Fig 1: adMSC-derived exosomes reduce DN symptoms in model rats. (A) Round nanoparticles observed under the TEM; (B) diameter of the collected particles measured by NTA; (C) levels of exosome-specific surface biomarkers TSG101, CD9 and CD63 determined by Western blot analysis; (D) changes in physiological parameters in each group of rats (one-way ANOVA, *p < 0.05 vs Control; #p < 0.05 vs PBS); (E) mesangial hyperplasia in rat kidney tissues determined by PAS staining (one-way ANOVA, *p < 0.05 vs Control; #p < 0.05 vs PBS); (F) pathological changes in rat kidneys examined by HE staining (one-way ANOVA, *p < 0.01 vs Control; #p < 0.05 vs PBS), (G) protein level of the fibrosis-related marker Col. I in rat kidney determined by IHC staining (one-way ANOVA, *p < 0.05 vs Control; #p < 0.05 vs PBS); (H) viability of GMCs after different concentrations of exosomes (1 µg, 2 µg, 3 µg, 4 µg and 5 µg) or PBS treatment determined by the CCK-8 method (one-way ANOVA, *p < 0.05 vs Control); (I) IL-6 mRNA expression in GMCs determined by RT-qPCR (one-way ANOVA, *p < 0.01 vs NG; #p < 0.05 vs PBS); (J) proliferation ability of GMCs determined using the CCK-8 method (one-way ANOVA, *p < 0.01 vs NG; #p < 0.05 vs PBS). N = 10 in each group. Data were exhibited as mean ± SEM from three independent experiments. Representative images are provided.
Fig 2: High glucose conditions inhibited SC proliferation, increased cell apoptosis, and downregulated the expression of miR-21 in cells and exosomes(A) The cell viability of SC was examined by CCK-8 assay, high glucose causes cell viability to decrease (n = 10-well, one-way ANOVA: ***p < 0.001 versus the low glucose group. ###p < 0.001 versus the normal glucose group).(B) The expression of miR-21 in SC and exosomes was detected by qPCR, and miR-21 was downregulated in the high glucose group compared with that of the low glucose group (n = 10 samples; t-test: ***p < 0.001 versus the low glucose group).(C) Exosome markers (CD9 and CD63) were detected by Western blot after exosomes were isolated by ultracentrifugation.(D and E) Protein expression of p-AKT, t-AKT, and GAPDH was detected by Western blot (D), and the expression of the AKT signaling pathway was decreased in the high glucose group compared with that of the low glucose group. (E) Gray value statistics of Western blot (n = 6 samples, nonparametric tests (Mann-Whitney Test, same below): **p < 0.01 versus the low glucose group).(F and G) TUNEL assay (F) was used to detect cell apoptosis (red), apoptosis increased in the high glucose group compared with the low glucose group. Scale bar: 50 µm.(G) Cell apoptosis statistics (n = 20 fields of view per group, nonparametric tests: ***p < 0.001 versus the low glucose group). For the above, data are represented as mean ± SD.
Fig 3: Characterization of exosomes derived from bone marrow mesenchymal stem cells (BMSCs). a Cellular morphology (P1, P3) of BMSCs observed under an inverted fluorescence microscope. Scale bar: 100 µm. b Flow cytometry was used to analyze the surface antigens (CD44, CD105, CD31) in BMSCs. c, d The morphology of FBS-derived exosomes and BMSC-derived exosomes was observed under transmission electron microscopy (TEM). Scale bar: 50 µm. e Western blot was used to examine the expression of CD9, CD63 in BMSCs, and BMSC-derived exosomes
Fig 4: The characterization and uptake of Exo derived from H9c2 cell lines cultured in normoxic or hypoxic environment. (A) Morphological characterization of Nor-Exo and Hypo-Exo by transmission electron microscopy (scale bar = 100 nm). (B) The surface makers (Alix, HSP70 and CD63) of Nor-Exo and Hypo-Exo were detected by western blotting. (C) The size distribution was measured using NanoSight analysis, which indicated that the diameter of most of the particles was within the range of 40–160 nm. ‘ns’ indicates p = 0.05. (D) A laser scanning confocal microscope captured the PKH26-labelled Exos (red fluorescence) taken up by RAW264.7 cells stained by DAPI (blue fluorescence) (scale bar = 20 µm)
Fig 5: Hypoxia-mediated apoptosis is associated with changes in PI3K and AKT. a Co-localization of miR-210 with exosome-specific surface marker CD81 and CD63 by the immunofluorescence. b The uptake of exosome by cardiomyocytes, and the localization of miR-210 molecule with Cy3 fluorescence in cardiomyocytes. c Dual-luciferase reporter assay showed that AIFM3 was a target of miR-210. F, Firefly luciferase; R, Renilla luciferase. d, e AIFM3, p-AKT/AKT, p-PI3K/PI3K, and p-p53/p53 levels were evaluated by Western blotting in cardiomyocytes from the indicated treatment groups ± hypoxia. Quantifications are shown in the lower panel. N = 3 independent experiments. **p < 0.01, ****p < 0.0001 for hypo vs. con. ##p < 0.01 for hypo + exo vs. hypo
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