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.