The Role Of P2Y₁₂ And P2Y₁₃ Receptors In Foot Burn Injury-Induced Mechanical Pain And Dorsal Horn Microglial Function In Rats

Burn injury pain makes great harm to human beings.The various inflammatory or chemokines were released in the skin injured area after noxious thermal stimulation.Then,the nociceptors were excited on the sensory nerve ending in the epidermis and intradermis.Following the integration of the information in the spinal cord,then,it is transmitted to the primary sensory cortex.Increased excitability of neurons in sensory pathways was observed at the development of burn injury pain.The activation of spinal microglia plays an important role in the hyperalgesia induced by II and III degree burn injury.In II degree burn injury rat model,microglial inhibitor minocycline can significant suppress microglia activition and the expression of phosphorylated P38（phospho-P38）in the dorsal horn.The III degree burn induces an increased expression of COX-2,iNOS,p-NF-κB and the production of inflammatory factor in spinal cord,which suggest that the inflammatory reaction may involved in the occurrence and maintenance of burn injury pain.Previous studies have shown that P2Y₁₂2 and P2Y₁₃3 receptors are highly selectively expressed in dorsal horn microglia.However,it is unknown whether P2Y₁₂2 and P2Y₁₃receptors play a key role in microglia activation,phenotype transformation and inflammatory cytokine release in dorsal spinal cord of I degree burn injury rats.In the present study,we will use some scientific research methods to investigate the role of P2Y₁₂2 and P2Y₁₃3 receptors in I degree foot burn injury-induced mechanical pain and dorsal horn microglial activation in rats.Objective:1.The aim of this study is to explore the possible role of dorsal spinal cord P2Y₁₂2 and P2Y₁₃3 receptor in I degree burn injury-induced mechanical allodynia and dorsal spinal cord microglia functional changes.Methods:1.All rats were randomized into five groups（n=6）:sham group,burn injury group（intrathecal injection of 0.005%DMSO in saline）,MRS2395-treated group（intrathecal injection of MRS2395,100 nmol/L）,MRS2211-treated group（intrathecal injection of MRS2211,100 nmol/L）,MRS2395-and MRS2211-treated group（intrathecal co-injection of MRS2211 and MRS2395,100 nmol/L）.The right hind limb plantar skin of rats sustained a force of 1000g for 5 seconds at 85°C,resulting in I degree burn injury（confirmed by pathological section）.After 24 hours,the MWT（mechanical withdrawal threshold）decreased significantly,which suggest that the model was successful.The MRS2395,MRS2211 or 005%DMSO in saline were intrathecally administrated on 2h before burning and on 5,11,23 and 47h after burning.The MWT was evaluated on 1h after treating.The dorsal horn of lumbar enlargement of spinal cord(L_4-6)was harvested on24h after burn injury.The expression of Iba-1,IL-1β,IL-6,TNF-α,iNOS,CD86,Arg-1and CD206 mRNA were examined by RT-PCR.At the same time,the content of IL-1β,IL-6and TNF-αwere examined by ELISA.In addition,the expression of iNOS,Arg-1,ROCK-1,ROCK-2,P38MAPK and NF-κBp65 were examined by western blot.2.Primary cultures of microglia were prepared from dorsal spinal cord of new born SD rats（<3d）.The cells were seeded on 6-well plates（5×10⁷/well）for 48h.These cells were randomized into three groups（n=3）:normal group（high glucose medium）;ADPβS（10μmol/L,3h）group;MRS2395（10μmol/L,20min）+MRS2211（10μmol/L,20min）+ADPβS（10μmol/L,3h）group.The expression of iNOS,Arg-1,ROCK-1,ROCK-2,P38MAPK and NF-κBp65 were examined by western blot analysis.Result:1.Compare with sham group,burn injury induce the decreased MWT at 6,12 and 24h（P<0.05）,without affecting the MWT at 48h.Repeated intrathecal administration of MRS2395 or MRS2211 significantly ameliorated the decrease in MWT at 6,12 and 24h after burning injury（12h:P<0.05;24h:P<0.01）.Co-administration of MRS2395 and MRS2211 obvious ameliorated bury injury-induced mechanical hypersensitivity（12h and24h:P<0.01）.2.The RT-PCR results showed that,compared with sham group,the expression of Iba-1,IL-1β,IL-6,TNF-α,iNOS and CD86 mRNA were significantly increased in dorsal spinal cord at 24h after burn injury（P<0.01）,without affecting the expression of Arg-1 and CD206 mRNA.Compared with foot burn injury rats,intrathecal administration of MRS2395 or MRS2211 partially inhibited the increased expression of Iba-1,IL-1β,IL-6,TNF-α,iNOS and CD86 mRNA after burn injury（MRS2395:P<0.05;MRS2211:P<0.05）.After treatment with MRS2395 or MRS2211,the expression of Arg-1 and CD206 mRNA are increased（P<0.05）.Co-administration of MRS2395 and MRS2211 almost completely blocked the increased expression of Iba-1,IL-6,TNF-α,iNOS and CD86 mRNA after bury injury（P<0.01）.However,the expression of IL-1βmRNA was only partially inhibited after MRS2395 and MRS2211 co-administration（P<0.05）.At the same time,the expression of Arg-1 and CD206 mRNA were significantly up-regulated（P<0.01）.3.The ELISA results showed that,compared with sham group,the content of IL-1β,IL-6and TNF-αwere significantly increased in dorsal spinal cord of burn-injured rats（24h:P<0.01）.Compared with burn group,repeated intrathecal administration of MRS2395 or MRS2211 partially inhibited the increased content of IL-1β,IL-6 and TNF-αat 24h after burn injury（MRS2395:P<0.05;MRS2211:P<0.05）.Co-administration of MRS2395 and MRS2211 almost completely blocked the increased content of IL-6 and TNF-αafter bury injury（P<0.01）.However,the increased content of IL-1βwas only partially inhibited after MRS2395 and MRS2211 Co-administration（P<0.05）.4.WB results showed that foot burns induce the increased expression of iNOS,ROCK-1,ROCK-2,P38MAPK（P<0.05）and NF-κBp65（P<0.01）,without affecting the expression of Arg-1 in dorsal spinal cord of foot burn injury rats.Co-administration of MRS2395 and MRS2211 almost completely blocked bury injury-induced the increased expression of NF-κBp65（P<0.01）.However,the increased expression of iNOS,ROCK-1,ROCK-2,P38MAPK was only partially inhibited after MRS2395 and MRS2211 Co-administration（P<0.05）.Compared with foot burn injury rats,after Co-administration with MRS2395 and MRS2211,the expression of Arg-1 was significantly increased（P<0.01）.5.In vitro,WB results showed that ADPβS induced the increased expression of iNOS,ROCK-1,ROCK-2,P38MAPK（P<0.05）and NF-κBp65（P<0.01）,without affecting the expression of Arg-1 in cultured rat dorsal spinal cord microglia cells.Co-pretreatment of MRS2395 and MRS2211 almost completely blocked ADPβS-induced the increased expression of NF-κBp65（P<0.01）.However,the increased expression of iNOS,ROCK-1,ROCK-2 and P38MAPK was only partially inhibited after MRS2395 and MRS2211Co-administration（P<0.05）.Compared with ADPβS group,after Co-pretreatment with MRS2395 and MRS2211,the expression of Arg-1 was significantly increased（P<0.01）.Conclusion:1.The activation of P2Y₁₂/P2Y₁₃3 receptor in dorsal horn of spinal cord is involved in mechanical hyperalgesia induced by foot burns in rats.Intrathecal injection of MRS2395and/or MRS2211 exerts analgesic effect by inhibiting P2Y₁₂/P2Y₁₃3 receptor activation in dorsal spinal cord.2.The activation of P2Y₁₂/P2Y₁₃3 receptor in cultured dorsal spinal cord microglia cells can promote microglia activation and M1 polarization,which may be mediated through the activation of the ROCK/P38MAPK/NF-κB pathway.