Fig 1.
Representative histological findings of liver tissue after intraperitoneal injection of alpha-galactosylceramide.
Hematoxylin and eosin staining showed evident inflammatory cell infiltration from day 2. Azan staining revealed no apparent fibrosis at any stage. Galectin-3-positive cells showed an increasing trend from day 1, forming characteristic clusters by day 2 and reaching a peak on day 4. Galectin-3-positive cells had almost disappeared by day 7; however, Iba1-positive cells were still observed. Scale bars = 100 μm.
Table 1.
Number of galectin-3-positive cell clusters, serum levels of galectin-3, degree of fibrosis, and inflammation score in liver tissue after alpha-galactosylceramide injection.
Fig 2.
Immunofluorescent image confirming the co-localization of galectin-3 and ionized calcium-binding adaptor molecule 1 (Iba1) immunoreactivity 4 days after alpha-galactosylceramide injection.
Galectin-3 and Iba1 are expressed in the same cells, indicating that galectin-3-positive cells are macrophages. Scale bar = 100 μm.
Fig 3.
Relationship between the number of galectin-3-positive cells and degree of fibrosis.
(A) The number of galectin-3-positive cell clusters (determined using immunohistochemistry) in the liver tissue was counted at each time point. Statistical difference between day 0 and day 2, p = 0.023; between day 0 and day 4, p = 0.034. (B) The degree of fibrosis of the liver tissue (determined by Azan staining) was quantified as the percentage of fibrotic areas in the total liver tissue at each time point. The sample numbers were as follows: n = 5 for day 0, n = 6 for day 1, n = 6 for day 2, n = 3 for day 4, n = 2 for day 7. *Statistical difference between each group determined using analysis of variance (p < 0.05) (One-way analysis of variance).
Fig 4.
Association between alanine aminotransferase (ALT) and serum galectin-3 levels.
(A) Serum ALT levels. ALT levels markedly increased 1 day after alpha-galactosylceramide (GalCer) injection and peaked at 2 days. Statistical difference between day 2 and day 0: p < 0.001; between day 2 and day 1: p = 0.025. (B) Serum galectin-3 levels. Serum galectin-3 levels markedly increased and peaked 2 days after alpha-GalCer injection. Statistical difference between day 0 and day 2: p = 0.006; between day 1 and day 2: p < 0.001. (C) A weak positive correlation between ALT and serum galectin-3 levels was observed. (r = 0.366, p = 0.043) (Pearson product-moment correlation coefficient). The sample numbers were as follows: n = 12 for day 0, n = 9 for day 1, n = 8 for day 2, n = 8 for day 4, n = 4 for day 7. *Statistical difference between groups determined by analysis of variance (p < 0.05) (One-way analysis of variance).
Fig 5.
Representative histological findings in mice models of non-alcoholic steatohepatitis (NASH) and primary sclerosing cholangitis (PSC).
(A) Fibrosis is only observed in the PSC model; no clusters of galectin-3-positive cells are observed in either the NASH or PSC models; galectin-3-positive cells are observed in fibrotic areas in the PSC model. Scale bars = 100 μm. (B) The degree of fibrosis in liver tissue (determined using Azan staining) is quantified as a percentage of fibrotic areas in total liver tissue. (C) The number of galectin-3-positive cell aggregation foci (determined using immunohistochemistry) in the liver tissue is counted at each time point. The sample numbers were as follows: n = 5 for day 0, n = 6 for day 1, n = 6 for day 2, n = 3 for day 4, n = 2 for day 7, n = 5 for NASH, n = 4 for PSC. *Statistical difference between each group determined using analysis of variance (p < 0.05) (One-way analysis of variance).