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Updated: Aug 1, 2025
Two-photon Imaging of Microglial Processes' Attraction Toward ATP or Serotonin in Acute Brain Slices
Published on: January 31, 2019
P2X and P2Y receptor antagonists reduce inflammation in ATP-induced microglia
Amer Imraish1, Tuqa Abu-Thiab2, Hana Hammad3
1Department of Biological Sciences, School of Science, The University of Jordan, Amman, Jordan. a.imraish@ju.edu.jo.
Background:
P2 receptors have been implicated in the release of neurotransmitter and pro-inflammatory cytokines due to their response to neuro-excitatory substances in the microglia. The P2X4, P2X7 and P2Y12 receptors are involved in the development of pain behavior induced by peripheral nerve injury. However, it is not known if blocking P2X4, P2X7 and P2Y12 receptors is associated with the expression and the release of interleukin-1B (IL-1β), interleukin-6 (IL-6), or tumor necrosis factor-α (TNF-α) in cultured neonatal spinal cord microglia.
Objective:
For this reason, we examined the effects of P2X4, P2X7 and P2Y12 antagonists on the expression and the release of IL-1β, IL-6, and TNF-α in ATP-stimulated microglia.
Methods:
In this study, we observed the effect of A-740003, PSB-12062 and MRS 2395 (P2X4, P2X7 and P2Y12 receptors antagonist, respectively), 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).
Results:
ATP induced the increased expression of IL-1β, IL-6 and TNF-α at the level of messenger RNA (mRNA). ATP-evoked increase in IL-1β, IL-6 and TNF-α mRNA expression was inhibited by the P2X4 receptor antagonist A-740003 or P2X7 receptor antagonist PSB-12062, respectively. Similarly, ATP-evoked release of IL-1β, IL-6 and TNF-α was inhibited by A-740003 and PSB-12062. Furthermore, ATP-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 A-740003 plus PSB-12062. Finally, ATP-evoked increased gene expression and release of IL-1β, IL-6 and TNF-α were also inhibited by MRS 2395 (P2Y12 antagonist).
Conclusion:
These observations suggest a new clue for therapeutic strategies to treat the neuro-inflammation.
Insights
Blocking P2X4, P2X7, and P2Y12 receptors in microglia reduces the release of inflammatory cytokines like IL-1β, IL-6, and TNF-α. This finding offers new therapeutic strategies for neuro-inflammation.
Area of Science:
- Neuroscience
- Immunology
- Pharmacology
Background:
- P2 receptors on microglia are involved in neurotransmitter and pro-inflammatory cytokine release.
- P2X4, P2X7, and P2Y12 receptors play a role in pain behavior following nerve injury.
- The impact of blocking these specific P2 receptors on cytokine expression and release in microglia remains unclear.
Purpose of the Study:
- To investigate the effects of P2X4, P2X7, and P2Y12 antagonists on the expression and release of IL-1β, IL-6, and TNF-α in cultured microglia stimulated with ATP.
- To determine if specific receptor antagonists can modulate inflammatory responses in microglia.
Main Methods:
- Utilized real-time fluorescence quantitative PCR to measure mRNA expression of cytokines.
- Employed enzyme-linked immunosorbent assay (ELISA) to quantify cytokine release.
- Tested specific antagonists: A-740003 (P2X4), PSB-12062 (P2X7), and MRS 2395 (P2Y12).
Main Results:
- ATP stimulation increased IL-1β, IL-6, and TNF-α mRNA expression and release.
- P2X4 and P2X7 antagonists (A-740003 and PSB-12062) significantly inhibited ATP-evoked cytokine mRNA expression and release.
- The P2Y12 antagonist (MRS 2395) also inhibited ATP-evoked cytokine expression and release, with combined P2X4/P2X7 antagonism showing near-complete blockade.
Conclusions:
- P2X4, P2X7, and P2Y12 receptors mediate ATP-induced inflammatory cytokine production in microglia.
- Targeting these P2 receptors presents a potential therapeutic strategy for neuro-inflammatory conditions.
- Findings provide a basis for developing novel treatments for diseases involving microglial activation and neuro-inflammation.
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