Salvianolic acid B protects against acute and chronic liver injury by inhibiting Smad2C/L phosphorylation

Abstract

Salvianolic acid B (Sal B) has strong antioxidant and anti-fibrosis effects, which are related to the transforming growth factor β/Smad signaling pathway. However, how Sal B affects this antioxidant pathway and the phosphorylation (p-) of Smad2 at both the COOH-terminal (pSmad2C) and linker region (pSmad2L) are unknown. The aims of the present study were to investigate the underlying mechanisms of Sal B on acute and chronic liver injury induced by CCl₄ and H₂O₂, and its effects on p-Smad2C/L. In in vivo experiments, acute and chronic liver injury models were induced by CCl₄, and the oxidative damage cell model was established in vitro with H₂O₂. Liver histopathology was assessed using hematoxylin and eosin and Van Gieson's staining. Moreover, serum biochemical indicators were analyzed using specific assay kits. Furthermore, the present study evaluated the oxidant/antioxidant status in acute and chronic liver injury models by oxidative stress parameters such as malondialdehyde, glutathione and superoxide dismutase. In addition, western blot analysis was performed to analyze the protein expression levels of pSmad2C, pSmad2L, nuclear factor erythroid-2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1). It was found that Sal B improved liver histology, decreased the levels of aminotransferase and attenuated oxidative stress in acute and chronic liver injury models. Additionally, the protein expression levels of pSmad2C and pSmad2L were decreased, but Nrf2 and HO-1 expression levels were increased both in vivo and in vitro. Collectively, the present results suggested that Sal B may protect against acute and chronic liver injury via inhibition of Smad2C/L phosphorylation, and the Nrf2/HO-1 signaling pathway may play an important role in this process.

Keywords: liver injury; nuclear factor erythroid-2-related factor 2/heme oxygenase-1; phosphorylated Smad2 at COOH-terminal/linker region; salvianolic acid B.

Figure 1

Effects of Sal B on pathological changes in CCl₄-induced acute and chronic liver injury by HE and VG staining. Representative liver sections from (A) acute liver injury and (B) chronic liver injury. n=6 mice per group. These representative figures were randomly selected from each group. Magnification, x400 in HE staining and x100 in VG staining. Scale bar, 50 µm. HE, hematoxylin and eosin; VG, Van Gieson's; Sal B, salvianolic acid B.

Figure 2

Effects of Sal B on changes of ALT and AST in CCl₄-induced acute and chronic liver injury mice. Levels of ALT and AST in (A) acute liver injury and (B) chronic liver injury. The data were obtained from three independent experiments and presented as the mean ± SD. n=6. ^*P<0.05; ^**P<0.01 vs. model group; ^#P<0.05 vs. control group. Sal B, salvianolic acid B; ALT, alanine transaminase; AST, aspartate aminotransferase.

Figure 3

Effects of Sal B on the activities of antioxidant enzymes in CCl₄-induced acute and chronic liver injury. Levels of SOD, MDA and GSH in (A) acute liver injury and (B) chronic liver injury. The data were obtained from three independent experiments and presented as the mean ± SD. n=6. ^*P<0.05 vs. model group; ^#P<0.05 vs. control group. Sal B, salvianolic acid B; SOD, superoxide dismutase; MDA, malondialdehyde; GSH, glutathione.

Figure 4

Effects of Sal B on the Nrf2/HO-1 signaling pathway in CCl₄-induced acute and chronic liver injury mice. Protein expression levels of Nrf-2 and HO-1 and immunohistochemical staining of Nrf2 were evaluated in CCl₄-induced (A) acute injury and (B) chronic liver injury. Samples were derived from the same experiment and the blots were processed in parallel, all the experiments were repeated ≥3 times. Data are presented as the mean ± SD. n=6. ^*P<0.05 vs. model group; ^#P<0.05 vs. control group. Sal B, salvianolic acid B; HO-1, heme oxygenase-1; Nrf2, nuclear factor erythroid-2-related factor 2.

Figure 5

Effects of Sal B on pSmad2C and pSmad2L in CCl₄-induced acute and chronic liver injury mice. Protein expression levels of pSmad2L and pSmad2C were evaluated in (A) CCl₄-induced acute injury and (B) chronic liver injury. Samples derive from the same experiment and the blots were processed in parallel, all the experiments were repeated ≥3 times. Data are presented as the mean ± SD. n=6. ^*P<0.05 and ^**P<0.01 vs. model group; ^#P<0.05 vs. control group. P-, phosphorylated; Smad2C, Smad2 at COOH-terminal; Smad2L, Smad2 at linker region.

Figure 6

Effects of Sal B on Nrf2 and HO-1 in H₂O₂ induced HSC-T6 cells injury. Protein expression levels of Nrf2 and HO-1 were evaluated in H₂O₂ induced cells injury. Samples derive from the same experiment and the blots were processed in parallel, all the experiments were repeated ≥3 times. Data are presented as the mean ± SD. n=6. ^*P<0.05 vs. model group. Sal B, salvianolic acid B; HO-1, heme oxygenase-1; Nrf2, nuclear factor erythroid-2-related factor 2.

Figure 7

Effects of Sal B on pSmad2C and pSmad2L in H₂O₂ induced HSC-T6 cells injury. Protein expression levels of pSmad2L and pSmad2C were evaluated in H₂O₂ induced cells injury. Samples derive from the same experiment and the blots were processed in parallel, all the experiments were repeated ≥3 times. Data are presented as the mean ± SD. n=6. ^*P<0.05 vs. model group; ^#P<0.05 vs. control group. P-, phosphorylated; Smad2C, Smad2 at COOH-terminal; Smad2L, Smad2 at linker region.