The Analgesic Effect And Mechanism Of AM1241 To CCI Rats By P2Y₁₃ Receptor In The Spinal Cord

Neuropathic pain is a maladaptive and chronic condition that can develop after a lesion or disease affecting the somatosensory system.After peripheral nerve injury,spinal cord microglia cells are activated and realease a variety of cytokines and chemokines.The endocannabinoid system exists in mammals and it consists of cannabiniod receptors and the endogenous ligands.Cannabinoid receptor mainly includes CB1 receptor and CB2 receptor subtype.The CB2 receptor is highly expressed in spinal cord microglia and is responsible for cannabinoid-mediated analgesic effect.ATP and its receptor play an important role in the transmission and modulation of nociceptive information.Among them,P2Y₁₃ receptor is highly expressed in spinal cord microglia and participates in the pain symptom after peripheral nerve injury.It is not clear whether activated CB2 receptor exert analgesic effect by inhibiting the activation of p38 MAPK and NF-κB signaling pathway and thereby downregulating the expression of P2Y₁₃ receptor.Chronic constriction injury（CCI）of the sciatic nerve is one of the most commonly used methods to induce chronic neuropathic pain.Thererore,CCI models in rats are made,to speculate:1)pain behavior in CCI rats;2)the effect of intrathecal injection of AM1241 on the pain behavior of CCI rats and the expression of P2Y₁₃ receptor,phosphorylation of p38-mitogen activated protein kinases（P38MAPK）,nuclear factor κBp65（NF-κBp65）in dorsal spinal cord.3)The effect of intrathecal injection of P38 MAPK phosphorylation inhibitor SB203580 or the NF-κB signal inhibitor PDTC on the pain behavior and the expression of P2Y₁₃ receptor in dorsal horn spinal cord of ADPβS-treated rats.Our findings may provide open new sight for deep understanding the mechanism that CB2 receptor are involved in the antiallodynic effect induced by AM1241 and will provide a theoretical basis for the analgesic drugs targeting CB2 receptor.Objective:1.To explore whether P2Y₁₃ receptor in dorsal horn microglia is involved in AM1241 analgesic effect on CCI rats.2.To explore whether CB2 receptor exert analgesic effect by inhibiting P38 MAPK phosphorylation or the expression of NF-κBp65,which suppress the upregulation of P2Y₁₃ receptor in dorsal spinal cord after nerve injury（CCI）.Methods:1.All rats were randomized into three groups（n=8）: sham group（sham operation）,CCI group（CCI+intrathecal 0.005%DMSO in saline）,CCI+AM1241（100p M）.For the rats after ligation,AM1241 or 0.005% DMSO in saline（10μL）were intrathecally administered twice a day for 14 d.TWL were evaluated before the surgey and on 1,3,5,7,10 d and 14 d after surgery.The expression of P2Y₁₃ receptor in dorsal horn was assessed by using realtime fluorescence quantitative PCR（RTFQ-PCR）and WB（western blots）on 3,7,14 post-operative days and the expression of p-P38 MAPK,NF-κBp65 by using WB.2.All rats were randomized into four groups（n=8）: sham group（sham operation）,CCI group（CCI+intrathecal 0.005%DMSO in saline）,CCI+SB203580（50μM）,CCI+PDTC（5μg/10μL）.For the rats after ligation,SB203580,PDTC or 0.005% DMSO in saline（10μL）were intrathecally administered twice a day for 14 d.TWL were evaluated before the surgey and on 1,3,5,7,10,14 d after surgery.The expression of P2Y₁₃ receptor in dorsal horn was assessed by RTFQ-PCR and WB on 3,7d post-operative days.3.All rats were randomized into four groups（n=8）: normal group（intrathecal 0.01 M PBS）,ADPβS-treated group（intrathecal 100μM ADPβS）,ADPβS+SB203580 group,ADPβS+PDTC group.SB203580（50μM）、 PDTC（5μg/10μL）or saline（10μL）were intrathecally administered twice a day for 7d.TWL were evaluated before intrathecal ADPβS and on 1,3,5,7d after intrathecal ADPβS.The expression of P2Y₁₃ receptor in dorsal horn was assessed by using RTFQ-PCR and WB on 7d after ADPβS administration.Results:1.TWL in CCI+0.005%DMSO group were significantly lower than sham group after surgey.Repeated intrathecal administration of AM1241 markedly suppressed this decrease in TWL after nerve injury.RTFQ-PCR and WB results showed that the expression of P2Y₁₃ receptor were markedly enhanced in the ipsilateral dorsal horn on 3,7,14 days after nerve injury.Compared with CCI+0.005%DMSO group,after AM1241 adiministration,the expression of P2Y₁₃ receptor were significantly suppressed on days3,7 and 14.2.WB results showed that the expression of p-P38 MAPK and NF-κBp65 in CCI+0.005%DMSO group were markedly enhanced in the ipsilateral dorsal horn on 3,7 days after nerve injury.Compared with CCI+0.005%DMSO group,after AM1241 adiministration,the expression of P2Y₁₃ receptor were significantly suppressed on days3 and 7.3.Compared with CCI+0.005%DMSO group,repeated intrathecal administration of SB203580 and PDTC markedly suppressed this decrease in TWL after nerve injury.RTFQ-PCR and WB results showed that the expression of P2Y₁₃ receptor were significantly suppressed on days 7 after repeated intrathecal administration of SB203580 and PDTC.4.TWL in ADPβS-treated group were significantly lower than normal group after surgey.Compared with ADPβS-treated group,repeated intrathecal administration of AM1241,SB203580 or PDTC markedly suppressed this decrease in TWL.RTFQ-PCR and WB results showed that the expression of P2Y₁₃ receptor were markedly enhanced on days 7in ADPβS-treated group.Compared with ADPβS-treated group,after AM1241,SB203580 or PDTC adiministration,the expression of P2Y₁₃ receptor were significantly suppressed on days 7.Conclusion:1.P2Y₁₃ receptor in dorsal horn microglia is involved in AM1241 analgesic effect on CCI rats.2.CB2 receptor stimulation inhibits the expression of P2Y₁₃ receptor via P38MAPK/NF-κB signaling.