Fig. 5

The effects of short-term activated HSC-sEV-specific miR-99a-5p on primary rat liver macrophage (KC) and human THP-1 macrophage differentiation. (A, E) The expression of miR-99a-5p and the mRNAs of 8 genes associated with HSC-sEV-induced macrophage differentiation in miR-99a-5p mimic-treated primary rat KCs (A) and human THP-1 macrophages (E) was determined by RT-qPCR. The relative miR-99a-5p expression was normalized to that of U6snRNA, and the relative gene expression was normalized to that of 18 S rRNA (or β-Actin) and then to miRNA mimic negative control (mimic-NC). Red font, highly expressed in 3dHSC-sEV cocultured KCs, green font, highly expressed in 14dHSC-sEV cocultured KCs. (B, F) Surface marker expression in miR-99a-5p mimic-treated primary rat KCs (B) and human THP-1 macrophages (F) was determined by flow cytometry. Representative flow cytometry images and statistical histograms for CD86- and CD206-positive cells in each group are provided. (C, G) The protein expression of INOS and CD206 in miR-99a-5p mimic-treated primary rat KCs (C) and human THP-1 macrophages (G) was detected by western blotting, β-Actin served as a loading control and was normalized to mimic-NC treated cells. (D, H) Cytokine array analyses for culture media from primary KCs (D) and human THP-1 macrophages (H) treated by miR-99a-5p mimic and control (mimic-NC). Inflammatory factors are listed on the right side. Each row represents an individual inflammatory factor, and each column represents an individual sample. Red font, proinflammatory factors; green font, anti- inflammatory factors. The scale represents normalized log2 inflammatory factor expression levels. Statistical significance was determined by Student’s t test, *** p < 0.001, ** p < 0.01, * p < 0.05