Enhanced anti-inflammatory efficacy of MV-DEX in vitro. GECs were stimulated with LPS and treated with the indicated concentrations of MV-DEX or free DEX for 12 h. (A) Effects of MV-DEX or free DEX on NF-κB luciferase reporter activity in LPS-induced GECs. Compared with free DEX, MV-DEX showed a 5-fold increase in inhibiting NF-κB activity. (B, C) Real-time PCR analysis of inflammatory cytokines (TNF, IL-6, IL-β and CCL-2) in GECs. TNF (D) and IL-6 (E) levels in the supernatants were detected by ELISA. (F) The expression of p65 and p-p65 were analyzed by western blot. (G) Immunofluorescent staining against p-p65 was observed under confocal microscope (scale bar, 10 μm). Both MV-DEX and free DEX exhibited anti-inflammatory efficacy, but MV-DEX was much more superior. (H-J) MV-DEX and free DEX were stored at 37 ℃ for 10 days to destroy most MVs, whereas the drug concentration between two groups remained the same (with 5 μmol/L DEX). NF-κB luciferase reporter activity was shown as fold change to renilla luciferase activity (H). ELISA detection of TNF (I) and IL-6 (J) levels in supernatants. Following MV destruction, the MV-DEX group no longer showed a superior inhibition of inflammation compared with free DEX. Data are presented as mean ± SD, ** p<0.01, *** p<0.001 vs. LPS-treated group, # p<0.05, ## p<0.01, ### p<0.001, N.S., not significant, one-way ANOVA.