Delivery of miR-26a-5p by Subcutaneous Adipose Tissue-Derived Extracellular Vesicles Alleviates Acute Lung Injury in Mice Through CHUK/NF-κB Pathway

Figures

• 

  Figure 1. Characterization of SAT and SAT-EVs. (A) Schematic illustration of SAT-EVs extraction. (B) Subcutaneous adipose tissue was isolated from the abdominal region of mice. (C) NTA of SAT-EVs showing the size distribution and concentration. (D) TEM visualization showing the morphology of SAT-EVs (Scale bar = 100 nm). (E) WB analysis of SAT-EVs and SAT showing EV markers (Alix, CD9, Tsg101) and negative marker (Calnexin).

• 

  Figure 2. SAT-EVs in a mouse model of ARDS. (A) Schematic illustration of SAT-EVs administration in ARDS mice. (B) DID-labeled SAT-EVs were imaged in mouse lung tissue. (C) Representative immunofluorescence images of CD31, CD68, and EPCAM in mouse lung tissue (n = 5 per group). (D) Quantification of Dil-labeled SAT-EVs in mouse lung tissue, showing uptake by three types of lung cells: CD31 (endothelial cells), CD68 (macrophages), and EPCAM (epithelial cells). n = 5 per group. **p < 0.01 (CD31 vs CD68), *p < 0.05 (CD68 vs EPCAM). (E) Kaplan–Meier survival analysis demonstrating significant differences among groups (n = 12 per group; Log rank test). **p < 0.01 (Sham vs LPS), #p < 0.05 (LPS vs LPS + SAT-EVs).

• 

  Figure 3. SAT-EVs alleviate lung injury and inflammation in LPS-induced ARDS mice. (A) Histological changes in lung tissue were assessed by H&E staining at ×200 and ×400 magnification.Green arrows highlight inflammatory cell infiltration, while red arrows indicate alveolar septal thickening. (B) Lung injury scores in different groups (n = 5). (C) Lung wet-to-dry weight ratio (n = 4). (D) Total protein concentration in BALF (n = 4). (E) MPO activity in lung tissue (n = 4). (F–I) Concentrations of TNF-α, IL-6, IL-1β, and CCL2 in BALF were measured by ELISA (n = 3–4). (J–M) mRNA expression levels of TNF-α, IL-6, IL-1β, and CCL2 in lung tissue were determined by qPCR (n = 3). *P < 0.05, **p < 0.01, ***p < 0.001.

• 

  Figure 4. SAT-EVs suppress LPS-induced inflammation in RAW 264.7 cells. (A) Schematic of the experimental workflow for SAT-EV treatment. (B) Uptake of PKH26-labeled SAT-EVs by RAW 264.7 cells. (C–F) Protein levels of TNF-α, IL-6, IL-1β, and CCL2 in cell supernatants measured by ELISA (n = 3). (G–J) Relative mRNA expression of TNF-α, IL-6, IL-1β, and CCL2 analyzed by qPCR (n = 3). **p < 0.01, ***p < 0.001.

• 

  Figure 5. miR-26a-5p is enriched in SAT-EVs and targets Chuk to regulate the NF-κB signaling pathway. (A) Relative expression level of miR-26a-5p in SAT-EVs determined by qRT-PCR. (B) Evolutionary conservation of miR-26a-5p across species. (C) Venn diagram showing the overlap between predicted miR-26a-5p target genes and ARDS-related genes. (D) Relative expression analysis of miR-26a-5p and Chuk. (E) Predicted binding site between miR-26a-5p and the 3′UTR of Chuk. (F) Dual-luciferase reporter assay confirming direct targeting of Chuk by miR-26a-5p (n = 5; **p < 0.01). (H) CHUK protein levels following siRNA-mediated knockdown. (G) Western blot analysis of CHUK expression following siRNA-mediated knockdown. (H and I) Volcano plot and heatmap showing differentially expressed genes between si-Chuk and si-control groups. (J) KEGG pathway analysis showing enrichment of NF-κB signaling–related genes among DEGs.

• 

  Figure 6. SAT-EVs modulate the CHUK/NF-κB signaling pathway in vitro and in vivo. (A) miR-26a-5p expression in RAW 264.7 cells following LPS and/or SAT-EV treatment. (B–D) Western blot analysis of CHUK, IκB-α, p-IκB-α, p65, p-p65, iNOS, IL-1β, and PTGS2 in RAW 264.7 cells. (E) miR-26a-5p expression in lung tissues from each experimental group. (F–H) Protein expression of CHUK/NF-κB signaling–related molecules and inflammatory markers in lung tissues. (I) Immunofluorescence staining of iNOS, IL-1β, and PTGS2 in lung tissue sections. n = 3 per group. **p < 0.01, ***p < 0.001.

• 

  Figure 7. Inhibition of miR-26a-5p abolishes the anti-inflammatory effects of SAT-EVs in LPS-stimulated RAW 264.7 cells. (A–D) ELISA analysis of TNF-α, IL-6, IL-1β, and CCL2 protein levels in cell supernatants. (E–H) Relative mRNA expression of TNF-α, IL-6, IL-1β, and CCL2 in cells. (I) miR-26a-5p expression following inhibitor transfection. (J–P) Western blot analysis of CHUK, IκB-α, p-IκB-α, p65, p-p65, iNOS, IL-1β, and PTGS2 in cells treated with LPS, SAT-EVs, and/or miR-26a-5p inhibitor. n = 3–5 per group. **p < 0.01, ***p < 0.001.

• 

  Figure 8. The protective effects of SAT-EVs in ARDS mice are dependent on miR-26a-5p. (A) Kaplan–Meier survival analysis of ARDS mice treated with SAT-EVs and/or miR-26a-5p antagomir (n = 12). (B and C) Representative H&E staining and lung injury scores, Green arrows highlight inflammatory cell infiltration, while red arrows indicate alveolar septal thickening. (D–F) Lung wet/dry ratio, BALF total protein, and MPO activity concentration. (G–J) BALF concentrations of TNF-α, IL-6, IL-1β, and CCL2 measured by ELISA. (K–N) mRNA expression levels of TNF-α, IL-6, IL-1β, and CCL2 assessed by qPCR. (O) miR-26a-5p expression in lung tissue. (P and Q) Western blot analysis of CHUK, IκB-α, p-IκB-α, p65, p-p65, iNOS, IL-1β, and PTGS2. (R) Immunofluorescence staining of iNOS, IL-1β, and PTGS2 in lung tissues. n = 3 per group unless otherwise specified. **p < 0.01, ***p < 0.001.

• 

  Figure 9. Schematic illustration of SAT-EVs–mediated regulation of ARDS via miR-26a-5p targeting the CHUK/NF-κB signaling pathway. SAT-EVs mitigate inflammation in ARDS by delivering miR-26a-5p to macrophages. miR-26a-5p targets CHUK, thereby inhibiting the phosphorylation of IκB-α and preventing NF-κB nuclear translocation. This pathway suppresses the expression of pro-inflammatory cytokines, including TNF-α, IL-1β, IL-6, and iNOS, ultimately alleviating the cytokine storm and attenuating ARDS-related lung injury.