Research Paper Volume 13, Issue 5 pp 6752—6764

Extracellular vesicle-encapsulated microRNA-23a from dorsal root ganglia neurons binds to A20 and promotes inflammatory macrophage polarization following peripheral nerve injury

Delivery of decreased miR-23a by DRG neuron-derived EVs reduces neuropathic hypersensitivity and recruitment of M1 macrophages in vivo. SNI mice were treated with intrathecal administration of EVs-treated miR-23a antagomir-transfected DRG neurons. (A) effect of intrathecal injection of EVs-miR-23a antagomir on mechanical hypersensitivity for 7 days, which was expressed as mean ± SD of 50% PWT; n (vehicle mice) = 6, n (oligomer-treated mice) = 12; * p vs. vehicle mice; # p vs. oligomer-treated mice. Values obtained from three independent experiments in triplicate are expressed as mean ± SD and analyzed by two-way ANOVA, followed by Tukey’s test among multiple groups. (B) miR-23a expression in DRG determined using RT-qPCR, normalized to U6; (C) Immunostaining of macrophages (F4/80+ cells, red), and nuclei (DAPI, blue) in SNI DRG (scale bar = 25 μm); (D) Macrophage phenotyping [M1 macrophages (CD206-CD11c+) and M2 macrophages (CD206+CD11c-)] in DRG analyzed using flow cytometry; (E) levels of TNF-α and IL-6 in L4-L5 DRG tissues measured using ELISA. Values obtained from three independent experiments are expressed as mean ± SD and analyzed by unpaired t-test between two groups in panel (B–E). * p vs. mice treated with EVs-miR-23a-NC or vehicle. Cell experiment was independently repeated for three times.

Figure 6. Delivery of decreased miR-23a by DRG neuron-derived EVs reduces neuropathic hypersensitivity and recruitment of M1 macrophages in vivo. SNI mice were treated with intrathecal administration of EVs-treated miR-23a antagomir-transfected DRG neurons. (A) effect of intrathecal injection of EVs-miR-23a antagomir on mechanical hypersensitivity for 7 days, which was expressed as mean ± SD of 50% PWT; n (vehicle mice) = 6, n (oligomer-treated mice) = 12; * p < 0.05 vs. vehicle mice; # p < 0.05 vs. oligomer-treated mice. Values obtained from three independent experiments in triplicate are expressed as mean ± SD and analyzed by two-way ANOVA, followed by Tukey’s test among multiple groups. (B) miR-23a expression in DRG determined using RT-qPCR, normalized to U6; (C) Immunostaining of macrophages (F4/80+ cells, red), and nuclei (DAPI, blue) in SNI DRG (scale bar = 25 μm); (D) Macrophage phenotyping [M1 macrophages (CD206-CD11c+) and M2 macrophages (CD206+CD11c-)] in DRG analyzed using flow cytometry; (E) levels of TNF-α and IL-6 in L4-L5 DRG tissues measured using ELISA. Values obtained from three independent experiments are expressed as mean ± SD and analyzed by unpaired t-test between two groups in panel (BE). * p < 0.05 vs. mice treated with EVs-miR-23a-NC or vehicle. Cell experiment was independently repeated for three times.