Fig 1: Ihh signaling is crucial for macrophage β-catenin-mediated hepatic fibrosis in HFD-induced MASH. To restore Ihh expression in macrophages, mice were administered the Ihh plasmid via polyethylenimine nanoparticles, namely in vivo jetPEI-Man. A Representative Sirius Red staining images of liver sections from HFD-fed β-cateninFL/FL and β-cateninM−KO mice. These mice were either treated or not treated with jetPEI-Man-Ihh (jetPEI-Ihh) or jetPEI-Man-GFP (jetPEI-GFP) (n = 5 mice per group). Scale bars are 100 μm. B Immunofluorescence images of α-SMA+ myofibroblasts in liver sections from HFD-fed β-cateninFL/FL and β-cateninM−KO mice. These mice were either treated or not treated with jetPEI-Ihh or jetPEI-GFP (n = 5 mice per group). Scale bars measure 50 μm. C Relative mRNA levels of fibrogenic genes such as Acta2, Col1α1, Col3α1, and Timp1 in the livers of HFD-fed β-cateninFL/FL and β-cateninM−KO mice. These mice were either treated or not treated with jetPEI-Ihh or jetPEI-GFP (n = 5 mice per group). Data are expressed as the mean ± standard deviation (SD). *P < 0.05, **P < 0.01
Fig 2: Schematic representation of the macrophage β-catenin-Ihh axis in modulating the activation of hepatic stellate cells and fibrosis in MASH. The consumption of a high-fat diet (HFD) led to the activation of β-catenin in liver macrophages. The activation of macrophage β-catenin triggered the expression and secretion of Ihh, which in turn facilitated the activation of hepatic stellate cells and fibrosis in MASH
Fig 3: Deficiency of myeloid β-catenin inhibits Ihh expression in macrophages in HFD-induced MASH. A Relative mRNA levels of Shh and Ihh in liver macrophages isolated from HFD-fed β-cateninFL/FL and β-cateninM−KO mice were determined. B Western blot was performed to analyze the expression of Ihh in liver macrophages from HFD-fed β-cateninFL/FL and β-cateninM−KO mice. C Double immunofluorescence staining was carried out on liver sections using antibodies against F4/80 (green) and Ihh (red). The nuclei were stained with DAPI (blue). There were five mice in each group. The scale bar represents 200 μm. The data are shown as the mean ± standard deviation (SD). **P < 0.01
Fig 4: Macrophage β-catenin-induced Ihh promotes HSC activation. A The schematic illustration of the co-culture model that utilizes bone marrow-derived macrophages (BMDMs) and primary murine hepatic stellate cells (HSCs). B The relative mRNA levels of fibrogenic genes such as Acta2, Col1α1, and Col3α1 in HSCs were measured after co-culturing with BMDMs from β-cateninFL/FL and β-cateninM−KO mice. C Immunofluorescence pictures of α-SMA expression in HSCs were obtained after co-culturing with BMDMs from β-cateninFL/FL and β-cateninM−KO mice. The scale bars represent 100 μm. D Immunofluorescence images of α-SMA expression in HSCs were captured after co-culturing with BMDMs transfected with the Lv-β-catenin plasmid or the control vector (Lv-GFP). The scale bars are 100 μm. E Immunofluorescence images of α-SMA expression in HSCs were taken after co-culturing with Lv-β-catenin-transfected BMDMs, either in the presence or absence of Ihh neutralizing antibody. The scale bars measure 100 μm. F The mRNA levels of Gli1, Gli2, and Gli3 in HSCs were determined after co-culturing with BMDMs from β-cateninFL/FL and β-cateninM−KO mice. The data are presented as the mean ± standard deviation (SD). *P < 0.05, **P < 0.01
Fig 5: Macrophage β-catenin induces Ihh expression and secretion. Bone marrow-derived macrophages (BMDMs) were treated with LPS (100 ng/ml) and PA (250 µM) or PBS for 12 h. A The mRNA level of Ctnnb1 in BMDMs following treatment with PA/LPS or PBS. B Western blot analysis was carried out to assess the expression of A-β-catenin in BMDMs after treatment with PA/LPS or PBS. C BMDMs underwent a ChIP-PCR assay using an anti-β-catenin or IgG antibody to detect the potential β-catenin binding site within the Ihh intron 1. D The mRNA level of Ihh in BMDMs isolated from β-cateninFL/FL and β-cateninM−KO mice. E The concentration of Ihh in the media of BMDMs isolated from β-cateninFL/FL and β-cateninM−KO mice was measured by ELISA. The data are presented as the mean ± standard deviation (SD). **P < 0.01
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