P2Y₁₂ and P2Y₁₃ 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 P2Y₁₂ and P2Y₁₃ receptors are involved in the development of pain behavior induced by peripheral nerve injury. However, it is not known whether P2Y₁₂ and P2Y₁₃ 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 P2Y₁₂ receptor antagonist MRS2395 or P2Y₁₃ 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 P2Y₁₂ and P2Y₁₃ 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, P2Y₁₂ and P2Y₁₃ 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 P2Y₁₂ and P2Y₁₃ receptor-evoked gene expression and release of IL-1β, IL-6 and TNF-α are associated with ROCK/P38MAPK/NF-κb signaling pathway.

Keywords:

• glial activation
• P2 receptor
• NF-kB
• p38 mitogen-activated protein kinase

Acknowledgments

This work is supported by the National Natural Science Foundation of China (No. 31460266 and 31640040). Program for New Century Excellent Talents in University (NCET-13–1070), Scientific Research Foundation for Excellent Talents in Guizhou Province (2012–93). We are very grateful to the other staff of Department of Physiology.

Disclosure

The authors report no conflicts of interest in this work.