Xu Yin, Liu Xinli, Chen Hui, Zhao Yun, Zhao Yubai, Xie Yu, Pang Jiahui, Zeng Hui, Zeng Yanyan, Peng Weiwei, Zheng Manxu, Wu Wen
Center of Rehabilitation Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou, 510282, China.
Department of Clinical and Rehabilitation Medicine, Guiyang Healthcare Vocational University, Guizhou, 550081, China.
J Headache Pain. 2025 Jul 10;26(1):158. doi: 10.1186/s10194-025-02089-x.
Microglia are essential in mediating responses to nerve injury, with neuroinflammation and engulfment of interneuron inhibitory synapses driving heightened neuronal excitability and pain hypersensitivity. Spi1 is the main regulator of microglial homeostasis in the central nervous system. However, its function in neuropathic pain remains unclear. The aim of this study was to investigate the effect of Spi1 on neuropathic pain and the underlying mechanism.
RNA-seq analysis identified Spi1 as a central hub gene in neuropathic pain. The spared nerve injury (SNI) mice and LPS induced BV2 cells were employed to detect the expression of Spi1. Silencing Spi1 was performed to determine their roles in SNI progression. Mechanical allodynia and thermal allodynia were assessed using von Frey filaments and hot plate, respectively. The expression of pro-inflammation marker CD86, anti-inflammation marker CD206 and inflammatory cytokines (TNF-α, IL-1β, IL-6) were qualified by western blot, immunofluorescence or qPCR. Additionally, the microglia morphological changes, the volume of CD68 or VGAT in microglia were examined by immunofluorescence to reflect microglial activation and phagocytosis. Patch clamp was performed to evaluate neuronal excitability. Luciferase assays and ChIP-qPCR were conducted to pinpoint the binding between Spi1 and the promoter of Clec7a. Subsequently, rescue experiments in vivo and in vitro were implemented to clarify whether Spi1 exerted itself through Clec7a.
Elevated Spi1 expression was observed in the spinal cords of SNI mice and in LPS-stimulated BV2 cells. Spi1 knockdown alleviated SNI-induced mechanical and thermal allodynia, diminished the release of inflammatory cytokine and the expression of proinflammation marker CD86, decreased the volume of CD68 or VGAT in microglia and increased the frequency of neuronal spontaneous inhibitory postsynaptic potentials(sIPSCs). Mechanistically, Spi1 was found to bind the promoter of Clec7a by luciferase assays and ChIP-PCR. And rescue experiments in vivo and in vitro showed that Spi1 aggravated pain hypersensitivity, promoted neuroinflammation and enhanced microglial phagocytosis by Clec7a.
Our results highlight that Spi1-Clec7a axis was a key contributor to neuropathic pain through promoting neuroinflammation and microglial phagocytosis, thereby implicating it as a promising therapeutic target for mitigating neuropathic pain.
The online version contains supplementary material available at 10.1186/s10194-025-02089-x.
小胶质细胞在介导对神经损伤的反应中至关重要，神经炎症和中间神经元抑制性突触的吞噬会导致神经元兴奋性增强和疼痛超敏反应。Spi1是中枢神经系统中小胶质细胞稳态的主要调节因子。然而，其在神经性疼痛中的作用仍不清楚。本研究旨在探讨Spi1对神经性疼痛的影响及其潜在机制。
RNA测序分析确定Spi1为神经性疼痛中的一个核心枢纽基因。采用 spared nerve injury（SNI）小鼠和LPS诱导的BV2细胞来检测Spi1的表达。通过沉默Spi1来确定其在SNI进展中的作用。分别使用von Frey细丝和热板评估机械性异常性疼痛和热异常性疼痛。通过蛋白质免疫印迹、免疫荧光或qPCR检测促炎标志物CD86、抗炎标志物CD206和炎性细胞因子（TNF-α、IL-1β、IL-6）的表达。此外，通过免疫荧光检查小胶质细胞形态变化、小胶质细胞中CD68或VGAT的体积，以反映小胶质细胞的激活和吞噬作用。进行膜片钳实验以评估神经元兴奋性。进行荧光素酶报告基因检测和染色质免疫沉淀-qPCR以确定Spi1与Clec7a启动子之间的结合。随后，进行体内和体外的挽救实验，以阐明Spi1是否通过Clec7a发挥作用。
在SNI小鼠的脊髓和LPS刺激的BV2细胞中观察到Spi1表达升高。敲低Spi1可减轻SNI诱导的机械性和热异常性疼痛，减少炎性细胞因子的释放和促炎标志物CD86的表达，减小小胶质细胞中CD68或VGAT的体积，并增加神经元自发性抑制性突触后电位（sIPSCs）的频率。机制上，通过荧光素酶报告基因检测和染色质免疫沉淀-PCR发现Spi1与Clec7a的启动子结合。体内和体外的挽救实验表明，Spi1通过Clec7a加重疼痛超敏反应，促进神经炎症并增强小胶质细胞吞噬作用。
我们的结果表明，Spi1-Clec7a轴通过促进神经炎症和小胶质细胞吞噬作用，是神经性疼痛的关键促成因素，因此表明它是减轻神经性疼痛的一个有前景的治疗靶点。
在线版本包含可在10.1186/s10194-025-02089-x获取的补充材料。
