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P2X7 Receptor-Induced Bone Cancer Pain by Regulating Microglial Activity via NLRP3/IL-1beta Signaling

Ping Wu¹, Xiaoqi Wu¹, Guohua Zhou²

¹Department of Anesthesiology, The First Affiliated Hospital of Dalian Medical University, Dalian, China.

|November 14, 2022

Abstract

Background:

Bone cancer pain (BCP) is the most severe and intractable type of cancer pain. Emerging evidence has demonstrated that activated microglia in the spinal cord could release a series of neurotoxic substances to stimulate neurons and form neuronal sensitization. The P2X7 receptor (P2X7R) is a nonselective ATP-gated ion channel predominantly present in microglia in the spinal cord as the key modulator of microglial activity. However, the specific effect and underlying molecular mechanism of P2X7R in BCP have not yet been elucidated.

Objectives:

This study aimed at investigating whether P2X7R-induced BCP by regulating microglial activity through NLRP3/IL-1beta signaling involvement in BCP.

Study Design:

Controlled animal study.

Methods:

A BCP animal model was established by injecting Walker-256 breast cancer cells into the tibia of female rats. Fifty percent paw withdrawal thresholds (50% PWTs), number of spontaneous flinches (NSF), and limb use scores were used to evaluate behavior in rats. P2X7R inhibitor brilliant blue G (BBG) was used to assess the role of P2X7R in BCP-induced NLRP3 inflammasome activation. Western blot, RT-PCR, and immunofluorescence were used for quantitative comparison. In vitro, BV2 cells were treated with lipopolysaccharide (LPS) and BzATP, in the presence or absence of P2X7 siRNA, with nigericin (an agonist of the NLRP3 inflammasome) to further study the mechanism of P2X7R regulate NLRP3/IL-1beta signaling.

Results:

The inhibition of spinal P2X7R with BBG could effectively inhibit BCP due to suppressing the expression of NF-kappaB p-p65, NLRP3 inflammasome formation, and downstream pain factors IL-1beta. Furthermore, P2X7 siRNA could reduce microglial activity, the nuclear translocation of NF-kappaB, and the synthesis of NLRP3 and IL-1beta in BV2 cells. In addition, nigericin partially reversed the ameliorating effect of P2X7 siRNA on BV2 cells induced by LPS and BzATP.

Limitations:

BBG could relieve BCP but not improve the destruction of bone, which may be related to the specificity of inoculated cells. Further mechanisms should be investigated.

Conclusion:

These findings suggest that targeting the microglial P2X7R activated NLRP3/IL-1beta signaling pathway could serve as a potential strategy for BCP treatment.

Insights

Targeting spinal P2X7 receptor (P2X7R) with brilliant blue G (BBG) effectively reduced bone cancer pain (BCP) by inhibiting microglial NLRP3/IL-1beta signaling. This suggests P2X7R as a potential therapeutic target for BCP.

Area of Science:

Neuroscience
Immunology
Oncology

Background:

Bone cancer pain (BCP) is severe and difficult to treat.
Activated spinal microglia release neurotoxic substances, causing neuronal sensitization.
The P2X7 receptor (P2X7R) on microglia modulates spinal cord activity, but its role in BCP is unclear.

Purpose of the Study:

To investigate the role of P2X7R in BCP.
To determine if P2X7R regulates microglial activity via NLRP3/IL-1beta signaling in BCP.

Main Methods:

Established a rat model of BCP by injecting Walker-256 cells into the tibia.
Administered P2X7R inhibitor brilliant blue G (BBG) and assessed pain behaviors.
Utilized Western blot, RT-PCR, and immunofluorescence to analyze molecular changes.
Investigated P2X7R's mechanism in BV2 microglial cells in vitro.

Main Results:

BBG treatment inhibited BCP by reducing NF-kappaB p-p65, NLRP3 inflammasome, and IL-1beta expression.
P2X7R inhibition in BV2 cells decreased microglial activity and NLRP3/IL-1beta synthesis.
Nigericin partially reversed the protective effects of P2X7R inhibition in BV2 cells.

Conclusions:

Targeting microglial P2X7R activation of NLRP3/IL-1beta signaling is a potential strategy for BCP treatment.
P2X7R inhibition alleviates BCP but does not affect bone destruction.

Read full analysis

Keywords:

NLRP3 brilliant blue G interleukin 1beta P2X7 receptor

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