METHODS: The RNA sequencing results of migraineurs and the panglaodb database were used to obtain differentially expressed genes (DEGs) in migraine related to microglia. A migraine rat model was established for validating and localizing of the MRGs, and subsequent screening for target genes was conducted. A shRNA was designed to interference the expression of target genes and administered into the trigeminal ganglion (TG) of rats. Pain sensitivity in rats was evaluated via the hot water tail-flick (HWTF) and formalin-induced pain (FIP) experiments. ELISA was used to quantify the levels of inflammatory cytokines and CGRP. WB and immunofluorescence assays were applied to detect the activation of microglia.
RESULTS: A total of five DEGs in migraine related to microglia were obtained from RNA sequencing and panglaodb database. Animal experiments showed that these genes expression were heightened in the TG and medulla oblongata (MO) of migraine rats. The gene S100A8 co-localized with microglia in both TG and MO. The HWTF and FIP experiments demonstrated that interference with S100A8 alleviated the sense of pain in migraine rats. Moreover, the levels of TNFα, IL-1β, IL-6, and CGRP in the TG and MO of rats in the model rats were increased, and the expression of microglia markers IBA-1, M1 polarization markers CD86 and iNOS was upregulated. Significantly, interference with S100A8 reversed these indicators.
CONCLUSIONS: Interference with S100A8 in microglia increased the pain threshold during migraine attacks, and inhibited neuroinflammation and microglia activation.
方法:使用偏头痛患者的RNA测序结果和panglaodb数据库获得与小胶质细胞相关的偏头痛中的差异表达基因(DEGs)。建立了偏头痛大鼠模型,用于验证和定位MRGs,随后进行靶基因的筛选。设计了一个shRNA来干扰靶基因的表达,并将其施用到大鼠的三叉神经节(TG)中。通过热水甩尾(HWTF)和福尔马林诱导的疼痛(FIP)实验评估大鼠的疼痛敏感性。ELISA用于定量炎性细胞因子和CGRP的水平。应用WB和免疫荧光法检测小胶质细胞的活化。
结果:从RNA测序和panglaodb数据库中获得了与小胶质细胞相关的偏头痛中总共五个DEGs。动物实验表明,这些基因在偏头痛大鼠的TG和延髓(MO)中的表达增强。基因S100A8与小胶质细胞共同定位在TG和MO中。HWTF和FIP实验表明,S100A8的干扰减轻了偏头痛大鼠的疼痛感。此外,TNFα的水平,IL-1β,IL-6和CGRP在模型大鼠的TG和MO均升高,小胶质细胞标志物IBA-1、M1极化标志物CD86和iNOS的表达上调。重要的是,干扰S100A8逆转了这些指标。
结论:干扰小胶质细胞中的S100A8可增加偏头痛发作时的痛阈,并抑制神经炎症和小胶质细胞活化。