目的:本研究旨在探讨豆甾醇治疗神经性疼痛的潜在治疗应用。
方法:通过DRG单细胞测序分析和在细胞实验中使用特异性抑制剂研究了相关机制。在动物实验中,32只雄性SD大鼠随机分为假手术组,CCI组,布洛芬组,和豆甾醇组。我们做了行为测试,ELISA,H&E染色和免疫组织化学,和西方印迹。
结果:通过单细胞测序进行的细胞通讯分析显示,周围神经损伤后,雪旺氏细胞分泌IL-34以作用于巨噬细胞中的CSF1R。周围神经损伤后,DRG中巨噬细胞CSF1R通路和NLRP3炎性体的mRNA表达水平升高。体外研究表明,豆甾醇可以减少LPS诱导的RSC96雪旺细胞IL-34的分泌;豆甾醇处理LPS诱导的雪旺细胞条件培养基(L-S-CM)不诱导RAW264.7巨噬细胞的增殖和迁移;L-S-CM降低CSF1R信号通路(CSF1R,P38MAPK,和NFκB)激活,NLRP3炎性体激活,和ROS生产。体内实验证实,在大鼠慢性压迫性神经损伤(CCI)模型中,豆甾醇能降低热痛觉和冷痛觉过敏,IL-6,TNF-α,CCL2,SP,和PGE2在CCI大鼠血清中的表达;免疫组化和免疫印迹证实,豆甾醇可降低CCI大鼠DRG中IL-34/CSF1R信号通路和NLRP3炎性体的水平。
结论:豆甾醇通过调节IL-34/CSF1R轴来减少DRG中的雪旺细胞-巨噬细胞级联反应,从而减轻神经性疼痛。
This study aimed to investigate the potential therapeutic applications of
stigmasterol for treating neuropathic pain.
Related mechanisms were investigated by DRG single-cell sequencing analysis and the use of specific inhibitors in cellular experiments. In animal experiments, 32 male Sprague-Dawley rats were randomly divided into the sham operation group, CCI group, ibuprofen group, and stigmasterol group. We performed behavioral tests, ELISA, H&E staining and immunohistochemistry, and western blotting.
Cell communication analysis by single-cell sequencing reveals that after peripheral nerve injury, Schwann cells secrete IL-34 to act on CSF1R in macrophages. After peripheral nerve injury, the mRNA expression levels of CSF1R pathway and NLRP3 inflammasome in macrophages were increased in DRG. In vitro studies demonstrated that
stigmasterol can reduce the secretion of IL-34 in LPS-induced RSC96 Schwann cells;
stigmasterol treatment of LPS-induced Schwann cell-conditioned medium (L-S-CM) does not induce the proliferation and migration of RAW264.7 macrophages; L-S-CM reduces CSF1R signaling pathway (CSF1R, P38MAPK, and NFκB) activation, NLRP3 inflammasome activation, and ROS production. In vivo experiments have verified that stigmasterol can reduce thermal and cold hyperalgesia in rat chronic compressive nerve injury (CCI) model;
stigmasterol can reduce IL-1β, IL-6, TNF-α, CCL2, SP, and PGE2 in serum of CCI rats; immunohistochemistry and western blot confirmed that
stigmasterol can reduce the levels of IL-34/CSF1R signaling pathway and NLRP3 inflammasome in DRG of CCI rats.
Stigmasterol alleviates neuropathic pain by reducing Schwann cell-macrophage cascade in DRG by modulating IL-34/CSF1R axis.