Department of Neurology, Henry Ford Health System, 2799 West Grand Boulevard, Detroit, MI, 48202, USA.
Department of Physics, Oakland University, Rochester, MI, USA.
Diabetologia. 2020 Feb;63(2):431-443. doi: 10.1007/s00125-019-05043-0. Epub 2019 Nov 19.
AIMS/HYPOTHESIS: Diabetic peripheral neuropathy (DPN) is one of the major complications of diabetes, which contributes greatly to morbidity and mortality. There is currently no effective treatment for this disease. Exosomes are cell-derived nanovesicles and play an important role in intercellular communications. The present study investigated whether mesenchymal stromal cell (MSC)-derived exosomes improve neurological outcomes of DPN.
Exosomes were isolated from the medium of cultured mouse MSCs by ultracentrifugation. Diabetic mice (BKS.Cg-m+/+Lepr/J, db/db) at the age of 20 weeks were used as DPN models. Heterozygous mice (db/m) of the same age were used as the control. MSC-exosomes were administered weekly via the tail vein for 8 weeks. Neurological function was evaluated by testing motor and sensory nerve conduction velocities, and thermal and mechanical sensitivity. Morphometric analysis was performed by myelin sheath staining and immunohistochemistry. Macrophage markers and circulating cytokines were measured by western blot and ELISA. MicroRNA (miRNA) array and bioinformatics analyses were performed to examine the exosomal miRNA profile and miRNA putative target genes involved in DPN.
Treatment of DPN with MSC-exosomes markedly decreased the threshold for thermal and mechanical stimuli and increased nerve conduction velocity in diabetic mice. Histopathological analysis showed that MSC-exosomes markedly augmented the density of FITC-dextran perfused blood vessels and increased the number of intraepidermal nerve fibres (IENFs), myelin thickness and axonal diameters of sciatic nerves. Western blot analysis revealed that MSC-exosome treatment decreased and increased M1 and M2 macrophage phenotype markers, respectively. Moreover, MSC-exosomes substantially suppressed proinflammatory cytokines. Bioinformatics analysis revealed that MSC-exosomes contained abundant miRNAs that target the Toll-like receptor (TLR)4/NF-κB signalling pathway.
CONCLUSIONS/INTERPRETATION: MSC-derived exosomes alleviate neurovascular dysfunction and improve functional recovery in mice with DPN by suppression of proinflammatory genes.
目的/假设：糖尿病周围神经病变（DPN）是糖尿病的主要并发症之一，极大地增加了发病率和死亡率。目前对此病尚无有效治疗方法。外泌体是细胞衍生的纳米囊泡，在细胞间通讯中发挥重要作用。本研究探讨了间充质基质细胞（MSC）衍生的外泌体是否能改善 DPN 的神经学预后。
通过超速离心法从培养的鼠 MSC 培养基中分离出外泌体。将 20 周龄的糖尿病小鼠（BKS.Cg-m+/+Lepr/J，db/db）用作 DPN 模型。同年龄的杂合子小鼠（db/m）用作对照。每周通过尾静脉给予 MSC 外泌体治疗 8 周。通过测试运动和感觉神经传导速度以及热和机械敏感性来评估神经功能。通过髓鞘染色和免疫组织化学进行形态计量学分析。通过 Western blot 和 ELISA 测量巨噬细胞标记物和循环细胞因子。通过 microRNA (miRNA) 阵列和生物信息学分析检查外泌体 miRNA 谱和参与 DPN 的 miRNA 潜在靶基因。
用 MSC 外泌体治疗 DPN 可显著降低糖尿病小鼠的热和机械刺激阈值，并提高神经传导速度。组织病理学分析表明，MSC 外泌体显著增加了 FITC-葡聚糖灌注血管的密度，并增加了表皮内神经纤维（IENFs）、髓鞘厚度和坐骨神经轴突直径。Western blot 分析显示，MSC 外泌体处理分别降低和增加了 M1 和 M2 巨噬细胞表型标记物。此外，MSC 外泌体大量抑制促炎细胞因子。生物信息学分析表明，MSC 外泌体含有丰富的 miRNA，可靶向 Toll 样受体（TLR）4/NF-κB 信号通路。
结论/解释：MSC 衍生的外泌体通过抑制促炎基因，减轻 DPN 小鼠的神经血管功能障碍并改善其功能恢复。
