P2Y12 and P2Y13 receptors involved in ADPβs induced the release of IL-1β, IL-6 and TNF-α from cultured dorsal horn microglia

Objective: P2 receptors have been implicated in the release of neurotransmitter and pro-inflammatory cytokines due to their response to neuroexcitatory substances in the microglia. Dorsal horn P2Y(12) and P2Y(13) receptors are involved in the development of pain behavior induced by peripheral nerve injury. However, it is not known whether P2Y(12) and P2Y(13) receptors activation is associated with the expression and the release of interleukin-1B (IL-1 beta), interleukin-6 (IL-6), tumor necrosis factor-a (TNF-alpha) in cultured dorsal spinal cord microglia. For this reason, we examined the effects of ADP beta s (ADP analog) on the expression and the release of IL-1 beta, IL-6, and TNF-alpha. Methods and results: In this study, we observed the effect of P2Y receptor agonist ADP beta s on the expression and release of IL-1 beta, IL-6 and TNF-alpha by using real-time fluorescence quantitative polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). ADP beta s induced the increased expression of Iba-1, IL-1 beta, IL-6 and TNF-alpha at the level of messenger RNA (mRNA). ADPs-evoked increase in I beta a-1, IL-1 beta, IL-6 and TNF-a mRNA expression was inhibited only partially by P2Y(12) receptor antagonist MRS2395 or P2Y(13) receptor antagonist MRS2211, respectively. Similarly, ADP beta s-evoked release of IL-1 beta, IL-6 and TNF-alpha was inhibited only partially by MRS2395 or MRS2211. Furthermore, ADP beta s-evoked increased expression of Iba-1, IL-1 beta, IL-6 and TNF-alpha mRNA, and release of IL-1 beta, IL-6 and TNF-alpha were nearly all blocked after co-administration of MRS2395 plus MRS2179. Further evidence indicated that P2Y(12) and P2Y(13) receptor-evoked increased gene expression of IL-1 beta, IL-6 and TNF-alpha were inhibited by Y-27632 (ROCK inhibitor), SB203580 (P38MAPK inhibitor) and PDTC (NF-kappa b inhibitor), respectively. Subsequently, P2Y(12) and P2Y(13) receptor-evoked release of IL-1 beta, IL-6 and TNF-alpha, were also inhibited by Y-27632, SB203580 and PDTC, respectively. Conclusion: These observations suggest that P2Y(12) and P2Y(13) receptor-evoked gene expression and release of IL-1 beta, IL-6 and TNF-alpha are associated with ROCK/P38MAPK/NF-kappa b signaling pathway.