The Role And Related Mechanisms Of IL-18/IL-18R Signaling Pathway In Experimental Models Of Migraine

BackgroudsMigraine is a severe and disabling neurological disorder.It affected about 15% of the world’s population,among which female morbidity was higher than male morbidity.The incidence and disability rate of migraine are increasing year by year,bringing a great detrimental influence on individuals and society.The exact molecular mechanisms of migraine were not fully understood,and there was a lack of effective treatment for migraine.About half of migraine sufferers could not achieve satisfactory results after treatment.Therefore,it is of great significance to elucidate the pathogenesis of migraine.Recently,emerging evidence indicates that inflammation has a significant role in pathophysiological mechanisms of migraine.The trigeminal neurovascular system is activated and releases calcitonin gene-related peptide(CGRP),which induces increased vascular permeability,destruction of blood-brain barrier,leukocyte infiltration,activation of glial cells and production of inflammatory mediators,amplifying the central inflammatory cascade and playing a key role in the pathogenesis of migraine.Glial cells play an important role in the development of pain,in which microglia and astrocytes are mainly activated.However,the effect of glial cells on the pathogenesis of migraine and its specific mechanism are still unclear.Interleukin is a kind of important inflammatory cytokines.Glial cells are the main source of interleukin(IL)in the central nervous system(CNS).Glial cells were found in close proximity to each other,and activated each other.They facilitated cell-cell communication through ligand-receptor pairings elevated in target tissues.IL-18 is a member of the IL-1 family and plays a key role in inflammatory and immune responses.In various rodent models,IL-18 was found primarily expressed in microglia,while its receptor,IL-18 R,was expressed in astrocytes.With the further elucidation of the biological characteristics and functions of IL-18,its role in the development of pain has been gradually recognized.On the period of the migraine outbreaks,the expression levels of various inflammatory cytokines in plasma were significantly up-regulated,such as tumor necrosis factor(TNF),IL-1β and IL-6,etc.Therefore,inflammatory cytokines may play an important role in the pathophysiological mechanism of migraine.How does the expression of IL-18 change in peripheral blood of migraine? Is IL-18 / IL-18 R signaling involved in migraine?Is glial cell interaction mediated by IL-18 / IL-18 R signaling pathway associated with migraine? Is IL-18 / IL-18 R signaling pathway a possible new therapeutic target for migraine? There has been no study investigating the above scientific questions.Toll-like receptor 4(TLR4)/mitogen-activated protein kinase(MAPK)signaling pathway is involved in the activation of microglia to release IL-18,while nuclear factor-κB(NF-κB)signaling pathway is involved in the activation of astrocytes.Activation of the TLR4 / p38 MAPK signaling pathway induces the activation of microglia to release IL-18 during nerve injury.IL-18 acts on IL-18 R of astrocytes in the form of paracrine,and further activates NF-κB signaling pathway,thereby inducing astrocyte activation to participate in the occurrence of neuropathic pain.More and more evidences indicate that TLR4,MAPK and NF-κB play an important role in the development of migraine.But how does it interact with the IL-18 / IL-18 R signaling pathway to participate in the occurrence of migraine? No research has been reported.The maintenance of glutamate homeostasis in the central nervous system is mainly dependent on the astrocyte excitatory amino acid transporter 2(EAAT2).EAAT2 dysfunction leads to abnormal aggregation of glutamate in synaptic cleft,which continues to act on neuronal N-methyl-D-aspartic acid receptor(NMDAR),inducing high expression of c-fos,namely neuronal hyperexcitability,and thus inducing central sensitization.Central sensitization is considered as one of the important pathogenesis of migraine.IL-18 / IL-18 R signaling pathway may regulate the expression of NMDAR and participate in the occurrence of neuropathic pain.Is EAAT2 dysfunction involved in the occurrence of episodic migraine? Does the IL-18/ IL-18 R signaling pathway regulate the glutamate signaling pathway involved in migraine? No research has been reported.The present study aims to investigate the role of IL-18-mediated microglia/astrocyte interaction in the medullary dorsal horn on the development of hyperpathia or allodynia induced by migraine,and further explore the exact molecular mechanism,and aims to detect the expression changes of IL-18 and IL-18 binding protein(IL-18BP)in peripheral blood of migraine.Objectives(1)To comfirm the activation of IL-18 / IL-18 R signaling in experimental models of migraine and observe the effect of interference on IL-18 / IL-18 R signaling on migraine.(2)To investigate the involvement and exact molecular mechanism of IL-18 /IL-18 R signaling-mediated microglia/astrocyte interaction in experimental models of migraine.(3)To explore the expression of IL-18 and IL-18 BP in peripheral blood of migraine preliminarily.Methods(1)Establishment of experimental models of migraine and related detection:The experimental model of migraine was established by repetitive inflammatory soup dural infusions.After the establishment of the model,headache behavior,c-fos and CGRP expression were detected.Headache behavior included the number of face rubbing and nociception threshold.(2)Detection of glial cell activation: The expression of Iba1(a marker of microglial activation)and GFAP(a marker of astrocyte activation)was examined by immunofluorescence and Western blot.(3)Detection of the expression of IL-18,IL-18 R,EAAT2 and NMDAR: The expression of IL-18 and IL-18 receptor(IL-18R)was examined by quantitative real-time polymerase chain reaction or Western blot.To identify the cell types expressing IL-18 and IL-18 R after inflammatory soup dural infusions,we performed double Immuno-fluorescence staining of the IL-18 or IL-18 R.The expression of EAAT2,NR1,NR2 A and NR2 B were detected by Western blot.Double Immuno-fluorescence staining of the EAAT2 and GFAP was performed to identify the expression localization of EAAT2.Double Immuno-fluorescence staining of the NMDAR and Neu N was performed to identify the expression localization of NMDAR.(4)Intervention of TLR4 signaling pathway and related detection: TAK-242 was used to inhibit the activation of toll-like receptor 4(TLR4)and the expression of IL-18 was detected by Western blot.Double Immuno-fluorescence staining was performed to observe the p-p38 entries in the nucleus of IL-18-immunopositive cells.(5)Intervention of IL-18 / IL-18 R signaling pathway and related detection:Anti-IL-18 antibody was used to inhibit the IL-18 / IL-18 R signaling.The headache behavior of rats was observed and the expression of GFAP,c-fos、CGRP、p-p65、EAAT2 、 NR1 、 NR2 A and NR2 B was detected by Western blot.Double Immuno-fluorescence staining of the IL-18 R and p-p65 was performed to observe the co-localization of IL-18 R and NF-κB.IL-18 dural infusions were performed.The headache behavior of rats was observed.The expression of GFAP and Iba1 was detected by immunofluorescence and Western blot.The expression of EAAT2、NR1、NR2A and NR2 B was detected by Western blot.(6)Detection of IL-18 and IL-18 BP in peripheral blood: Ten migraine patients and 8 healthy controls were recruited at the same time.Elbow venous blood was extracted and serum was separated,and the expression levels of IL-18 and IL-18 BP were detected by ELISA.Results(1)Experimental models of migraine were successfully established:Inflammatory soup dural infusions significantly induced the development of face rubbing and tactile allodynia(P < 0.05).Inflammatory soup dural infusions significantly induced the upregulation of c-fos and CGRP in the medullary dorsal horn(P < 0.05).(2)Glial cells and IL-18 / IL-18 R signaling pathway were activated:Inflammatory soup dural infusions significantly induced the activation of microglia and astrocyte(P < 0.05).Inflammatory soup dural infusions significantly induced the upregulation of IL-18 and IL-18R(P < 0.05).(3)IL-18 / IL-18 R signaling pathway positively regulated headache behavior:IL-18 dural infusions produced nociceptive behavior(P < 0.05).IL-18 blockade attenuated nociceptive behavior induced by repeated inflammatory soup dural infusions,and inhibited the activation of c-fos and CGRP(P < 0.05).(4)IL-18/ IL-18 R signaling pathway mediated cross talk between microglia and astrocyte: Double immunofluorescence showed that IL-18 was expressed in microglia and IL-18 R was expressed in astrocytes.IL-18 dural infusions induced the activation of microglia and astrocyte(P < 0.05).IL-18 blockage significantly inhibited the activation of astrocytes(P < 0.05).IL-18 induced by inflammatory soup dural infusions was significantly alleviated by blocking TLR4.Double-labelling immunofluorescence revealed p-p38 entries in the nucleus of IL-18-immunopositive cells.Inflammatory soup dural infusions significantly induced the activation of NF-κB(P < 0.05).IL-18 blockage significantly inhibited NF-κB p65 activation induced by inflammatory soup dural infusions(P < 0.05).Double-labelling immunofluorescence showed that NF-κB was colocalized with IL-18 R.(5)IL-18/ IL-18 R signaling pathway regulated the expression of EAAT2 and NMDAR: Inflammatory soup dural infusions significantly reduced the expression of EAAT2(P < 0.05).EAAT2 was mainly expressed in astrocyte.NMDAR was expressed in neuron.Inflammatory soup dural infusions significantly induced the upregulation of NR1,NR2 A and NR2B(P < 0.05).IL-18 blockage significantly inhibited the down-regulation of EAAT2 and upregulation of NR1,NR2 A and NR2 B induced by inflammatory soup dural infusions(P < 0.05).IL-18 dural infusions reduced the expression of EAAT2 and induced the upregulation of NR1,NR2 A and NR2B(P < 0.05).(6)Il-18 and IL-18 BP expression levels in peripheral blood: ELISA showed no significant difference in IL-18 and IL-18 BP expression levels in peripheral blood between the migraine group and healthy controls.Conclusions(1)IL-18 / IL-18 R signaling pathway was involved in the pathogenesis of migraine.IL-18 dural infusions produced nociceptive behavior,while IL-18 blockade attenuated nociceptive behavior induced by repeated inflammatory soup dural infusions.It provided possible therapeutic targets of the migraine.(2)IL-18-mediated microglia/astrocyte interactions in the medullary dorsal horn might contribute to the development of hyperpathia or allodynia induced by migraines.Inflammatory soup dural infusions induced microglia-derived IL-18 upregulation via TLR4 / p38 MAPK.Microglia-derived IL-18 mediated the activation of astrocytes in a NF-κB-dependent manner.(3)EAAT2 was down-regulated in the activated astrocyte,resulting in excessive accumulation of glutamate in synaptic cleft and hyperactivation of neuron NMDAR.It in turn induced the formation of neuronal hyperexcitability and central sensitization,thus leading to migraine.(4)The clinical significance of peripheral blood IL-18 and IL-18 BP in migraine needed to be further explored by enlarging the sample size.