The Deficiency of Indoleamine 2,3-Dioxygenase Aggravates the CCl4-Induced Liver Fibrosis in Mice.

In the present study, we examined the role of indoleamine 2,3-dioxygenase (IDO) in the development of CCl4-induced hepatic fibrosis. The liver fibrosis induced by repetitive administration with CCl4 was aggravated in IDO-KO mice compared to WT mice. In IDO-KO mice treated with CCl4, the number of several inflammatory cells and the expression of pro-inflammatory cytokines increased in the liver. In the results, activated hepatic stellate cells (HSCs) and fibrogenic factors on HSCs increased after repetitive CCl4 administration in IDO-KO mice compared to WT mice. Moreover, the treatment with l-tryptophan aggravated the CCl4-induced hepatic fibrosis in WT mice. Our findings demonstrated that the IDO deficiency enhanced the inflammation in the liver and aggravated liver fibrosis in repetitive CCl4-treated mice

Time-course analysis of liver and serum galectin-3 in acute liver injury after alpha-galactosylceramide injection.

Galectin-3 is a beta-galactoside-binding lectin that plays important roles in diverse physiological functions, such as cell proliferation, apoptosis, and mRNA splicing. This protein is expressed on inflammatory cells and acts as a local inflammatory mediator. Recently, galectin-3 has been detected in several diseases, such as chronic liver, heart, and kidney diseases, diabetes, viral infection, autoimmune and neurodegenerative diseases, and tumors, and its role as a biomarker has attracted attention. Alpha-galactosylceramide is an artificially synthesized sphingolipid that can induce acute liver injury via the natural killer T pathway. However, the pathophysiological roles and kinetics of galectin-3 in acute liver injury are not fully understood. This study aimed to elucidate the expression and time course of galectin-3 in liver tissues during acute liver injury following alpha-galactosylceramide injection. Animals were histologically examined on days 1, 2, 4, and 7 after intraperitoneal injection of alpha-galactosylceramide, and the expressions of galectin-3 and ionized calcium-binding adaptor molecule 1 were analyzed. Notably, galectin-3 formed characteristic cluster foci, particularly on day 2 after injection. Cluster formation was not observed in chronic liver disease. Simultaneously, ionized calcium-binding adaptor molecule 1-positive cells were observed in the cluster foci. Serum galectin-3 levels increased on day 2 of treatment and correlated well with the number of galectin-3-positive cell clusters in the liver. Moreover, galectin-3 expression was an important mediator of the early phase of liver injury after alpha-galactosylceramide injection. These results suggest that serum galectin-3 may be a biomarker for the early diagnosis of acute liver injury and that clusters of galectin-3-positive cells may be a specific finding in acute liver injury

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.</p

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).</p

Minimum data.

Galectin-3 is a beta-galactoside-binding lectin that plays important roles in diverse physiological functions, such as cell proliferation, apoptosis, and mRNA splicing. This protein is expressed on inflammatory cells and acts as a local inflammatory mediator. Recently, galectin-3 has been detected in several diseases, such as chronic liver, heart, and kidney diseases, diabetes, viral infection, autoimmune and neurodegenerative diseases, and tumors, and its role as a biomarker has attracted attention. Alpha-galactosylceramide is an artificially synthesized sphingolipid that can induce acute liver injury via the natural killer T pathway. However, the pathophysiological roles and kinetics of galectin-3 in acute liver injury are not fully understood. This study aimed to elucidate the expression and time course of galectin-3 in liver tissues during acute liver injury following alpha-galactosylceramide injection. Animals were histologically examined on days 1, 2, 4, and 7 after intraperitoneal injection of alpha-galactosylceramide, and the expressions of galectin-3 and ionized calcium-binding adaptor molecule 1 were analyzed. Notably, galectin-3 formed characteristic cluster foci, particularly on day 2 after injection. Cluster formation was not observed in chronic liver disease. Simultaneously, ionized calcium-binding adaptor molecule 1-positive cells were observed in the cluster foci. Serum galectin-3 levels increased on day 2 of treatment and correlated well with the number of galectin-3-positive cell clusters in the liver. Moreover, galectin-3 expression was an important mediator of the early phase of liver injury after alpha-galactosylceramide injection. These results suggest that serum galectin-3 may be a biomarker for the early diagnosis of acute liver injury and that clusters of galectin-3-positive cells may be a specific finding in acute liver injury.</div

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).</p

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).</p

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.</p

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.

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.</p