Fig. 4

NAFLD hepatic sEVs induce microvascular endothelial hyperpermeability by activating Cathepsin B-dependent NLRP3 inflammasome axis. A MVECs were pretreated with or without 20 μmol/L dynasore for 40 min, followed by incubation of 120 μg/mL DiI-labeled hepatic sEVs for 8 h. Representative fluorescent confocal images of DiI-labeled hepatic sEVs (red) with DAPI (blue), n = 4 per group. The pellet derived from the ultracentrifugation of DiI alone was used as a vehicle control. Scale bar, 10 μm. B Representative western blot bands and the summarized data determined by densitometric analysis, n = 4 per group. C Representative flow cytometry images of caspase-1 FLICA staining and the summarized data of the positive cells, n = 4 per group. The fold changes were obtained by calculating the ratio of the positive cells of the treated groups to the MCS or ND group. D Representative Western blot bands and the summarized data determined by densitometric analysis, n = 4 per group. E Relative permeability of endothelial monolayer to FITC-dextran, n = 4 per group. F Representative images of acridine orange (AO) and magic red (MR) staining, n = 4 per group. Scale bar, 20 μm. G MVECs were incubated with 120 μg/mL hepatic sEVs for 24 h with or without the presence of 5 μmol/L CA-074. Representative flow cytometry images of caspase-1 FLICA staining, n = 4 per group. The fold changes were obtained by calculating the ratio of the positive cells of the treated groups to the MCS or ND group. H Relative permeability of endothelial monolayer to FITC-dextran, n = 4 per group. Data are expressed as the mean ± SEM. Statistics: Student t-test (B, D) and One-way ANOVA (C, E, G, H), *P < 0.05, **P < 0.01 vs. MVECs incubated with MCS hepatic sEVs; ^#P < 0.05, ^##P < 0.01 vs. MVECs incubated with MCD hepatic sEVs; ^&P < 0.05, ^&&P < 0.01 vs. MVECs incubated with ND hepatic sEVs; ^$P < 0.05, ^$$P < 0.01 vs. MVECs incubated with HFD hepatic sEVs