The Mechanisms Of Protease Activated Receptor 2 In Mediating Pain Signaling Through Nonselective Cation Channels

Proteinase-activated receptor 2(PAR2)is a widely distributed G protein-coupled receptor(GPCR)whose activation triggers a series of signal transduction pathways.Transient receptor potential cation channel vanilloid 1(TRPV1)and transient receptor potential ankyrinl(TRPA1)are both classical physical and chemical stimuli receptors that play an important role in the process of pain transmission.Purinergic receptor P2X3 is a new pain-related molecule in recent years.To determine the relationship between the activation of PAR2 and its downstream signaling pathways and the expression of nonselective cation channel P2X3,TRPV1,and TRPA1 in dorsal root ganglion(DRG)neurons in the development of the pain,neuropathic pain rat model induced by PAR2 agonist SL-NH2 and cultured DRG neurons were used to approach these questions.The results of the present study will provide new clues for interpreting the mechanisms of neuropathic pain and new evidence for relieving neuropathic pain.Part Ⅰ The actions of nonselective cation channels in PAR2-mediated neuropathic pain in ratsTo explore the relationship between the activation of PAR2 and its downstream signaling pathways and nonselective cation channel P2X3,TRPV1,and TRPA1 in DRG neurons in the development of the neuropathic pain,the neuropathic pain rat model was established by injection of PAR2 agonist SL-NH2 in the hindpaw.The mechanical allodynia and thermal hyperalgesia were examined.The expression of nonselective cation channel P2X3,TRPV1,and TRPA1 in DRG neurons and c-Fos in spinal dorsal horn(SDH)were detected at mRNA level,protein level,and in situ in DRG neurons.The phosphorylation of PAR2’s downstream key molecule PLCβ3 and PKCε in DRG neurons were also determined.The results showed that the thresholds of mechanical stimulation and thermal stimulation were reduced after injection of PAR2 agonist SL-NH2 from 0.5 h to 3 d.The c-Fos expression was upregulated in SDH at 3 d after SL-NH2 injection.The expression of P2X3,TRPV1,TRPA1,and PAR2 was upregulated in DRG at 3 d after SL-NH2 injection.And also,the proportion of P2X3,TRPV1,TRPA1,and PAR2 positive neurons elevated in DRG.Interestingly,the proportion of P2X3,TRPV1,and TRPA1 positive neurons in PAR2 positive neurons also elevated.The protein levels of pPLCβ3 and pPKCεwere also upregulated in DRG at 3 d after SL-NH2 injection.These results imply that activation of PAR2 and its downstream signaling pathways could induce persistent neuropathic pain.The development of neuropathic pain is related to the expression of nonselective cation channel P2X3,TRPV1,and TRPA1 in DRG neurons.These data provide new clues and evidence for relieving neuropathic pain by interfering nonselective cation channel P2X3,TRPV1,and TRPA1 in the status of PAR2 activation.Part Ⅱ The effects of PAR2 activation on expression of nonselective cation channels in cultured DRG neuronsAccording to the research progress and the results from the rat model of the present study,the relationship between PAR2 and nonselective cation channel P2X3,TRPV1,and TRPA1 need to be further investigated.In this situation,primary cultured DRG neurons with PAR2 agonist SL-NH2 challenge was studied by determining the expression of nonselective cation channel P2X3,TRPV1,and TRPA1.The results showed that the expression of P2X3,TRPV1,TRPA1,and PAR2 was upregulated after SL-NH2 stimulation.The proportion of P2X3,TRPV1,TRPA1,and PAR2 positive neurons elevated in DRG neurons.The proportion of P2X3,TRPV1,and TRPA1 positive neurons in PAR2 positive neurons also elevated.The protein levels of pPLCβ3 and pPKCε in DRG neurons were also upregulated.These results imply that activation of PAR2 and its downstream signaling pathways involved in the expression of nonselective cation channels.These results imply that activation of PAR2 may be through its downstream signaling pathway PIP2 to induce P2X3,TRPV1,and TRPA1 expression.By targeting one of these molecules may be a novel therapeutic approach.