Journal: Neural Regeneration Research
Article Title: Small extracellular vesicles derived from hair follicle neural crest stem cells enhance perineurial cell proliferation and migration via the TGF-β/SMAD/HAS2 pathway
doi: 10.4103/NRR.NRR-D-25-00127
Figure Lengend Snippet: miR-21-5p in hfNCSC-sEVs augments cell proliferation and migration by enhancing HAS2 expression in PCs. (A, B) Western blot (A) and statistical analyses (B) revealed the relative protein expression levels of HAS2, proliferating cell nuclear antigen (PCNA), and vimentin in PCs across the –/–, –/si- Has2 , hfNCSC-sEVs/–, and hfNCSC-sEVs/si- Has2 groups on day 5 of in vitro culture (normalized to β-actin, n = 3 per group). (C, D) The wound healing assay (C) and statistical analysis (D) demonstrated the migration rates of PCs in the aforementioned groups ( n = 3 per group). (E) The Cell Counting Kit-8 assay was used to assess cell viability in PCs across the same groups on day 5 of in vitro culture ( n = 5 per group). (F, G) Western blot (F) and statistical analyses (G) indicated the relative protein expression levels of HAS2, PCNA, and vimentin in PCs treated with phosphate-buffered saline (PBS), hfNCSC-sEVs, or hfNCSC-sEVs + miR-21-5p inhibitor on day 5 of in vitro culture (normalized to β-actin, n = 3 per group). (H–J) Immunofluorescence staining visualized the expression of HAS2 (red) and 5-ethynyl-2′-deoxyuridine (EdU; green) in PCs (H), and statistical analysis revealed the integrated optical density (IOD) of zonula occludens 1 (ZO1; I) and the cell proliferation rates (J) in the PBS, hfNCSC-sEVs, and hfNCSC-sEVs + miR-21-5p inhibitor groups on day 5 of in vitro culture ( n = 3 per group). (K, L) Western blot (K) and statistical analyses (L) showed the relative protein expression levels of HAS2, PCNA, and vimentin in regenerated tissue from the PBS, hfNCSC-sEVs, and hfNCSC-sEVs + miR-21-5p inhibitor groups on day 5 post-operation (normalized to β-tubulin, n = 3 per group). Data are expressed as the mean ± SEM. ** P < 0.01, *** P < 0.001 (one-way analysis of variance and Tukey’s multiple comparison test for B, D, E, G, I, J, and L). The data were from at least three separate and independent studies. CCK-8: Cell counting kit-8; EdU: 5-ethynyl-2′-deoxyuridine; HAS2: hyaluronan synthase 2; hfNCSCs: hair follicle neural crest stem cells; IOD: integrated optical density; PCNA: proliferating cell nuclear antigen; PCs: perineurial cells; sEVs: small extracellular vesicles; ZO1: zonula occludens 1.
Article Snippet: The following primary antibodies were used: rabbit polyclonal anti-p75 neurotrophin receptor (p75) antibody (1:100, Cat# 55014-1-AP, Proteintech), mouse monoclonal anti-nestin antibody (1:100, Cat# MAB353, Sigma), rabbit polyclonal anti-claudin-1 antibody (1:250, Cat# 13050-1-AP, Proteintech), rabbit polyclonal anti-ZO1 antibody (1:200, Cat# 21773-1-AP, Proteintech), rabbit polyclonal anti-glucose transporter 1 (GLUT1) antibody (1:500, Cat# 21829-1-AP, Proteintech), rabbit monoclonal anti-S100 antibody (1:800, Cat# MAB353, Abcam), mouse monoclonal anti-neurofilament 200 (NF200) antibody (1:800, Cat# N5389, Sigma), rabbit polyclonal anti-myelin basic protein (MBP) antibody (1:400, Cat# 10458-1-AP, Proteintech), mouse monoclonal anti-β-tubulin antibody (1:1000, Cat# M20005 , Abmart), and rabbit polyclonal anti-HAS2 antibody (1:200, Cat# DF13702, Affinity).
Techniques: Migration, Expressing, Western Blot, In Vitro, Wound Healing Assay, Cell Counting, Saline, Immunofluorescence, Staining, Comparison, CCK-8 Assay
![hfNCSC-sEVs are taken up by PCs in vitro and enhance their proliferation and migration. (A) Primary cultures of hfNCSCs were established from male Sprague–Dawley rats. (B) Immunofluorescence staining of the neural crest cell marker p75 (red) and the stem cell marker nestin (green) in hfNCSCs, with 4′,6-diamidino-2-phenylindole (DAPI) staining indicating the nuclei. (C) Western blot analysis demonstrated the presence of surface markers (cluster of differentiation [CD]9, CD81, and tumor susceptibility gene 101 protein [TSG101]) and the absence of an endoplasmic reticulum marker (calnexin) in hfNCSC-sEVs. (D) Nanoparticle tracking analysis was used to quantify the concentration and size distribution of hfNCSC-sEVs. (E) Transmission electron microscopy was used to visualize the characteristic morphology of hfNCSC-sEVs. (F) Immunofluorescence staining indicated that the third-generation PCs cultured in vitro were positive for claudin-1, zonula occludens 1 <t>(ZO1),</t> and glucose transporter 1 (GLUT1) but negative for S100, with DAPI staining marking the nuclei. (G) The internalization of PKH26-labeled hfNCSC-sEVs (red) by ZO1-positive PCs (green) was visualized using immunofluorescence staining, with DAPI staining to mark the nuclei. (H) The Cell Counting Kit-8 assay was used to evaluate the cell viability of PCs across concentrations of 0, 2 × 10 8 , 5 × 10 8 , and 10 × 10 8 particles/mL hfNCSC-sEVs at 3, 5, and 7 days of in vitro culture ( n = 5 per group). (I) The Transwell assay was used to quantify the number of migrating PCs at 6, 12, and 18 hours post-treatment with the aforementioned concentrations of hfNCSC-sEVs, in in vitro culture ( n = 6 per group). (J) Western blot and (K) statistical analyses revealed the relative protein expression levels of proliferating cell nuclear antigen (PCNA) and vimentin in PCs from the phosphate-buffered saline (PBS) and hfNCSC-sEVs groups on day 5 of in vitro culture (normalized to β-actin, n = 3 per group). Data are expressed as the mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 (one-way analysis of variance and Tukey’s multiple comparison test for H and I; Student’s t -test for K). The data were from at least three separate and independent studies. CCK-8: Cell counting kit-8; GLUT1: glucose transporter 1; hfNCSCs: hair follicle neural crest stem cells; ns: not significant; PCNA: proliferating cell nuclear antigen; PCs: perineurial cells; sEVs: small extracellular vesicles; ZO1: zonula occludens 1.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_4726/pmc12694726/pmc12694726__NRR-21-2060-g002.jpg)
![WB600/ZD-FMT and AHR activation alleviate ileal barrier damage caused by Salmonella enterica subsp . enterica serovar Infantis( S. Infantis) infection. (A) Immunohistochemical detection of mucins2 (MUC2) expression in the ileum (the black arrow). (B) Immunofluorescence detection of zonula occludens-1 <t>(ZO-1)</t> expression in the ileum. (C) Claudin-1 and occludin expression in the ileum by Western blot assays. (D-F) RT-qPCR detection of the expression of MUC2 and mucin-related genes trefoil factor 3 ( TFF3 ) and Galactose-3-O-sulfotransferase 2 ( GAL3ST2 ) in the ileum. All data are presented as means ± SD from at least three independent experiments. The P -values shown denote statistical significance for all pairwise comparisons between groups. WB600/ZD is an engineered Bacillus subtilis strain (derived from the protease-deficient parental strain WB600) expressing the antimicrobial defensin peptide from Zophobas atratus , where “ZD” denotes the Zophobas atratus defensin. FMT = fecal microbiota transplantation; SI = S . Infantis infection; CH CH223191. ABX = antibiotic mixture; FICZ = 6-formylindolo[3,2-b]carbazole; PWD = post-weaning diarrhea; AHR = aryl hydrocarbon receptor; CYP1A1 = cytochrome P450 family 1 subfamily A member 1.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_8522/pmc12828522/pmc12828522__gr6.jpg)
