The Effect Of CB2 Receptors Activation On Purine P2X₄ And P2X₇ Receptors Expression In Dorsal Spinal Cord Of CCI Rats

Objective:1.To explore whether P2X₄ and P2X₇ receptors in dorsal spinal cord are involved in AM1241-mediated analgesic effect in CCI rats.2.To explore the role of miR-124-3p in CB2 receptors activation-induced the decreased P2X₄ and P2X₇ receptors expression in dorsal spinal cord of CCI rats.Methods:1.All rats were randomized into groups（n=8）:sham group,CCI group,CCI+intrathecal 0.005%DMSO group,AM1241 group,Y-27632 group,SB203580 group,PDTC group.For the rats after ligation,AM1241,Y-27632,SB203580 and PDTC were intrathecally administered twice a day for 7d.MWT were evaluated before the surgey and on 1,3,5,7d after surgery.Evaluate the expression of CB2,P2X₄,P2X₇,RhoA,ROCK2,p-P38MAPK,p-NF-κBp65, BDNF,IL-18 and TNF-αon 7 post-operative days.2.All rats were randomized into groups（n=8）:sham group,sham+intrathecal agomir NC group,sham+intrathecal antagomir NC group,CCI+intrathecal saline group,miR-124-3p agomir group,miR-124-3p antagomir group,AM1241 group,miR-124-3p agomir+AM1241 group,miR-124-3p antagomir+AM1241 group.For the rats after ligation,agomir/antagomir NC,miR-124-3p agomir/antagomir and AM1241 were intrathecally administered twice a day for 7d.Evaluate the expression of miR-124-3p,P2X₄,P2X₇,RhoA,ROCK2,p-NF-κBp65,BDNF,IL-18 and TNF-α.3.All rats were randomized into groups（n=6）:sham group,CCI+intrathecal 0.005%DMSOgroup,AM1241 group,AM630+AM1241 group.AM1241 and AM630 were intrathecally administered twice a day for 7d.Evaluate the expression of miR-124-3p,P2X₄ and P2X₇ receptors.Results:1.MWT in CCI group were significantly lower than sham group after surgey（P<0.05）.Repeated intrathecal administration of AM1241,Y-27632,SB203580 and PDTC markedly suppressed this decrease in MWT than CCI group on 1,3,5,7 days after nerve surgey（P<0.05）.Compared with sham group,the expression of CB2,P2X₄,P2X₇,RhoA,ROCK2,p-P38MAPK, p-NF-κBp65,BDNF,IL-18 and TNF-αin CCI group were markedly enhanced（P<0.05）. Compared with CCI group,the expression of CB2 receptors were enhanced after AM1241 adiministration（P<0.05）,however,the expression of P2X₄ and P2X₇ receptors were suppressed after AM1241,Y-27632,SB203580 and PDTC adiministration（P<0.05）.Compared with CCI group,the expression of RhoA,ROCK2,p-P38MAPK,p-NF-κBp65,BDNF,IL-18 and TNF-α were significantly suppressed after AM1241 adiministration（P<0.05）.2.MWT in CCI group were significantly lower than sham group after surgey（P<0.05）.After miR-124-3p agomir treatment,the MWT measured on days 3,5 and 7 were significantly higher than these vehicle-treated CCI rats（P<0.05）.After miR-124-3p antagomir treatment,the MWT measured on days 5 and 7 were significantly lower than these miR-124-3p agomir-treated CCI rats（P<0.05）.AM1241-treated（100pM）CCI rats displayed significantly increased MWT compared with CCI group（P<0.05）,which was markedly enhanced by pretreatment with miR-124-3p agomir（P<0.05）.AM1241-induced the increased MWT was significantly reversed by pretreatment with miR-124-3p antagomir（P<0.05）.CCI-induced the increased expression of P2X₄,P2X₇,RhoA,ROCK2,p-P38MAPK and p-NF-κBp65 were significantly attenuated in rats treated with miR-124-3p agomir on day 7 after CCI（P<0.05）.MiR-124-3p antagomir application slightly enhanced the increased expression of P2X₄,P2X₇,RhoA,ROCK2, p-P38MAPK and p-NF-κBp65 in CCI rats than the miR-124-3p agomir group（P<0.05）. MiR-124-3p agomir pretreatment obvious enhanced AM1241-induced the decreased expression of P2X₄,P2X₇,RhoA,ROCK2,p-P38MAPK,p-NF-κBp65,BDNF,IL-18 and TNF-αin dorsal spinal cord on day 7 after CCI（P<0.05,compare with AM1241-treated CCI rats）.On the contrast,AM1241-induced the decreased expression of P2X₄,P2X₇,RhoA,ROCK2, p-P38MAPK,p-NF-κBp65,BDNF,IL-18 and TNF-αwere significantly reversed after miR-124-3p antagomir pretreatment（P<0.05,compare with AM1241-treated CCI rats）.3.MWT in CCI group were significantly lower than sham group after surgey（P<0.05）.Compared with CCI group,repeated intrathecal administration of AM1241 suppressed this decrease in MWT（P<0.05）,and the effect was reversed by the AM630（P<0.05,compare with AM1241-treated CCI rats）.AM1241 treatment increased the expression of miR-124-3p and suppressed the expression of P2X₄ and P2X₇ receptors on day 7 after CCI（P<0.05,compare with CCI rats）.Besides,repeated AM630 pretreatment obviously reversed the AM1241-induced the increased expression of miR-124-3p and the decreased expression of P2X₄ and P2X₇ receptors（P<0.05,compare with AM1241-treated CCI rats）.Conclusion:1.CB2 receptor activation significantly inhibited peripheral nerve injury-induced mechanical hyperalgesia and the increased expression of P2X₄ and P2X₇ receptors may through RhoA/ROCK/P38MAPK/NF-κB signaling in dorsal spinal cord of CCI rats.2.CB2 receptor/miRNA-124-3p pathway may involved in AM1241-induced the down regulation of RhoA/ROCK/P38MAPK and NF-κB activation and the decreased expression of P2X₄ and P2X₇ receptors expression.