Extracellular histones stimulate collagen expression in vitro and promote liver fibrogenesis in a mouse model via the TLR4-MyD88 signaling pathway

Figure 1 Circulating histones are elevated in ICR mice treated with CCl₄. Typical western blots of histone H3 standard and histone H3 in plasma from mice treated with the first dose of CCl₄ (A) and CCl₄ + non-anticoagulant heparin (NAHP) (B), and a typical H3 blot before and 24 h after the last dose of CCl₄ (C); D: The mean ± SD of circulating histones at different time points, including 0 h (before the experiment), and 4, 8, 24, 48 and 84 h after first dose of CCl₄ (orange) and CCl₄ + NAHP (blue) of each week are presented. ANOVA test, ^aP < 0.05 compared to time 0; E: The mean ± SD of peak circulating histones (8 and 24 h after first CCl₄ injection of each week). ANOVA test, ^aP < 0.05 compared to time 0 (before first injection). The mean ± SE of blood alanine aminotransferase levels (F) and liver injury scores (G) from nine mice in each group following 4 wk of treatment. ANOVA test, ^aP < 0.05 compared to mice without treatment (before). ^bP < 0.05 compared to CCl₄ group. ANOVA: Analysis of variance; NAHP: Non-anticoagulant heparin; ALT: Alanine aminotransferase.

Figure 2 Extracellular histones induced collagen I and α-smooth muscle actin production in LX2 cells. A: Typical western blots of collagen I in medium (upper panel) and lysates (middle panel) of LX2 cells treated with different concentrations of histones at day 6. Lower panel: β-actin in the cell lysates; B: The mean ± SD of the relative percentages of collagen/β-actin ratios with untreated LX2 cells set at 100% from five independent experiments. Analysis of variance test, ^aP < 0.05 compared to untreated cells; C: Immunofluorescent staining of LX2 cells with anti-collagen I and anti-α- smooth muscle actin (SMA) antibodies. Control: Cells were treated with culture medium without histones for 6 d. Histones: Cells were treated with culture medium + 5 μg/mL histones. Typical images are shown. White arrows indicate staining for collagen I, orange arrows indicate staining for α-SMA. Bar = 50 m. α-SMA: α-smooth muscle actin.

Figure 3 Effect of anti-histone reagent, non-anticoagulant heparin, on histone-enhanced collagen I production in LX2 cells and CCl₄-induced fibrosis in mice. A: Typical western blots of collagen I in LX2 cells treated with histones or histones + non-anticoagulant heparin (NAHP). Beta-actin is shown as a loading reference; B: The mean ± SD of relative percentage of collagen/actin ratios in untreated LX-2 cells set at 100% from three independent experiments. ANOVA test, ^aP < 0.05 compared to untreated cells. ^bP < 0.05 compared to cells treated with 5 μg/mL histones; C: Typical images of stained liver sections (hematoxylin and eosin staining: a-c; immunohistochemical staining with anti-α-SMA: d-f; and Sirius red staining: g-i) from normal mice (a, d, g); mice treated with CCl₄ (b, e, h) and mice treated with CCl₄ + NAHP (CCl₄ + H) (c, f, i). Arrows in b and c: Black indicate hepatocyte swelling and white indicate necrosis and immune cell infiltration. Arrows in e and f: Indicate staining for smooth muscle actin. Arrows in h and i: Indicate collagen deposition. The mean ± SD of percentage of areas of staining for α-SMA (D) and Sirius red staining for collagen (E) (nine mice per group, and six randomly selected sections per mouse). ANOVA test, ^aP < 0.05 compared to controls. ^bP < 0.05 compared to CCl₄ alone; F: The mean ± SD of hydroxyproline levels in liver tissues from nine mice per group. ANOVA test, ^aP < 0.05 compared to controls. ^bP < 0.05 compared to CCl₄ alone. α-SMA: α-smooth muscle actin; ANOVA: Analysis of variance; NAHP: Non-anticoagulant heparin.

Figure 4 TLR4 is involved in histone-enhanced collagen I production and CCl₄-induced mouse liver fibrosis. A: LX2 cells were treated with 5 μg/mL histones (Hist) in the presence or absence of TLR4 neutralizing antibody (TLR4i). The mean ± SD of the relative percentage of collagen Ι/β-actin ratios are presented with control (UT) set at 100% from three independent experiments. ANOVA test, ^aP < 0.05 compared to UT. ^bP < 0.05 compared to histone alone; B: Typical images of Sirius red staining of liver section from untreated wt C57BL/6j mice (a), CCl₄-treated wt mice (b), and TLR4 and MyD88 knockout mice; C: The mean ± SD of stained areas of liver sections from untreated wt mice (UT), CCl₄-treated wt mice (WT), CCl₄-treated TLR4^-/- and MyD88^-/- mice. Eight mice were in each group and six sections from each mouse were analyses. ANOVA test, ^aP < 0.05 compared to untreated wt mice (UT). ^bP < 0.05 compared to CCl₄-treated wt mice. ANOVA: Analysis of variance; wt: Wild type; UT: Control.