Protective effect of danhong injection on acute hepatic failure induced by lipopolysaccharide and d-galactosamine in mice

Abstract

Acute hepatic failure (AHF), which leads to an extremely high mortality rate, has become the focus of attention in clinic. In this study, Danhong injection (DHI) was investigated to evaluate the preventive and protective effect on AHF induced by lipopolysaccharide (LPS) and D-galactosamine (GalN) in mice. For AHF induction, ICR mice were intraperitoneally injected with D-GalN (700 mg/kg) and LPS (20 μ g/kg). DHI was administrated twice, at 12 and 1 h, respectively, before D-GalN/LPS injection. After stimulation with D-GalN/LPS for 1 and 6 h, serum and livers were collected for analysis. We found that mice administrated with DHI displayed a higher survival rate, lower serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBil), glutathione S-transferase (GST), and tumor necrosis factor (TNF)- α . DHI inhibited the elevations of hepatic lipid peroxidation (malondialdehyde), caspase-8 activity, and mRNA expression levels of inflammatory cytokines (interleukin-1 β and interleukin-6) increased by D-GalN/LPS in the liver. Furthermore, liver histopathological analysis indicated that the DHI group showed markedly fewer apoptotic (TUNEL positive) cells and less pathological changes than those in the AHF model group. These results provide a novel insight into the pharmacological actions of DHI as a potential candidate for treating AHF.

Figure 1

Effects of DHI on the survival rate of mice. Mice were subjected to sterile PBS or DHI (1.5, 3, and 6 mL/kg) at 12 and 1 h before D-GalN/LPS injection. Then, D-GalN (700 mg/kg)/LPS (20 μg/kg) was intraperitoneally injected to mice. The survival rate was assessed for 24 h.

Figure 2

Effects of DHI on serum TNF-α levels, caspase-8 activity, and inflammatory mediators expressed in liver. Serum for measuring TNF-α level (a) was collected at 1 h after D-GalN/LPS injection. The mRNA expressions of IL-1β (b) and IL-6 (d) were analyzed by real-time RT-PCR. Caspase-8 (c) activity was determined in liver homogenates at 6 h after D-GalN/LPS injection using the commercial caspase-8 colorimetric assay kit. Data show mean ± SEM (n = 8). ^† P < 0.05 versus the control group; *P < 0.05 and **P < 0.01 versus the GalN/LPS group.

Figure 3

Effects of DHI on the liver. Mice were treated with D-GalN/LPS with or without DHI. Representative images are shown for the different experimental groups (original magnification, 400x). (a) Control group, (b) D-GalN/LPS-stimulated group, (c) NAC-treated group, (d) DHI (6 mL/kg) + D-GalN/LPS-stimulated group, (e) DHI (3 mL/kg) + D-GalN/LPS-stimulated group, and (f) DHI + D-GalN/LPS-stimulated (1.5 mL/kg) group.

Figure 4

Effects of DHI on apoptosis of hepatocytes. TUNEL assay of apoptotic hepatocytes in the liver was performed 6 h after D-GalN/LPS injection with or without DHI. DHI (1.5, 3, and 6 mL/kg body weight) or sterile PBS was intraperitoneally administrated at 12 and 1 h before D-GalN/LPS injection (original magnification, 400x). (a) Control group, (b) D-GalN/LPS-stimulated group, (c) NAC-treated group, (d) DHI (6 mL/kg) + D-GalN/LPS-stimulated group, (e) DHI (3 mL/kg) + D-GalN/LPS-stimulated group, and (f) DHI + D-GalN/LPS-stimulated (1.5 mL/kg) group.