Role of activin A in carbon tetrachloride-induced acute liver injury

Abstract

Aim: To investigate the expression and role of activin A in a mouse model of acute chemical liver injury.

Methods: Acute liver injury in C57BL/6 male mice was induced by intraperitoneal injection with carbon tetrachloride (CCl4) (0.5 mL/kg, body weight) dissolved in olive oil (1:19 v/v). Mice were sacrificed 1, 3, 5 and 7 d after the treatment. The levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum were examined and pathological changes of liver observed by hematoxylin and eosin staining to evaluate the liver injury. Activin A protein levels in serum and hepatic tissue homogenate of mice were detected by enzyme-linked immunosorbent assay, and the expression pattern of activin A protein in livers of mice was examined by immunohistochemistry. Activin type IIA receptor (ActRIIA) and Smad3 expressions in the liver were analyzed by real-time quantitative reverse transcription-polymerase chain reaction. In order to further investigate the role of activin A, we also utilized activin A blocking experiment by anti-activin A antibody (500 μg/kg, body weight) injection into mouse tail vein.

Results: In CCl4-treated mice, serum ALT and AST levels were significantly increased, compared with that in control mice (P < 0.01). Furthermore, the serious necrosis was observed around hepatic portal areas in CCl4-treated mice. Simultaneously, activin A levels in serum and hepatic tissue homogenate of mice treated with CCl4 for 1, 3 and 5 d increased significantly, compared with that in control mice (P < 0.01). Activin A protein expression in hepatocytes not within the necrotic area was also upregulated in mice following CCl4 treatment. Not only activin A, but also ActRIIA and activin signaling molecule Smad3 mRNA expressions in injury liver induced by CCl4 were significantly higher than that in control liver. In addition, levels of serum ALT and AST in CCl4-treated mice were significantly decreased by injection of anti-activin A antibody to block endogenous activin A action, compared with that in CCl4-treated mice by injection of immunoglobulin G instead of anti-activin A antibody (P < 0.01), and the severity of liver injury was also reduced remarkably.

Conclusion: These data show that activin A is involved in CCl4-induced acute liver injury. Blocking activin A actions may be a therapeutic approach for acute liver injury.

Keywords: Activin A; Carbon tetrachloride; Immunohistochemistry; Liver injury.

Figure 1

Examination of serum alanine aminotransferase and aspartate aminotransferase levels and pathological changes of liver in carbon tetrachloride-treated mice. A: Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were detected by enzyme-linked immunosorbent assay in olive oil control mouse and carbon tetrachloride (CCl₄)-treated mouse. ^bP < 0.01 vs control; B: Pathological change of liver was analyzed by hematoxylin and eosin staining. Arrows represent lesion area (magnification, × 100).

Figure 2

Detection of levels of activin A in serum and hepatic homogenates of mouse treated with carbon tetrachloride by enzyme-linked immunosorbent assay. CCl₄: Carbon tetrachloride. ^bP < 0.01 vs control.

Figure 3

Expression of activin A protein in liver of mouse assessed by immunohistochemical staining. A, B: Activin A expression was examined by using anti-activin A antibody in the same liver tissues on day 1 after olive oil treatment; C, D: Activin A expression was examined by using anti-activin A antibody in the same liver tissues on day 1 after carbon tetrachloride (CCl₄) treatment; E, F: Activin A expression was examined by using anti-activin A antibody in the same liver tissues on day 3 after CCl₄ treatment; G, H: The procedural background control staining was represented by using normal mouse immunoglobulin G instead of anti-activin A antibody in livers of olive oil-treated and CCl₄-treated mice. Red arrows represent lesion area and black arrows represent positive staining for activin A. A, C, E: Magnification × 100; B, D, F, G, H: Magnification × 200.

Figure 4

Assay of mRNA expressions of activin βA and activin signal molecules in liver of mouse by real-time quantitative reverse transcription-polymerase chain reaction. The mRNA levels in olive oil control group (Cont) were adjusted to 100%. All values (mean ± SD) were expressed as % of that in control. ^aP < 0.05, ^bP < 0.01 vs control.

Figure 5

Effects of anti-activin A antibody in vivo on serum alanine aminotransferase and aspartate aminotransferase levels and pathological change of liver in carbon tetrachloride-treated mouse. A: Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were detected in mouse 3 d after carbon tetrachloride (CCl₄) treatment. Immunoglobulin G (IgG) + Cont, IgG control group; Anti-A + Cont, anti-activin A control group; IgG + CCl₄, IgG plus CCl₄ group; Anti-A + CCl₄, anti-activin A plus CCl4 group. ^bP < 0.01 vs IgG + CCl₄ group; B: Pathological change of liver in mouse 3 d after CCl₄ treatment was analyzed by hematoxylin and eosin staining. Arrows represent lesion area (magnification × 40).