinal firing amplitude was increased and the time need-to-be normalization was lengthened after external stimuli. Besides, CSF MIF increased time-dependently, but intrathecal ISO-1 could dose-dependently improve mechanical and thermal behaviors which were similar to the effect produced by intrathecal MIF antibody. These results suggest MIF involves in the regulation of pain threshold in CCI-induced neuropathic animals. In addition, the expression of spinal CD74 showed a similar trend to MIF, and the immunofluorescence revealed MIF and NeuN colocalized and CD74 and CD11b colocalized. These findings indicate CD74 has similar changes with MIF after CCI nervous injury, but the production origin and the functional target of MIF in this context do not on the same cells. It so can say that spinal MIF is a sensitizer for pain by functional shuttling between spinal neurons and microglia. Further, the downstream effectors of p-p44/42 MAPK including IL-8, NR2B and CSF PGE2 were increased after CCI injury, but these changes could be inhibited by intrathecal ISO-1 administration; in addition, p44/42 MAPK inhibitor mimicked the effect of ISO-1 in decreasing the mechanical and thermal evoked pain threshold. These data reveal the p44/42 MAPK signaling pathway is activated in CCI-induced mice models, and then involves in the regulation of pain threshold. To identify further the role for MIF in CCI-induced animals, MIF gene modified mice (MIF knock-out, MIF(?)) were investigated and found the threshold of both mechanical and thermal stimuli elevated than that in the wild-type (WT) comparisons, and correspondingly the expression of CD74, p-p44/42 MAPK, IL-8 and NR2B in the spinal cord dorsal horn downregulated and the level of CSF PGE2 decreased. These results provide further evidence for MIF's role in the regulation of CCI-induced pain. Subsequently, intrathecal rMIF administered to normal mice evoked decrease in pain threshold of both mechanical and thermal stimuli, but these changes in pain behaviors could be prevented by prophylactic administration of p44/42 MAPK inhibitor; in concert with these, the expression of IL-8 and NR2B in the spinal cord dorsal horn displayed corresponding changes. These observations indicate CCI-induced downregulation of pain threshold correlates with the increased level of CSF MIF. Therefore, it can be said that MIF in both CSF and spinal cord dorsal horn produces a synergic role in the regulation of pain threshold evoked by CCI nerve injury. At last, the inhibiting role of ISO-1 in MIF tautomerase and the tautomerase activity of rMIF were tested in cultured microglial cells.Part III. MIF activates cultured spinal microglial cells in vitroLipopolysaccharide (LPS) stimulation of cultured spinal microglial cells was regarded as the reference of inflammation activation and corresponding release of inflammatory mediators, and then rMIF stimulation was performed and found cyclooxygenase 2 (COX 2) and microsomal prostaglandin E2 synthase-1 (mPGES-1) were activated and resulted in increase in PGE2 synthesis in a dose-dependent manner; these above responses could be blocked by ISO-1. Furthermore, both p38 and p44/42 MAPK signaling pathways were found involved in the regulation of rMIF evoked COX 2/mPGES-1/PGE2 inflammatory responses. These results showed external MIF itself can produce mimetic role of LPS-induced activation of inflammation in cultured microglial cells.In conclusion, spinal MIF regulates pain threshold through activating different signaling pathways in both acute inflammatory and chronic neuropathic pain animals, this effect is strongly associated with inflammatory responses produced by microglia in the spinal cord dorsal horn. By using intrathecal administration of MIF tautomerase inhibitor as an interventional tool, the feasibility of whether or not MIF tautomerase inhibition could be used for inhibiting MIF biological activity was testified, and then provides basic evidence for MIF-associated pain therapy.