Fig 2: EA upregulates miR-34a-3p to promote post-SCI axonal myelination. (a–d) Western blotting analysis and quantification of GFAP, NF-200, and MBP protein levels under EA stimulation and miR-34a-3p agomir treatment following SCI. Six experiments were performed in triplicate from six animals (n = 6 animals/group)), and the representative images are shown. The gray value of bands from each band relative to ß-actin is presented in the graphs. (e–g) Immunofluorescence assay of NF200 and GFAP and quantitative analysis of NF200- and GFAP-positive ratios under EA stimulation and miR-34a-3p agomir treatment following SCI. Representative images of stained tissues sections from six different animals per group are shown. The quantitative analysis showed the positive ratio of each protein; scale bars = 50 µm. (h) Toluidine blue staining showing that the number of myelinated axons was significantly elevated in response to either EA stimulation or treatment with miR-34a-3p agomir following SCI, scale bars = 50 µm. Representative images of stained tissues sections from six different animals per group (n = 6 animals/group) are shown. (i) Quantification of the myelinated axons; values from each of the six animals in each group are presented. (j) Viable myelinated axons in the spinal cord under the scanning electron microscope, scale bar = 20 µm. Representative images of stained tissues sections from six different animals per group are shown. One-way ANOVA followed by the Tukey's multiple comparisons test was utilized to identify differences. The arrows indicated the injury parts; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and ns: nonsignificant among the compared groups.

Fig 3: AQP2 overexpression-induced pro-inflammation secretion is partially caused by TLR4 signaling activation in astrocyte. Astrocyte with overexpressed AQP2 (pcDNA3.1-AQP2 plasmid) were exposed to TLR inhibitor (TAK-242) for 24 h. (A) Western blot analysis of AQP2, GFAP, TLR4, NF-?B, phospho- NF-?B and IL-1ß in astrocyte transfected with pcDNA3.1 empty vector or pcDNA3.1-AQP2 plasmid treated with or without TAK-242 (n = 3/group). (B–E, G) Quantification of the relative protein expression levels of AQP2, GFAP, TLR4, NF-kB and IL-1ß. (#p < 0.05, empty vector + vehicle vs. empty vector+ TAK-242; *p < 0.05, empty vector + vehicle vs. pcDNA3.1-AQP2 plasmid + vehicle; **p < 0.05, pcDNA3.1-AQP2 plasmid + vehicle vs. pcDNA3.1-AQP2 plasmid + TAK-242). The data are presented as the mean ± s.d. of three independent experiments. (F) Immunofluorescence of AQP2 (red signal) and GFAP (green signal) in astrocyte with AQP2 overexpression treated with or without TAK-242. Astrocyte cell nuclei were immunostained (blue, DAPI). Scale bar: 10 µm. (H) Microglia were treated with medium of astrocyte for 24h transfected with pcDNA3.1 empty vector or pcDNA3.1-AQP2 plasmid (with or without TAK-242). CD68 and CD86 were assessed by western blotting (n = 3/group). Quantification of the relative protein expression level of CD68 (I) and CD86 (J) is shown. (#p < 0.05, empty vector + vehicle vs. empty vector+ TAK-242; * p < 0.05, empty vector + vehicle vs. pcDNA3.1-AQP2 plasmid + vehicle; **p < 0.05, pcDNA3.1-AQP2 plasmid + vehicle vs. pcDNA3.1-AQP2 plasmid + TAK-242). The data are presented as the mean ± s.d. of three independent experiments.

Fig 4: Hemin induced astrocyte activation and microglia polarization. Astrocyte was exposed to hemin for 24 h. (A) AQP2, GFAP, TLR4, NF-kB and IL-1ß protein levels in rat astrocyte cell line CTX-TNA2 were assessed by western blotting in the blank control group, and different groups were treated with several concentrations of hemin (n = 3/group). (B) Astrocyte exposed to hemin for 24 h were stained with antibodies to AQP2 (green signal) and visualized by immunofluorescence microscopy. Astrocyte were immunostained for nuclei (blue, DAPI). Representative images are shown. Scale bar: 10 µm. Quantification of the relative protein expression level of AQP2(C), GFAP (D), TLR4 (E), NF-kB (G) and IL-ß (H) after 24 h hemin treatment. *p < 0.05 vs. the blank group without hemin. (F) CD206, CD68 and CD86 protein levels in microglia in the blank control group were assessed by western blotting, and different groups were treated with several concentrations of hemin (n = 3/group). Quantification of the relative protein expression level of CD206 (I), CD68 (J) and CD86 (K) are shown. *p < 0.05 vs. the blank group without hemin. The data are presented as the mean ± s.d. of three independent experiments.

Fig 5: Validation of randomly selected DEPs (N = 5, data were expressed as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001). We randomly selected SNCA, SPP1, and SYN1 in Sham-a/CCI-a groups and VIM, GFAP, and S100A4 in Sham-s/CCI-s groups to verify. These were randomly selected from the top 20 up-regulated and down-regulated proteins, respectively. (A) Protein expression and relative level of SNCA. The experiment was repeated five times with similar results. (B) Protein expression and relative level of SPP1. The experiment was repeated five times with similar results. (C) Protein expression and relative level of SYN1. The experiment was repeated five times with similar results. (D) Protein expression and relative level of VIM. The experiment was repeated five times with similar results. (E) Protein expression and relative level of GFAP. The experiment was repeated five times with similar results. (F) Protein expression and relative level of S100A4. The experiment was repeated five times with similar results.