Fig 2: TGFBI orchestrates macrophages through enhancing PDGF-B expression.A Western blot analyses of PDGF-B in BMDM or RAW264.7 treated with rTGFBI (ng/mL) with the indicated concentrations (n = 3 independent experiments). B RT-qPCR analyses of PDGF-B in BMDM or RAW264.7 treated with rTGFBI (ng/mL) with the indicated concentrations (n = 3 independent experiments). C Western blot analysis of the indicated proteins in BMDM or RAW264.7 treated with or without rTGFBI (200 ng/mL) for 6 h. D RT-qPCR analysis of Pdgfb in BMDM or RAW264.7 treated with rTGFBI (200 ng/mL) alone or in combination with integrin αvβ3 or αvβ5 neutralizing antibody (n = 3 independent experiments). E Flow cytometry analysis and statistical analysis of the percentage of Trem2+CD9+ subpopulation in BMDM or RAW264.7 transfected with the indicated siRNAs for 24 h and treated with or without rTGFBI (200 ng/mL) for 24 h (n = 3 independent experiments). F RT-qPCR and Western blot analysis of α-SMA in HSCs cocultured with BMDMs transfected the indicated siRNAs for 24 h and treated with or without rTGFBI (200 ng/mL) for 24 h (n = 3 independent experiments). G RT-qPCR and Western blot analyses of α-SMA in HSCs treated with rTGFBI or in combination with SU11248 (n = 3 independent experiments). Mice samples (n = 4–6 per group). Data were presented as the mean ± SDs; scale bars, 50 µm; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns not significant (Student’s t test).

Fig 3: TGFBI can promote HSC proliferation and activation directly.A, B RT-qPCR (A) and Western blot (B) analyses of α-SMA expression in WT and Tgfbi−/− mice livers of CCl4 and BDL models. C IF staining of α-SMA and Ki67 in primary HSC from WT and Tgfbi−/− mice. D, E RT-qPCR (D) and Western blot (E) analysis of α-SMA in HSC or LX2 treated with rTGFBI (ng/mL) with the indicated concentrations (n = 3 independent experiments). F IF staining of α-SMA in primary HSC or LX2 after rTGFBI (200 ng/mL) treatment for 6 h (n = 3 independent experiments). G Western blot analysis of the indicated proteins in HSCs treated with rTGFBI (200 ng/mL) for 6 h (n = 3 independent experiments). H Western blot analysis of the indicated proteins in HSCs treated with rTGFBI (200 ng/mL) alone or in combination with integrin αvβ3 or αvβ5 neutralizing antibody (n = 3 independent experiments). I Western blot analysis of the indicated proteins in HSCs treated with rTGFBI (200 ng/mL) or in combination with the indicated inhibitors for 6 h (n = 3 independent experiments). J Relative mRNA levels of Acta2 in HSCs treated with rTGFBI (200 ng/mL) alone or in combination with integrin αvβ3 or αvβ5 neutralizing antibody for 6 h (n = 3 independent experiments). K Relative mRNA levels of Acta2 in HSCs or LX2 treated with rTGFBI (200 ng/mL) or in combination with the indicated inhibitors for 6 h (n = 3 independent experiments). Mice samples (n = 4–6 per group). Data were presented as the mean ± SDs; scale bars, 50 µm; *p < 0.05 **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig 4: TGFBI deficiency remarkably alleviates mice liver inflammation and fibrosis.A H&E, Sirius, Masson staining, and IHC staining of COL1A1 in the fibrotic livers of WT and Tgfbi −/− mice of CCl4 and BDL models. B The measurement of serum ALT and AST in mice. C TUNEL staining and quantitative analysis of apoptosis in CCl₄ and BDL model livers. D RT-qPCR analysis of Col1a1, Col3a1, and Timp-1 expression. E IF staining of F4/80 in mouse fibrotic livers. F IHC staining of CD68 in mouse livers of CCl4 and BDL models. G RT-qPCR analysis of Ccl2, Il6, and Tnf-a expression. Mice samples (n = 4–6 per group). Data were presented as the mean ± SDs; scale bars, 50 µm; *p < 0.05 **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig 5: TGFBI promotes HSC activation in a PDGFRβ dependent manner.A Heat map of differentially expressed genes drawn based on RNA sequencing data. Samples were obtained from oil-treated WT mice, CCl4-induced WT mice, and Tgfbi−/− mice liver tissue lysates. B GO gene analysis of the differential proteins. C IF staining and quantitative analysis of TGFBI and PDGFRβ in human fibrotic liver tissue. D Correlation analysis of expression between Tgfbi and Pdgfrb in the Human Chronic Liver Disease database (n = 58 independent samples). E Western blot analysis of PDGFRβ expression in WT and Tgfbi−/− mice livers of CCl4 and BDL models. F RT-qPCR analysis of PDGF-B in WT and Tgfbi−/− mice livers of CCl4 and BDL models. G Relative mRNA levels of PDGFRβ in primary liver parenchymal cells, HSCs, and macrophages isolated from mice livers of CCl4 and BDL models (n = 3 independent experiments). H Western blot and RT-qPCR analysis of PDGFRβ expression in HSC treated with rTGFBI (200 ng/mL) for 6 h (n = 3 independent experiments). I Western blot and RT-qPCR analysis of α-SMA and PDGFRβ in HSC treated with rTGFBI (200 ng/mL) alone or in combination with integrin αvβ3 or αvβ5 neutralizing antibody for 6 h (n = 3 independent experiments). J Western blot and RT-qPCR analyses of PDGFRβ in HSC treated with rTGFBI (200 ng/mL) alone or in combination with the indicated inhibitors for 6 h (n = 3 independent experiments). Mice samples (n = 4–6 per group). Data were presented as the mean ± SDs; scale bars, 50 µm; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns not significant (Student’s t test).