Journal: Materials Today Bio
Article Title: miR-19b-3p engineered adipose-derived stem cell exosomes attenuate acute liver failure via promoting tissue repair and microenvironment remodeling
doi: 10.1016/j.mtbio.2025.102697
Figure Lengend Snippet: Anti-inflammation and anti-oxidation effect of ADSC-EXO derived exosomal miR-19b-3p in D-GalN/LPS-challenged macrophages. (A) Flowchart of ADSCs-derived exosomes collection and isolation. (B) Transmission electron micrograph (TEM) of ADSCs-derived exosomes (ADSC-EXO), scale bar = 200 nm (left panel), scale bar = 100 nm (right panel). (C) Size distribution, concentration and intensity of ADSC-EXO were determined using Nano-sight tracking analysis (NTA). (D) Western blot analysis for identifying exosomal protein markers in ADSC-EXO, ADSCs protein as control. (E) Confocal microscopy image showing the cellular uptake of DiO- labeled exosomes by test cells in vitro . Blue: DAPI label nuclei, Green: DiO-labeled exosomes, Red: TRITC-phalloidin labeled cytoskeleton; scale bar = 25 μm. (F) The particle concentration, protein concentration, and particle-to-protein ratio of exosomes from three other batches were measured. (G)miRNA sequencing analysis of ADSC-EXO isolated ADSCs from 2 donors' adipose tissue. (H) Rat primary macrophages (RMa-bm cell) challenged with D-GalN (70 mmol/L) and LPS (100 ng/mL) were collected and detected the exosomal miRNA respond within 48 h once 4 h using RT-PCR assay. (I) ROS level of each group in RMa-bm cells was measured using flow cytometry assay after 48 h miRNA transfection and 24 h D-GalN/LPS treatment. (J) miR-19b-3p mimic/mimic NC or inhibitor/inhibitor NC were transfected to RMa-bm cells for 48 h. Then cells were challenged with D-GalN (70 mmol/L) and LPS (100 ng/mL) for 24 h and supernatant were collected. Cytokines level of TNF-α, IL-6, IL-1α and IL-10 in supernatant were measured using ELISA assay. Statistical differences were determined using unpaired Student's t -test between each 2 cohorts (ns = no significance, ∗p < 0.05, ∗∗p < 0.01). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Article Snippet: Blue: DAPI label nuclei, Green: DiO-labeled exosomes, Red: TRITC-phalloidin labeled cytoskeleton; scale bar = 25 μm. (F) The particle concentration, protein concentration, and particle-to-protein ratio of exosomes from three other batches were measured. (G)miRNA sequencing analysis of ADSC-EXO isolated ADSCs from 2 donors' adipose tissue. (H) Rat primary macrophages (RMa-bm cell) challenged with D-GalN (70 mmol/L) and LPS (100 ng/mL) were collected and detected the exosomal miRNA respond within 48 h once 4 h using RT-PCR assay. (I) ROS level of each group in RMa-bm cells was measured using flow cytometry assay after 48 h miRNA transfection and 24 h D-GalN/LPS treatment. (J) miR-19b-3p mimic/mimic NC or inhibitor/inhibitor NC were transfected to RMa-bm cells for 48 h. Then cells were challenged with D-GalN (70 mmol/L) and LPS (100 ng/mL) for 24 h and supernatant were collected.
Techniques: Derivative Assay, Isolation, Transmission Assay, Concentration Assay, Western Blot, Control, Confocal Microscopy, Labeling, In Vitro, Protein Concentration, Sequencing, Reverse Transcription Polymerase Chain Reaction, Flow Cytometry, Transfection, Enzyme-linked Immunosorbent Assay
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