Research About The Modulatory Role Of TRPM8 Membrane Trafficking And P2Y1R On NMDA Receptor Phosphorylation In Dorsal Root Ganglion In Remifentanil-induced Hyperalgesia

Objective Explore the modulatory role of TRPM8 membrane trafficking and P2Y1R on NMDA receptor phosphorylation in dorsal root ganglion(DRG)in remifentanil-induced hyperalgesia(RIH).Contents Investigate the changes of TRPM8 membrane trafficking,NMDA receptor phosphorylation and P2Y1R expression in DRG in rats with RIH,and demonstrate the concrete roles of TRPM8 membrane trafficking and P2Y1R in the mechanism of RIH and the mediating role of NMD A receptor phosphorylation.Methods Part Ⅰ:Remifentanil was infused for 60 min at a rate of 1 μg·kg-1·min-1 in group R and NS was infused in group C.MWT,TWL and NPL were measured at T0-4.L4-6 DRGs were dissected for detecting TRPM8,NR1,NR2B and P2Y1R mRNA by RT-qPCR,tTRPM8,mTRPM8,pNRl,NR1,pNR2B,NR2B and P2Y1R by western blot,and m/t TRPM8 was calculated.Experiment I of Part II:NS or RQ-00203078 was injected in group C and RQ,and NS was infused for 60 min.NS or RQ-00203078 were injected in group R and R+RQ,and remifentanil was infused for 60 min.MWT,TWL and NPL were measured at T0-4.L4-6 DRGs were dissected for detecting NR1 and NR2B mRNA by RT-qPCR,pNRl,NR1,pNR2B and NR2B by western blot,and percentages of pNRl and pNR2B-IR by immunofluorescence.Experiment Ⅱ of Part Ⅱ:NS was injected in group NS+NS.NS and RQ-00203078 were injected in group NS+RQ.NMD A and NS were injected in group NMDA+NS.NMD A and RQ-00203078 were injected in group NMDA+RQ.Remifentanil was infused.NPL was measured at T0-4.Experiment I of Part III:NS or MRS2179 was injected in group C and MRS,and NS was infused for 60 min.NS or MRS2179 was injected in group R and R+MRS,and remifentanil was infused for 60 min.MWT,TWL and NPL were measured at T0-4.L4-6 DRGs were dissected for detecting NR1 and NR2B mRNA by RT-qPCR,P2Y1R,pNRl,NR1,pNR2B and NR2B by western blot,and percentages of pNRl and pNR2B-IR by immunofluorescence.Experiment II of Part Ⅲ:NS was injected in group NS+NS.NS and MRS2179 were injected in group NS+MRS.NMD A and NS were injected in group NMDA+NS.NMD A and MRS2179 were injected in group NMDA+MRS.Remifentanil was infused.MWT,TWL and NPL were measured at T0-4.Results Part Ⅰ:Compared with group C,MWT was decreased,TWL was shortened,NPL was elevated,TRPM8,NR1,NR2B and P2Y1R mRNA were increased,tTRPM8,mTRPM8,m/t TRPM8,pNRl,NR1,pNR2B,NR2B and P2Y1R protein were upregulated at T1-4 in group R.In group R,MWT was gradually decreased,TWL was gradually shorten,NPL was gradually elevated,TRPM8,NR1,NR2B and P2Y1R mRNA were gradually upregulated,tTRPM8,mTRPM8,m/t TRPM8,pNR1,NR1,pNR2B,NR2B and P2Y1R protein were gradually increased.Experiment Ⅰ of Part Ⅱ:Compared with group C,MWT was decreased,TWL was shortened,and NPL was elevated in group R at T1-4.Compared with group R,NPL was decreased,but differences in MWT and TWL were not significant in group R+RQ at T1-4.Compared with group C,NR1 and NR2B mRNA,pNRl,NR1,pNR2B and NR2B protein,and percentages of pNR1 and pNR2B-IR were increased in group R.Compared with group R,NR1 and NR2B mRNA,pNRl,NR1,pNR2B and NR2B protein,and percentages of pNR1 and pNR2B-IR were decreased in group R+RQ.Experiment Ⅱ of Part Ⅱ:Compared with group NS+NS,NPL was decreased in group NS+RQ,NPL was increased in group NMDA+NS and NMDA+RQ at T1-4.Compared with group NMDA+NS,difference in NPL was not significant in group NMDA+RQ at T1-4.Experiment Ⅰ of Part Ⅲ:Compared with group C,MWT was decreased,TWL was shorten,NPL was elevated in group R at T1-4.Compared with group R,MWT was increased,TWL was prolonged and NPL was decreased in group R+MRS at T1-4.Compared with group C,NR1 and NR2B mRNA,pNRl,NR1,pNR2B and NR2B protein,and percentages of pNR1 and pNR2B-IR were increased in group R.Compared with group R,NR1 and NR2B mRNA,pNR1,NR1,pNR2B and NR2B protein,and percentages of pNR1 and pNR2B-IR were decreased in group R+MRS.Experiment Ⅱ of Part Ⅲ:Compared with group NS+NS,MWT was increased,TWL was prolonged and NPL was decreased in group NS+MRS,MWT was decreased,TWL was shortened and NPL was increased in group NMDA+NS and NMDA+MRS at T1-4..Compared with NMDA+NS,differences in MWT,TWL and NPL were not significant in group NMDA+MRS at T1-4.Conclusion Inhibiting TRPM8 membrane trafficking attenuated remifentanil-induced cold hyperalgesia by decreasing phosphorylation of NR1 and NR2B.P2Y1R antagonist attenuated remifentanil-induced mechanical allodynia,heat and cold hyperalgesia via downregulating phosphorylation of NR1 and NR2B.