Evaluation of the effect of recombinant thrombomodulin on a lipopolysaccharide-induced murine sepsis model

Abstract

To evaluate the effect of recombinant human thrombomodulin (rTM) on sepsis, the levels of nucleosome as well as high-mobility group box 1 (HMGB1) and cytokines in sera and peritoneal fluids were measured in a mouse model of lipopolysaccharide (LPS)-induced sepsis after administration of rTM. C57BL/6 mice were intraperitoneally injected with LPS (15 mg/kg; Escherichia coli O111:B4) with or without the intravenous administration of rTM (3 mg/kg; 30 min prior to or 2 h after LPS injection). The survival rates were evaluated and levels of tumor necrosis factor (TNF)-α, interleukin (IL)-6, monocyte chemoattractant protein (MCP)-1, HMGB1 and nucleosome in sera and peritoneal fluids were analyzed by ELISA. Administration of rTM prior to or after LPS improved the survival rate of septic mice. In addition, rTM administered prior to or after LPS suppressed the level of pro-inflammatory cytokine TNF-α in sera at 1-3 h after LPS injection, whereas only the administration of rTM after LPS suppressed the levels of HMGB1 and nucleosome (late-phase mediators of sepsis) (9-12 h) in sera after the LPS injection. Furthermore, administration of rTM prior to or after LPS suppressed the level of TNF-α in the peritoneal fluids at 1-3 h after LPS injection, whereas only the administration of rTM after LPS suppressed the levels of IL-6 and MCP-1 in the peritoneal fluids at 6-9 h after LPS injection. These observations indicated that administration of rTM significantly improves the survival rate and suppresses the increased levels of TNF-α, IL-6, MCP-1, HMGB1 and nucleosome in the LPS-induced septic shock model. Thus, rTM may exert a protective action on sepsis and reduce mortality, possibly by reducing not only the levels of cytokines and chemokine but also the levels of late-phase mediators of sepsis.

Keywords: cytokines; high-mobility group box 1; lipopolysaccharide; neutrophil extracellular traps; nucleosome; recombinant thrombomodulin; sepsis.

Figure 1.

Protective effect of rTM on survival of LPS-induced septic model mice. LPS (15 mg/kg) was injected into the peritoneal cavity (LPS group); 3 mg/kg of rTM was administered 30 min prior to or 2 h after the LPS-injection (pre-rTM group or post-rTM group, respectively). Survival of LPS, pre-rTM and post-rTM groups (n=8 mice each) was monitored for up to 3 days. Values are compared between LPS group and pre-rTM group or post-rTM group. **P<0.01. LPS, lipopolysaccharide; rTM, recombinant thrombomodulin.

Figure 2.

Effects of rTM on the levels of TNF-α, HMGB1 and nucleosome in sera. LPS (15 mg/kg) was injected into the peritoneal cavity (LPS group); 3 mg/kg of rTM was administered 30 min before or 2 h after the LPS injection (pre-rTM group or post-rTM group, respectively). As a control, physiological saline was administered instead of LPS and rTM (control group). At the indicated time-points (3, 6, 9 and 12 h after LPS administration in both groups and at 1 h after LPS administration in the rTM pre-administration group), blood was collected and sera were prepared. (A) TNF-α, (B) HMGB1 and (C) nucleosome were measured by ELISA. Values are expressed as the means ± standard deviation, and compared between control group and LSP group, and LPS group and rTM pre-administration group or rTM post-administration group. *P<0.05; **P<0.01. LPS, lipopolysaccharide; rTM, recombinant thrombomodulin; TNF, tumor necrosis factor; HMGB1, high-mobility group box 1.

Figure 3.

Effects of rTM on the levels of cytokines, HMGB1 and nucleosome in peritoneal fluids. LPS (15 mg/kg) was injected into the peritoneal cavity (LPS group); 3 mg/kg of rTM was administered 30 min before or 2 h after LPS injection (pre-rTM group or post-rTM group, respectively). As a control, physiological saline was administered instead of LPS and rTM (control group). At the indicated time-points (1, 3, 6, 9 and 12 h after LPS administration; except 1 h in the rTM post-administration group), ascites was collected and peritoneal fluids were prepared. (A) TNF-α, (B) IL-6 and (C) MCP-1 were measured by cytometric bead array and (D) HMGB1 and (E) nucleosome were measured by ELISA. Values are expressed as the means ± standard deviation, and compared between control group and LPS group, and LPS group and rTM pre-administration group or rTM post-administration group. *P<0.05 and **P<0.01. LPS, lipopolysaccharide; rTM, recombinant thrombomodulin; TNF, tumor necrosis factor; IL interleukin; MCP, monocyte chemoattractant protein.