Fig. 3. Splenic macrophages promote hepatic macrophage CCL2 secretion via the upregulation of SOCS3 signaling. (A) Hepatic and splenic macrophages were isolated from fibrotic or healthy control rats and cultured ex vivo. Hepatic macrophages were stimulated with conditioned medium derived from splenic macrophages. After stimulation, CCL2 mRNA and protein levels were measured by qPCR and ELISA, respectively. (B) Livers were sectioned and SOCS3 and CD68 co-staining was examined using immunofluorescence. Magnification: ×630 scale bar: 10 µm. (C) SOCS3 mRNA and protein levels were measured by qPCR and western blotting, respectively. (D) Hepatic macrophages were stimulated with conditioned medium from splenic macrophages transfected with SOCS3-specific or scrambled siRNA. SOCS3 and CCL2 mRNA levels were determined by qPCR. Statistics: two-way ANOVA (A,C,D: n = 5; B: n = 10). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, Ctrl SpMφ-CM group vs. CCl₄ SpMφ-CM group, Spx group vs. Sham group, or SOCS-siR group vs. scrambled siR group. #P<0.05, ##P<0.01, ###P<0.001, ####P<0.0001, Ctrl group vs. CCl₄ i.p. group. Abbreviations: HMφ, hepatic macrophage; SpMφ, splenic macrophage; CM, conditioned medium.