P2Y ₁₂ and P2Y ₁₃ receptors involved in ADPβs induced the release of IL-1β, IL-6 and TNF-α from cultured dorsal horn microglia

Abstract

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-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) in cultured dorsal spinal cord microglia. For this reason, we examined the effects of ADPβs (ADP analog) on the expression and the release of IL-1β, IL-6, and TNF-α.

Methods and results

In this study, we observed the effect of P2Y receptor agonist ADPβs on the expression and release of IL-1β, IL-6 and TNF-α by using real-time fluorescence quantitative polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). ADPβs induced the increased expression of Iba-1, IL-1β, IL-6 and TNF-α at the level of messenger RNA (mRNA). ADPβs-evoked increase in Iba-1, IL-1β, IL-6 and TNF-α mRNA expression was inhibited only partially by P2Y ₁₂ receptor antagonist MRS2395 or P2Y ₁₃ receptor antagonist MRS2211, respectively. Similarly, ADPβs-evoked release of IL-1β, IL-6 and TNF-α was inhibited only partially by MRS2395 or MRS2211. Furthermore, ADPβs-evoked increased expression of Iba-1, IL-1β, IL-6 and TNF-α mRNA, and release of IL-1β, IL-6 and TNF-α 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-1β, IL-6 and TNF-α were inhibited by Y-27632 (ROCK inhibitor), SB203580 (P38MAPK inhibitor) and PDTC (NF-κb inhibitor), respectively. Subsequently, P2Y ₁₂ and P2Y ₁₃ receptor-evoked release of IL-1β, IL-6 and TNF-α, 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-1β, IL-6 and TNF-α are associated with ROCK/P38MAPK/NF-κb signaling pathway.