P2X7Receptor Paiticipates In The Activation Of NALP3Inflammasome In Trigeminal Ganglia Of Dural Inflammatory Stimulirats

Background:Migraine is a disabling brain disorder affecting around12%of the general population. It recurs as attacks of unilateral throbbing headache, worsened by movements and routine daily activities. Associated symptoms include nausea, vomiting and increased sensitivity to light and sounds. The trigeminovascular system (TGVS) constituted by the trigeminal afferents innervating the meningeal blood vessels is a major site inmigraine pathophysiology. Pain in migraine results from activation of the TGVS, which also leads to releaseof neuropeptidescontained intheir peripheral nerve endings and the neurogenicinflammation. The inflammation can lead to the occurrence of long term potentiation in dural nociceptors. Patients show severe unilateral headache in clinical. This phenomenon is now considered as peripheral sensitization in the primary nociceptive neurons.The inflammasome, a multi-protein complex, whichcan induce maturation and secretion of inflammatory cytokines interleukin (IL)-1β and IL-18is assembled in cellswhen subjected to exogenous and endogenous stimulation. Mature IL-1β causes a series of inflammatory reactions which lead to peripheral sensitization of migraine by the activation of the trigeminal ganglion satellite glial cells. There are four types of inflammasome:NALP1, NALP3, NLRC4and AIM2.The most important type is NALP3. Studies have demonstrated that activation of inflammasome is mainly through P2X7receptor, reactive oxygen species(ROS)and cathepsin.But now researchs on NALP3inflammasome participated in peripheral sensitization of migraine and activation pathway involved are rare.Therefore,through establishment of the model of dural inflammatory stimuli rat (migraine model common used),we hypothesize that NALP3inflammasome can be observed in the model of dural inflammatory stimuli rat.And P2X7R may participates in the activation of NALP3.Objective:To explore whether purinergic receptorP2Xligand-gated ion channel7(P2X7R) is involved in NALP3activation intrigeminal ganglia of dural inflammatory stimuli rats by using the rat model of dura inflammatory stimulus.Methods:There are42male SD rats.27rats wereused forimmunoblottingexperiments.They were randomlydividedinto9groups(each group n=3):blank group、inflammatorysoup3h group、inflammatorysoup6h group、 inflammatorysoup1d group、inflammatorysoup2d group、inflammatorysoup3d group、saline group、inflammatorysoup2d control group、brilliant Blue G (BBG) treatment group.15rats wereused forimmunofluorescenceexperiments.They were randomly dividedinto3groups(each group n=5):blank group、saline group、 inflammatorysoup2dgroup.Observe the P2X7R and NALP3expression in trigeminal ganglion in each group.And aftertreatment of P2X7receptor specific antagonistBrilliant BlueG (BBG)、observe the P2X7R andNALP3expression in each group of rats.Result:①Immunofluorescence double staining showed that:NALP3was found to locatein trigeminal neurons.The P2X7R was expressed mainly in thesatellite glial cell.And compared with the blank group and saline group,fluorescence intensity ofNALP3and P2X7R in inflammatorysoup2d group was significantly higher.②Immunoblottingshowedthat:NALP3、P2X7Rproteinexpression of eachinflammatorygroup was significantlyhigher than the blankgroup(P<0.05).P2X7R and NALP3proteinexpression of BBG treatmentgroupwas significantlylower than that ofinflammatory2dgroup and saline group (P<0.05).Conclusion:In a rat model of dura inflammatory stimuli, activation ofP2X7Rand NALP3canbe observed.In addition, P2X7R specific antagonistBBG can significantlyinhibite NALP3inflammasome.This shows that P2X7R participates in the activation of NALP3.