人脐带间充质干细胞来源的外泌体通过 miR-146a-5p/TRAF6 轴调节小胶质细胞焦亡和自噬来减轻炎性疼痛。
Huc-MSCs-derived exosomes attenuate inflammatory pain by regulating microglia pyroptosis and autophagy via the miR-146a-5p/TRAF6 axis.
机构信息
Department of Anesthesiology, Changzheng Hospital, Naval Medical University, Shanghai, 200003, China.
Research Center of Developmental Biology, Department of Histology and Embryology, College of Basic Medicine, Naval Medical University, Shanghai, 200433, China.
出版信息
J Nanobiotechnology. 2022 Jul 14;20(1):324. doi: 10.1186/s12951-022-01522-6.
BACKGROUND
Chronic inflammatory pain significantly reduces the quality of life and lacks effective interventions. In recent years, human umbilical cord mesenchymal stem cells (huc-MSCs)-derived exosomes have been used to relieve neuropathic pain and other inflammatory diseases as a promising cell-free therapeutic strategy. However, the therapeutic value of huc-MSCs-derived exosomes in complete Freund's adjuvant (CFA)-induced inflammatory pain remains to be confirmed. In this study, we investigated the therapeutic effect and related mechanisms of huc-MSCs-derived exosomes in a chronic inflammatory pain model.
METHODS
C57BL/6J male mice were used to establish a CFA-induced inflammatory pain model, and huc-MSCs-derived exosomes were intrathecally injected for 4 consecutive days. BV2 microglia cells were stimulated with lipopolysaccharide (LPS) plus adenosine triphosphate (ATP) to investigate the effect of huc-MSCs-derived exosomes on pyroptosis and autophagy. Bioinformatic analysis and rescue experiments were used to demonstrate the role of miR-146a-5p/ TRAF6 in regulating pyroptosis and autophagy. Western blotting, RT-qPCR, small interfering RNA and Yo-Pro-1 dye staining were performed to investigate the related mechanisms.
RESULTS
Huc-MSCs-derived exosomes alleviated mechanical allodynia and thermal hyperalgesia in CFA-induced inflammatory pain. Furthermore, huc-MSCs-derived exosomes attenuated neuroinflammation by increasing the expression of autophagy-related proteins (LC3-II and beclin1) and inhibiting the activation of NLRP3 inflammasomes in the spinal cord dorsal horn. In vitro, NLRP3 inflammasome components (NLRP3, caspase1-p20, ASC) and gasdermin D (GSDMD-F, GSDMD-N) were inhibited in BV2 cells pretreated with huc-MSCs-derived exosomes. Western blot and Yo-Pro-1 dye staining demonstrated that 3-MA, an autophagy inhibitor, weakened the protective effect of huc-MSCs-derived exosomes on BV2 cell pyroptosis. Importantly, huc-MSCs-derived exosomes transfected with miR-146a-5p mimic promoted autophagy and inhibited BV2 cell pyroptosis. TRAF6, as a target gene of miR-146a-5p, was knocked down via small-interfering RNA, which increased pyroptosis and inhibited autophagy.
CONCLUSION
Huc-MSCs-derived exosomes attenuated inflammatory pain via miR-146a-5p/TRAF6, which increased the level of autophagy and inhibited pyroptosis.
背景
慢性炎症性疼痛显著降低了生活质量，且缺乏有效的干预措施。近年来，人脐带间充质干细胞（huc-MSCs）衍生的外泌体已被用于缓解神经病理性疼痛和其他炎症性疾病，作为一种很有前途的无细胞治疗策略。然而，huc-MSCs 衍生的外泌体在完全弗氏佐剂（CFA）诱导的炎症性疼痛中的治疗价值仍有待证实。在这项研究中，我们研究了 huc-MSCs 衍生的外泌体在慢性炎症性疼痛模型中的治疗效果和相关机制。
方法
使用 C57BL/6J 雄性小鼠建立 CFA 诱导的炎症性疼痛模型，并连续 4 天鞘内注射 huc-MSCs 衍生的外泌体。用脂多糖（LPS）加三磷酸腺苷（ATP）刺激 BV2 小胶质细胞，研究 huc-MSCs 衍生的外泌体对细胞焦亡和自噬的影响。采用生物信息学分析和挽救实验证明 miR-146a-5p/TRAF6 在调节细胞焦亡和自噬中的作用。通过 Western blot、RT-qPCR、小干扰 RNA 和 Yo-Pro-1 染色来研究相关机制。
结果
huc-MSCs 衍生的外泌体缓解了 CFA 诱导的炎症性疼痛中的机械性痛觉过敏和热痛觉过敏。此外，huc-MSCs 衍生的外泌体通过增加自噬相关蛋白（LC3-II 和 beclin1）的表达和抑制脊髓背角 NLRP3 炎性小体的激活，减轻了神经炎症。体外实验中，huc-MSCs 衍生的外泌体预处理后，BV2 细胞中的 NLRP3 炎性小体成分（NLRP3、caspase1-p20、ASC）和 gasdermin D（GSDMD-F、GSDMD-N）表达受到抑制。Western blot 和 Yo-Pro-1 染色表明，自噬抑制剂 3-MA 减弱了 huc-MSCs 衍生的外泌体对 BV2 细胞细胞焦亡的保护作用。重要的是，huc-MSCs 衍生的外泌体转染 miR-146a-5p 模拟物可促进自噬并抑制 BV2 细胞细胞焦亡。TRAF6 作为 miR-146a-5p 的靶基因，通过小干扰 RNA 敲低后，细胞焦亡增加，自噬受到抑制。
结论
huc-MSCs 衍生的外泌体通过 miR-146a-5p/TRAF6 减轻炎性疼痛，从而增加自噬水平并抑制细胞焦亡。
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