Role Of P2Y₁₂/P2Y₁₃ Receptor In ADPβs-induced The Increased Expression And Release Of Inflammatory Cytokines In Cultured Dorsal Horn Microglia Cells

External noxious stimulation can activate microglia in the dorsal spinal cord,and activated microglia can release a variety of active and pain-related substances.Experimental studies have shown that spinal cord microglia cell surface receptors and intracellular kinase activations play important roles in cell activation and chronic pain.Dorsal horn P2Y₁₂ and P2Y₁₃ receptor is involved in the development of pain behavior induced by peripheral nerve injury.In some rat peripheral nerve injury models,the increased expression of P2Y₁₂ and P2Y₁₃ receptor in spinal cord microglia results in the P38 MAPK phosphorylation and pain behaviors.However,it is not known whether P2Y₁₂ and P2Y₁₃ receptor activation can influence the expression and the release of IL-1β、IL-6、TNF-α in cultured dorsal spinal cord microglia.For this reason,we examined the effects of ADPβs（ADP analogue）on the expression and the release of IL-1β、IL-6、TNF-α by using RTFQ-PCR and ELISA.The role of ROCK/ P38MAPK/NF-κB signaling pathway in this action was also explored.The research works can help us better understand the role of purinergicreceptor in the development of in the development of painhypersensitivity following peripheral nerve injury.Objective: 1.To explore whether P2Y₁₂ and P2Y₁₃ receptor activation can lead to the the increased expression and release of IL-1β,IL-6 and TNF-α in cultured dorsal horn microglia cells.2.To explore whether ROCK/P38MAPK/NF-κB signaling pathway in volved in P2Y₁₂ and P2Y₁₃ receptor-induced the production and release of these inflammatory cytokines.Methods: 1.Primary cultures of microglia were prepared from dorsal spinal cord of new born SD rats（<3d）.The cells were seeded on 12-well plates at a cell density of 5×105/well for 48 h.These cells were randomized into four groups（n=5）: normal group（intrathecal normal saline）,ADPβS（10μmol/L,3h）group,MRS2395（10μmol/L,20min）+ ADPβS（10μmol/L,3h）group;MRS2211（10μmol/L,20min）+ ADPβS;MRS2395（10μmol/L,20min）+ MRS2211（10μmol/L,20min）+ ADPβS（10μmol/L,3h）group.The effects of ADPβs（ADP analogue）on the expression and the release of IL-1β、IL-6、TNF-α by using RTFQ-PCR and ELISA.2.Primary cultures of microglia were prepared from dorsal spinal cord of newborn SD rats（<3d）.The cells were seeded on 12-well plates at a cell density of 5×105/well for 48 h.These cells were randomized into four groups（n=5）: normal group（intrathecal normal saline）,ADPβS（10μmol/L,3h）group,Y-27632（10μmol/L,1h）+ ADPβS（10μmol/L,3h）group;SB203580（20μmol/L,2h）+ ADPβS;PDTC（20μmol/L,4h）+ADPβS（10μmol/L,3h）group.The expression of Iba-1,IL-1β,IL-6 and TNF-α m RNA in cultured dorsal spinal cord microglia were observed by using realtime fluores-cence quantitative PCR（FQRT,PCR）.ADPβs-induced IL-1β,IL-6 and TNF-α release from microglia cells were measured by ELISA assay.Results: 1.In cultured spinal dorsal horn microglia cells,stimulation of microglia cells with 10 μM ADPβS for 3h led to a robust increase of Iba-1 m RNA（P<0.01）.Both MRS2395 and MRS2211 show a moderate degree of inhibition with ADPβS stimulatory effect on the increased gene expression（MRS2395: P<0.05;MRS2211: P<0.05）.ADPβs-evoked the increased expression of Iba-1 were nearly all blocked after co-administration of MRS2395 plus MRS2211（P<0.01）.2.In cultured spinal dorsal horn microglia cells,stimulation of microglia cells with 10 μM ADPβS for 3h led to the increase of IL-1β,IL-6 and TNF-α m RNA（P<0.01）.Both MRS2395 and MRS2211 show a moderate degree of inhibition with ADPβS stimulatory effect on the increased gene expression（MRS2395:P<0.05;MRS2211:P<0.05）.ADPβs-evoked the increased expression of IL-1β,IL-6 and TNF-α m RNA were nearly all blocked after co-administration of MRS2395 plus MRS2211（P<0.01）.3.In cultured spinal dorsal horn microglia cells,stimulation of microglia cells with 10 μM ADPβS for 3h led to the release of IL-1β,IL-6 and TNF-α（P<0.01）.Both MRS2395 and MRS2211 show a moderate degree of inhibition with ADPβS stimulatory effect on the inflammatory cytokines release（MRS2395: P<0.05;MRS2211: P<0.05）.ADPβs-evoked the release of IL-1β,IL-6 and TNF-α were nearly all blocked after co-administration of MRS2395 plus MRS2211（P<0.01）.4.In cultured spinal dorsal horn microglia cells,RTFQ-PCR results show the ADPβS-induced the increased expression of IL-1β,IL-6 and TNF-α m RNA（P<0.01）。Administration of Y-27632（10μmol/L）、P38MAPK（20μmol/L）and PDTC（20μmol/L）significantly suppressed the increased expression of of expression of IL-1β,IL-6 and TNF-α m RNA,which indicats that ROCK/ P38MAPK/NF-κB signaling pathway maybe involved in this process（Y-27632: P﹤0.05;SB203580: P﹤0.05;PDTC: P﹤0.01）.5.In cultured spinal dorsal horn microglia cells,ELISA results show the ADPβS（10μM）-induced the IL-1β,IL-6 and TNF-α release from cultured spinal dorsal horn microglia cells by using ELISA technique.Administration of Y-27632（10μmol/L）、P38MAPK（20μmol/L）and PDTC（20μmol/L）significantly suppressed IL-1β,IL-6 and TNF-α release from microglia cells,which indicats that ROCK/P38MAPK/NF-κB signaling pathway maybe involved in this process（Y-27632: P﹤0.05;SB203580: P﹤0.05;PDTC: P﹤0.01）.Conclusion: 1.P2Y₁₂ and P2Y₁₃ receptor are participates in ADPβS-evoked the expression and the release of IL-1β、IL-6、TNF-α from cultured dorsal spinal cord microglia cells.2.P2Y₁₂ and P2Y₁₃ receptor-mediated ROCK/P38MAPK/NF-k B signaling leads to the increased expression and the release of IL-1β、IL-6 and TNF-α from cultured dorsal spinal cord microglia cells.