Fig. 4

Asperosaponin VI activates the PPAR-γ in hippocampal microglia of CMS mice and in primary microglia. A Levels of p-PPAR-γ, PPAR-γ 1 and PPAR-γ 2 in hippocampus of Ctrl or CMS mice after treatment with saline or ASA VI. Levels of PPAR-γ-1 and PPAR-γ-2 were normalized to those of β-actin. Levels of phosphorylated PPAR-γ were normalized to those of PPAR-γ-1 and PPAR-γ-2 (n = 3, each sample in triplicate). B Fluorescence micrographs showing PPAR-γ expression in microglia (as indicated by the white arrow) of hippocampus in CMS mice after treatment with ASA VI. PPAR-γ was stained with antibody (green), microglia were stained with an anti-Iba1 antibody (red), and nuclei were stained with DAPI (blue). The yellow arrow indicates the high-resolution cells inserted in the upper right. The blue arrow indicates that the nuclear transfer of PPAR-γ is in the hippocampal microglia. Scale bar, 20 μm. C Fluorescence micrographs showing PPAR-γ expression in Arg-1⁺ cells (as indicated by the white arrow) of hippocampus in CMS mice after treatment with ASA VI. Scale bar, 10 μm. D Schematic illustrating ASA VI switches activated microglia from a pro-inflammatory (iNOS⁺) to anti-inflammatory (Arg-1⁺) phenotype via the PPAR-γ activation in hippocampus of CMS mice. E Experimental scheme to monitor the effect of GW9662 on PPAR-γ in Ctrl, lipopolysaccharide (LPS), LPS + ASA VI and LPS + ASA VI + GW9662 in primary microglia. F Fluorescence micrographs showing nuclear translocation of PPAR-γ in Ctrl, LPS, LPS + ASA VI and LPS + ASA VI + GW9662 microglia. PPAR-γ was stained with PPAR-γ antibody (green), Arg-1 in microglia were stained with an anti-Arg-1 antibody (red), and nuclei were stained with DAPI (blue). Scale bar, 10 μm. G Effects of GW9662 treatment on levels of p-PPAR-γ in Ctrl, LPS, LPS + ASA VI and LPS + ASA VI + GW9662 microglia (n = 3, each sample in triplicate). H Levels of mRNAs encoding the pro-inflammatory cytokines (TNF-α, iNOS, IL-1β, and IL-6) and the anti-inflammatory cytokines (Arg-1, IL-10 and TGF-β) and BDNF in Ctrl, LPS, LPS + ASA VI and LPS + ASA VI + GW9662 microglia. Data are displayed with mean ± SEM (n = 3–5), A *p < 0.05, ***p < 0.001 (two-way ANOVA with Tukey’s multiple-comparisons test). G and H *p < 0.05, *p < 0.01, ***p < 0.001 vs. Ctrl group, ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 vs. LPS group, ^&p < 0.05, ^&&&p < 0.001 vs. LPS + ASA VI group (one-way ANOVA with Tukey’s multiple-comparisons test)