Figure 2.

Adipose-derived exosomal miR-122-5p promotes the pulmonary endothelial barrier by inhibiting EndMT and oxidative stress through down-regulating the TGF-β1/TGF-βR1/Smad2 pathway in mice with ARDS. (a) Dual-Luciferase reporter assay showed that the CDS of mouse TGF-βR1 is a direct target of miR-122-5p. The firefly luciferase activity of the reporter containing the CDS-WT of mouse TGF-βR1 was decreased to 80.62% by miR-122-5p, which was blocked by the reporter containing the CDS-MUT of TGF-βR1 (complementary mutation). (b) Alterations of lung tissue by H&E, Masson's trichrome staining, and ROS production showed that the recombinant mouse TGF-β1 (10 μg/mL) partially abolished the inhibitory effects of adipose-derived exosomal miR-122-5p on EndMT, whereas pretreatment with SB431542 (10 mg/kg) restored these favorable effects in mouse model of ARDS. The collagen content (c) PaO₂ (d), BALF protein (e), EBDA extravasation (f), wet/dry ratio (g), and free GSH concentration (h) in mice with ARDS treated with adipose-derived exosomal miR-122-5p in the presence or absence of recombinant mouse TGF-β1 and SB431542. (i) Western blot analysis of the protein expression of VE-cadherin, β-catenin, α-SMA, and Vimentin in lungs. Relative abundances of protein bands were normalized to β-actin as shown in the bar graphs. (j) Western blot analysis of the protein expression of TGF-β1, and the level of p-TGF-βR1 and p-Smad2 in lungs. Relative abundances of protein bands were normalized to β-actin as shown in the bar graphs. Relative phosphorylation levels of protein are expressed normalized to the corresponding total protein. Representative images are shown from three replicated independent experiments. n = 6 per group (a). n = 3 mice per group ((b), (i), (j)). n = 5 mice per group (c)–(h). Data are presented as mean ± S.D. Significant differences are shown by ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.