背景:间充质干细胞(MSC)来源的外泌体已显示出有望作为神经性疼痛的无细胞治疗策略。进行这项研究以探索MSC衍生的外泌体治疗神经性疼痛的镇痛作用的潜在机制。
方法:分离并鉴定人脐带MSCs(huc-MSCs)来源的外泌体。在存在或不存在外泌体的情况下,用脂多糖(LPS)刺激BV-2小胶质细胞。通过基于串联质量标签(TMT)的蛋白质组学分析鉴定差异表达的蛋白质。在慢性缩窄性损伤(CCI)的大鼠模型中评估了huc-MSC来源的外泌体的镇痛作用。通过流式细胞术研究了潜在的机制,RT-qPCR,西方印迹,免疫荧光染色,和小干扰RNA转染。
结果:体外,huc-MSCs来源的外泌体抑制LPS诱导的小胶质细胞活化,抑制TLR2/MyD88/NF-κB信号通路的活化。基于蛋白质组学分析,Rsad2被鉴定并证实被huc-MSC衍生的外泌体下调。重要的是,Rsad2的敲除也抑制了小胶质细胞的活化,并抑制了TLR2/MyD88/NF-κB信号通路的活化。在体内,鞘内注射外泌体改善CCI诱导的机械性异常性疼痛,脊髓小胶质细胞中Rsad2表达下调,抑制TLR2/MyD88/NF-κB信号通路的激活。
结论:Huc-MSCs来源的外泌体通过抑制脊髓小胶质细胞TLR2/MyD88/NF-κB信号通路的激活对神经病理性疼痛有镇痛作用。这些抗伤害作用的潜在机制涉及外泌体介导的Rsad2表达干扰,从而抑制小胶质细胞活化。
BACKGROUND: Mesenchymal stem cells (MSCs)-derived exosomes have shown promise as a cell-free therapeutic strategy for neuropathic pain. This study was conducted to explore the potential mechanisms underlying the analgesic effects of MSC-derived exosomes in treating neuropathic pain.
METHODS: Human umbilical cord MSCs (huc-MSCs)-derived exosomes were isolated and identified. BV-2 microglia were stimulated with lipopolysaccharide (LPS) in the presence or absence of exosomes. Differentially expressed proteins were identified by tandem mass tag (TMT)-based proteomic analysis. The analgesic effects of huc-MSCs-derived exosomes were evaluated in a rat model of chronic constriction injury (CCI). The underlying mechanism was investigated by flow cytometry, RT-qPCR, Western blotting, immunofluorescent staining, and small interfering RNA transfection.
RESULTS: In vitro, huc-MSCs-derived exosomes suppressed LPS-induced microglial activation and inhibited activation of the TLR2/MyD88/NF-κB signaling pathway. Based on the proteomic analysis,
Rsad2 was identified and confirmed to be down-regulated by huc-MSCs-derived exosomes. Importantly, knockdown of
Rsad2 also inhibited microglial activation and restrained activation of the TLR2/MyD88/NF-κB signaling pathway. In vivo, intrathecal injection of exosomes ameliorated CCI-induced mechanical allodynia, down-regulated
Rsad2 expression and restrained TLR2/MyD88/NF-κB signaling activation in the spinal microglia.
CONCLUSIONS: Huc-MSCs-derived exosomes exerted analgesic effects on neuropathic pain by inhibiting activation of the TLR2/MyD88/NF-κB signaling pathway in the spinal microglia. The mechanism underlying these antinociceptive effects involved exosome-mediated interference with
Rsad2 expression, thereby inhibiting microglial activation.