Journal: Frontiers in Pharmacology

Article Title: Effective delivery of osteopontin small interference RNA using exosomes suppresses liver fibrosis via TGF-β1 signaling

doi: 10.3389/fphar.2022.882243

Figure Lengend Snippet: Accumulation of OPN during progression of liver fibrosis murine model. (A) Volcano plots showing the number of differentially expressed genes (DEGs) in GSE55747; (B) Heatmap for the top 20 DEGs in GSE55747. (C) Protein-protein interaction networks (PPI) of OPN and ECM-related genes according to GSE71379. (D,H) Representative H&E staining (D) of liver sections from CCl 4 -treated and olive oil vehicle-treated mice. (H) Percentage of degenerated and necrotic hepatocytes per visual field. n = 4 mice per group; Scale bar: 50 μm. (E,I) Sirius red staining (E) in each group with indicated treatments. (I) Percent sirius red positive areas per visual field (%). n = 4 mice per group; Scale bar: 50 μm. (F,G,J,K) α−SMA (F) and OPN (G) immunofluorescence (IF) staining in mice (5 visual fields for each tissue analyzed). The average number of α−SMA (J) and OPN (K) positive cells. n = 4 mice per group; Scale bar: 200 μm. Data in are expressed as mean ± SD. Individual dots represent distinct mice in graphs. Unpaired t–test was used in fig (D–G,J,K) . p < 0.001 ***, p < 0.0001 ****.

Article Snippet: Frozen sections were blocked for one hour with 1% BSA and then incubated overnight with anti α -SMA (proteintech, 1:200), OPN (Servicebio, 1:50), HMGB1 (Servicebio, 1:100) and TGF-β1 (Servicebio, 1:100).

Techniques: Staining, Immunofluorescence

Journal: Frontiers in Pharmacology

Article Title: Effective delivery of osteopontin small interference RNA using exosomes suppresses liver fibrosis via TGF-β1 signaling

doi: 10.3389/fphar.2022.882243

Figure Lengend Snippet: Accumulation of OPN activated primary HSCs. (A) Primary HSCs were isolated from wild-type (WT) mice. Images of IF staining for α−SMA (red) and DAPI (blue) of HSCs. Scale bar: 200 μm. (B) OPN expression at protein level by western blot analysis. (C) qPCR analysis of OPN in primary HSCs after transfection with siRNA. (D) Proliferated cells detected by staining of EdU (left panel). The percentages of EdU-positive cells were quantified (right panel). Scale bar: 200 μm. Data are expressed as the mean ± SD. (C,D) unpaired t–test. P < 0.01 **, p < 0.0001 ****.

Article Snippet: Frozen sections were blocked for one hour with 1% BSA and then incubated overnight with anti α -SMA (proteintech, 1:200), OPN (Servicebio, 1:50), HMGB1 (Servicebio, 1:100) and TGF-β1 (Servicebio, 1:100).

Techniques: Isolation, Staining, Expressing, Western Blot, Transfection

Journal: Frontiers in Pharmacology

Article Title: Effective delivery of osteopontin small interference RNA using exosomes suppresses liver fibrosis via TGF-β1 signaling

doi: 10.3389/fphar.2022.882243

Figure Lengend Snippet: iExosomes targeting OPN inhibit liver fibrosis in vivo . (A,B,H) Relative Opn expression analyed by qPCR analysis (A) and IF staining (B) . (H) The average number of OPN positive cells in each visual field. Untreated, Exo, and iExo siRNA−Cntrl : n = 4 mice per group; iExo siRNA−OPN , siRNA-OPN: n = 5 mice per group. Scale bar: 200 μm (inserted scale bar: 50 μm). (C,D) Col1a1 (C) and Acta2 (D) expression by qPCR analysis in liver from mice subjected to exosomes electroporated with siRNA or nake siRNA. n = 4 mice per group. (E,I) Sirius red staining (E) of listed liver sections. (I) Percentage of sirius red positive area in each visual field. Untreated, Exo, and iExo siRNA−Cntrl : n = 4 mice per group; iExo siRNA−OPN , siRNA-OPN: n = 5. Scale bar: 200 μm. (F,J) α−SMA IF staining (F) in each group. (J) The average number of α−SMA positive cells in each visual field. Untreated, Exo, and iExo siRNA−Cntrl : n = 4 mice per group; iExo siRNA−OPN , siRNA-OPN: n = 5. Scale bar: 200 μm. (G,K) H&E staining (G) of liver with the indicated treatment. (K) Percentage of necrotic and degenerated hepatocytes. Untreated, Exo, and iExo siRNA−Cntrl : n = 4 mice per group; iExo siRNA−OPN , siRNA-OPN: n = 5. Scale bar: 200 μm. (L) Serum ALT (left panel) and AST (right panel) levels in the listed groups. Untreated, Exo, and iExo siRNA−Cntrl : n = 4 mice per group; iExo siRNA−OPN , siRNA-OPN: n = 5 mice per group. (M) Hydroxyproline content in rat livers. n = 3 mice per group. The data are expressed as Sidak’s post–hoc analysis. P < 0.05 *, p < 0.01 **, p < 0.001 ***, p < 0.0001 ****.

Article Snippet: Frozen sections were blocked for one hour with 1% BSA and then incubated overnight with anti α -SMA (proteintech, 1:200), OPN (Servicebio, 1:50), HMGB1 (Servicebio, 1:100) and TGF-β1 (Servicebio, 1:100).

Techniques: In Vivo, Expressing, Staining