Protective effect of Salvia miltiorrhiza and Carthamus tinctorius extract against lipopolysaccharide-induced liver injury

Figure 1 Effects of Danhong injection on hepatic histopathology and apoptosis. A: Hematoxylin-eosin staining (magnification × 200); B: TUNEL assay (magnification × 200), (a/f) Blank control group, (b/g) Lipopolysaccharide (LPS)-stimulated group, (c/h) Danhong injection (DHI) (0.75 g/kg) + LPS-stimulated group, (d/i) DHI (3 g/kg) + LPS-stimulated group, and (e/j) RGI (300 mg/kg) + LPS-stimulated group; C: Analysis of apoptotic cell numbers. The number of apoptotic cells per field (1 mm²) was determined using NIH Image J software (n = 6). ^bP < 0.01 vs blank control group, ^dP < 0.01 vs LPS-treated group. Arrows indicate fat degeneration.

Figure 2 Effects of Danhong injection on serum alanine transferase and aspartate transaminase activities and total bilirubin production in lipopolysaccharide-treated mice. Different groups of mice were pre-treated with glutathione for injection (RGI) or 0.9% saline for 30 min before lipopolysaccharide (LPS) or 0.9% saline injection for 6 h. Values are mean ± SE (n = 8). ^bP < 0.01 vs blank control group, ^cP < 0.05, ^dP < 0.01 vs LPS-treated group.

Figure 3 Effects of Danhong injection on malondialdehyde production and glutathione-S-transferase activity in liver homogenates of lipopolysaccharide-induced mice. Mice were pre-treated with Danhong injection (DHI), glutathione for injection (RGI) or 0.9% saline for 30 min before lipopolysaccharide (LPS) or 0.9% saline injection for 6 h. Values are mean ± SE (n = 8). ^bP < 0.01 vs blank control group, ^cP < 0.05, ^dP < 0.01 vs LPS-treated group.

Figure 4 Effects of Danhong injection on tumour necrosis factor-α and interleukin-6 expression in mouse liver. The protein expression levels of tumour necrosis factor (TNF)-α (A) and interleukin (IL)-6 (C) were measured by ELISA. Values are mean ± SE (n = 8). TNF-α (B) and IL-6 (D) mRNA expression was detected by real-time RT-PCR. Values are mean ± SE (n = 3) of three independent experiments. ^bP < 0.01 vs blank control group, ^cP < 0.05, ^dP < 0.01 vs lipopolysaccharide (LPS)-treated group.

Figure 5 Effects of Danhong injection on liver caspase-3 activity. A: Caspase-3 enzymatic activity was measured using a modified commercial kit. Values are mean ± SE (n = 8); B: Caspase-3 mRNA expression was detected by real-time reverse transcription-polymerase chain reaction. Values are mean ± SE (n = 3) of three independent experiments. ^bP < 0.05, ^dP < 0.01 vs blank control group, ^dP < 0.01 vs lipopolysaccharide (LPS)-treated group.

Figure 6 Effects of Danhong injection on liver Bcl-2 and Bax expression. The mRNA expression of Bax (A) and Bcl-2 (B) was detected by real-time RT-PCR. Values are mean ± SE (n = 3) of three independent experiments. Bcl-2 and Bax protein expression was detected by Western blot (E). The relative optical density is shown (C and D). ^bP < 0.01 vs blank control group, ^cP < 0.05, ^dP < 0.01 vs lipopolysaccharide (LPS)-treated group. β-actin was used as an internal control.

Figure 7 Effects of Danhong injection on liver nuclear factor-κB family protein expression. Phospho-IκB-α, IκBα, phospho-NF-κB p65 and NF-κB p65 protein expression in the mouse liver was examined by Western blot (A). The relative optical density is shown (B-E). ^bP < 0.01 vs blank control group, ^cP < 0.05, ^dP < 0.01 vs lipopolysaccharide (LPS)-treated group. β-actin was used as an internal control.

Figure 8 Pharmacological mechanism of Danhong injection on lipopolysaccharide-induced acute liver injury. Danhong injection (DHI) protected against lipopolysaccharide (LPS)-induced acute liver injury by regulating IκBα, and nuclear factor (NF)-κB p65 phosphorylation as well as NF-κB p65 translocation from the cytoplasm to the nucleus. Subsequently, TNF-α and IL-6 secretion, Bax and Bcl-2 expression, and caspase-3 activity were modified and balanced.