Fig. 7
Knocking down HMGB3 attenuates the inflammatory activation and migration induced by SiO2-Exo in macrophages. (A-B). RT–PCR and western blot analysis of HMGB3-silenced RAW264.7 macrophages. (C). Western blot analysis of HMGB3 expression in exosomes derived from macrophages transfected with siNC or siHMGB3. (D). ELISA analysis of the expression of IL-1β, IL-6 and TNF-α in the SN of RAW264.7 macrophages treated with PBS, SiO2 + siNC-Exo, or SiO2 + siHMGB3-Exo. n = 3 each group. (E). Transwell assay of the migration of RAW264.7 macrophages treated with PBS, SiO2 + siNC-Exo, or SiO2 + siHMGB3-Exo. The scale bar represents 50 μm. (F). Western blot analysis of the expression of p-p65, p-STAT3, p-ERK1/2, p-p38, CCR2 and pro-IL-1β in RAW264.7 macrophages treated with PBS, SiO2 + siNC-Exo, or SiO2 + siHMGB3-Exo. (G). Flow chart showing the process of exosome administration in mice. (H). The distribution of PKH26-labelled exosomes in mice was observed by an in vivo Xtreme II system, and PKH26-labelled exosomes in peripheral blood were observed by fluorescence microscopy. The scale bar represents 20 μm. (I). HE staining of lung tissue from mice treated with PBS, SiO2 + siNC-Exo, or SiO2 + siHMGB3-Exo. n = 5 mice per group. (J). Flow cytometry gating strategy for CD11b+/F4/80+ subsets. Flow cytometric analysis of the proportions of iNOS+ or CD206+ macrophages in the lung tissue of mice treated with PBS, SiO2 + siNC-Exo, or SiO2 + siHMGB3-Exo. n = 5 mice per group. The data are representative of three individual experiments and expressed as the mean ± SEM. The data were analysed by Student’s t test or two-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Abbreviations SiO2 = silica dust; SN = cell culture supernatant; SiO2 + siNC-Exo = exosomes derived from SiO2-exposed macrophages transfected with siNC; SiO2 + siHMGB3-Exo = exosomes derived from SiO2-exposed macrophages transfected with siHMGB3