Department of Anesthesiology, The First Affiliated Hospital of Dalian Medical University, Dalian, China.
Department of Anesthesiology, Ningbo First Hospital, Ningbo, China.
Pain Physician. 2022 Nov;25(8):E1199-E1210.
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.
This study aimed at investigating whether P2X7R-induced BCP by regulating microglial activity through NLRP3/IL-1beta signaling involvement in BCP.
Controlled animal study.
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.
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.
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.
These findings suggest that targeting the microglial P2X7R activated NLRP3/IL-1beta signaling pathway could serve as a potential strategy for BCP treatment.
骨癌痛（BCP）是最严重和最难治疗的癌症疼痛类型。新出现的证据表明，脊髓中激活的小胶质细胞可以释放一系列神经毒性物质来刺激神经元并形成神经元敏化。P2X7 受体（P2X7R）是一种非选择性 ATP 门控离子通道，主要存在于脊髓中的小胶质细胞中，是小胶质细胞活性的关键调节剂。然而，P2X7R 在 BCP 中的具体作用及其潜在的分子机制尚未阐明。
本研究旨在通过 NLRP3/IL-1β信号通路参与 BCP 来研究 P2X7R 是否通过调节小胶质细胞活性引起 BCP。
对照动物研究。
通过将 Walker-256 乳腺癌细胞注入雌性大鼠胫骨中建立 BCP 动物模型。使用 50%足底退缩阈值（50%PWT）、自发抽搐次数（NSF）和肢体使用评分来评估大鼠的行为。使用 P2X7R 抑制剂亮蓝 G（BBG）来评估 P2X7R 在 BCP 诱导的 NLRP3 炎性小体激活中的作用。使用 Western blot、RT-PCR 和免疫荧光进行定量比较。在体外，用脂多糖（LPS）和 BzATP 处理 BV2 细胞，在存在或不存在 P2X7 siRNA 的情况下，用 Nigericin（NLRP3 炎性小体的激动剂）进一步研究 P2X7R 调节 NLRP3/IL-1β 信号的机制。
用 BBG 抑制脊髓 P2X7R 可有效抑制 BCP，因为它抑制了 NF-κB p-p65、NLRP3 炎性小体形成和下游疼痛因子 IL-1β的表达。此外，P2X7 siRNA 可降低 BV2 细胞中小胶质细胞的活性、NF-κB 的核转位以及 NLRP3 和 IL-1β 的合成。此外，Nigericin 部分逆转了 P2X7 siRNA 对 LPS 和 BzATP 诱导的 BV2 细胞的改善作用。
BBG 可以缓解 BCP，但不能改善骨破坏，这可能与接种细胞的特异性有关。应进一步研究其他机制。
这些发现表明，靶向小胶质细胞 P2X7R 激活的 NLRP3/IL-1β 信号通路可能是治疗 BCP 的一种潜在策略。
