Figure 3.

Increased miR-9-5p in hepatocyte-EVs induced by PA induces macrophage inflammation. LO2 cells were treated with different concentrations of PA (200 μM and 400 μM PA) followed by extraction of EVs. A, Lipid droplets in LO2 cells detected by Oil red O staining. B, miR-9-5p level in LO2 cells-EVs measured by RT-qPCR. C, Uptake of PKH67-labeled PA-EVs by THP-1 cells. PKH67-labeled EVs were incubated with THP-1 cells at 37 ℃ for 12 h and then observed under a fluoresence microscope. Green represents PKH67-labeled PA-EVs, blue represents DAPI-stained nuclei, and red indicates phalloidin-labeled cytoskeleton. D, miR-9-5p level in PA-EVs from hepatocytes measured by RT-qPCR. E, Levels of IL-1β, IL-6, TNF-α and iNOS in PA-EVs from hepatocytes measured by RT-qPCR. F, Positive rate of CD86⁺ CD11b⁺ elevated in PA-EVs from hepatocytes detected by flow cytometry. G, IL-1β, IL-6, TNF-α and iNOS levels in the supernatant determined with ELISA. Macrophages were treated with miR-9-5p mimic or miR-9-5p inhibitor. H, miR-9-5p level in macrophages measured by RT-qPCR. I, Levels of IL-1 β, IL-6, TNF-α and iNOS in macrophages measured by RT-qPCR. J, Expression of inflammatory factors in cell supernatant of macrophages determined by ELISA. K, Positive rate of CD86⁺ CD11b⁺ in macrophages detected by flow cytometry. N = 3. * p < 0.05 vs. LO2 cells treated with Control/mimic-NC/Inhibitor-NC. Data are shown as the mean ± standard deviation of three independent experiments. Unpaired t-test was used for analysis differences between two groups. Data among multiple groups were compared using one-way ANOVA with Tukey post hoc tests used.