Danaparoid sodium inhibits systemic inflammation and prevents endotoxin-induced acute lung injury in rats

Abstract

Introduction: Systemic inflammatory mediators, including high mobility group box 1 (HMGB1), play an important role in the development of sepsis. Anticoagulants, such as danaparoid sodium (DA), may be able to inhibit sepsis-induced inflammation, but the mechanism of action is not well understood. We hypothesised that DA would act as an inhibitor of systemic inflammation and prevent endotoxin-induced acute lung injury in a rat model.

Methods: We used male Wistar rats. Animals in the intervention arm received a bolus of 50 U/kg of DA or saline injected into the tail vein after lipopolysaccharide (LPS) administration. We measured cytokine (tumour necrosis factor (TNF)alpha, interleukin (IL)-6 and IL-10) and HMGB1 levels in serum and lung tissue at regular intervals for 12 h following LPS injection. The mouse macrophage cell line RAW 264.7 was assessed following stimulation with LPS alone or concurrently with DA with identification of HMGB1 and other cytokines in the supernatant.

Results: Survival was significantly higher and lung histopathology significantly improved among the DA (50 U/kg) animals compared to the control rats. The serum and lung HMGB1 levels were lower over time among DA-treated animals. In the in vitro study, administration of DA was associated with decreased production of HMGB1. In the cell signalling studies, DA administration inhibited the phosphorylation of IkappaB.

Conclusion: DA decreases cytokine and HMGB1 levels during LPS-induced inflammation. As a result, DA ameliorated lung pathology and reduces mortality in endotoxin-induced systemic inflammation in a rat model. This effect may be mediated through the inhibition of cytokines and HMGB1.

Figure 1

Effect of danaparoid sodium (DA) on the survival rate of lipopolysaccharide (LPS)-treated rats. The survival rate of animals treated with a bolus of LPS (7.5 mg/kg) into the tail vein (LPS group, n = 10) is represented by black squares. The survival rate of animals that received DA (50 U/kg) in addition to the intravenous injection of LPS (7.5 mg/kg) into the tail vein (DA treated LPS groups, n = 10) is represented by black circles.

Figure 2

Effects of danaparoid sodium (DA) on lung histopathology in lipopolysaccharide (LPS)-administered rats. Rats were intravenously infused with either saline (control group), 7.5 mg/kg LPS (LPS group), or 7.5 mg/kg LPS with 50 U/kg DA (DA+LPS group). Lung tissue specimens were obtained from the negative control (a) magnification ×40, (d) magnification ×100, (g) magnification ×400); LPS (b) magnification ×40, (e) magnification ×100, (h) magnification ×400; and DA+LPS (c) magnification ×40, (f) magnification ×100, (i) magnification ×400 groups, respectively. Haematoxylin and eosin staining was used.

Figure 3

Effects of danaparoid sodium (DA) on lung histopathology score in lipopolysaccharide (LPS)-administered rats. The histological changes identified included congestion, oedema, inflammation, and haemorrhaging 12 h after the administration of LPS. White bars represent the non-injected control animals, black bars represent the animals injected with LPS, and slashed bars represent animals injected with DA and LPS. The data are expressed as the mean ± SD. *Denotes a significant difference compared with the LPS group (p < 0.05).

Figure 4

Temporal changes in the tumour necrosis factor (TNF)α, interleukin (IL)-6, IL-10, and high mobility group box 1 (HMGB1) serum concentrations following LPS administration. The IL-6 (a), TNFα (b), HMGB1 (c) and IL-10 (d) serum concentrations at the indicated times are shown for the lipopolysaccharide (LPS) (n = 6; squares) and danaparoid sodium (DA)-treated (n = 6; circles) groups. All data are expressed as mean ± SD. *Denotes a significant difference compared with the LPS group (p < 0.05).

Figure 5

Changes in high mobility group box 1 (HMGB1) protein expression in lung tissue after lipopolysaccharide (LPS) administration in rats. (a) The expression of HMGB1 protein in the lung 12 h following administration of LPS in untreated LPS and danaparoid sodium (DA)-treated LPS groups was detected by Western blot. Representative blots from three separate experiments are shown. (b) Signal intensities for HMGB1 expression in lung tissue were quantified using an image analyser. Black bars represent the negative control group, white bars represent the LPS group, mesh bars represent the DA-treated LPS group. The expression intensity of HMGB1 protein relative to that of the negative control group was calculated for each group.

Figure 6

Effect of danaparoid sodium (DA) on high mobility group box 1 (HMGB1) production by lipopolysaccharide (LPS)-stimulated murine macrophages. Murine macrophages treated without or with DA (1, 15, 50 U/ml) were stimulated with LPS (100 ng/ml) for 20 h. Supernatants and cell protein were prepared and examined by enzyme linked immunosorbent assay (ELISA). All data are expressed as means ± SD. *Denotes a significant difference compared with the LPS group (p < 0.05).

Figure 7

Effect of danaparoid sodium (DA) on interleukin (IL)-6 and tumour necrosis factor (TNF)α production by lipopolysaccharide (LPS)-stimulated murine macrophages. Murine macrophages treated with or without DA (50 U/ml) were stimulated with LPS (100 ng/ml) for the indicated time. Supernatants were collected and IL-6 and TNFα levels were determined by enzyme linked immunosorbent assay (ELISA). All data are expressed as mean ± SD. *Denotes a significant difference compared with the LPS-treated cells (p < 0.05).

Figure 8

Effect of danaparoid sodium (DA) on the lipopolysaccharide (LPS)-induced increase of p50/p65 binding to DNA. The DNA binding activity assay showed a marked decrease in the p50/p65 binding activity in nuclear fractions from RAW264.7 cells. All data are expressed as the mean ± SD. *Denotes a significant difference compared with LPS group at 1 h (p < 0.05). #Denotes a significant difference compared with LPS group at 2 h (p < 0.05).