Yonsei Med J.  2015 Jan;56(1):112-123. 10.3349/ymj.2015.56.1.112.

The Effect of Bortezomib on Expression of Inflammatory Cytokines and Survival in a Murine Sepsis Model Induced by Cecal Ligation and Puncture

• Han SH
• Kim JS
• Woo JH
• Jeong SJ
• Shin JS
• Ahn YS
• Kim JM

• KMID: 2352796
• DOI: http://doi.org/10.3349/ymj.2015.56.1.112

Abstract

PURPOSE
Although the proteasome inhibitor known as bortezomib can modulate the inflammatory process through the nuclear factor-kappa B signaling pathway, the immunomodulatory effect of pre-incubated bortezomib has not been fully evaluated for inflammation by infectious agents. Therefore, we evaluated the effect of bortezomib on the expression of inflammatory cytokines and mediators in macrophage cell lines and on survival in a murine peritonitis sepsis model.
MATERIALS AND METHODS
Bortezomib was applied 1 hr before lipopolysaccharide (LPS) stimulation in RAW 264.7 cells. The cecal ligation and puncture (CLP) experiments were performed in C57BL/6J mice.
RESULTS
Pre-incubation with bortezomib (25 nM or 50 nM) prior to LPS (50 ng/mL or 100 ng/mL) stimulation significantly recovered the number of viable RAW 264.7 cells compared to those samples without pre-incubation. Bortezomib decreased various inflammatory cytokines as well as nitric oxide production in LPS-stimulated cells. The 7-day survival rate in mice that had received bortezomib at 0.01 mg/kg concentration 1 hr prior to CLP was significantly higher than in the mice that had only received a normal saline solution of 1 mL 1 hr prior to CLP. In addition, the administration of bortezomib at 0.01 mg/kg concentration 1 hr before CLP resulted in a significant decrease in inflammation of the lung parenchyma. Collectively, pretreatment with bortezomib showed an increase in the survival rate and changes in the levels of inflammatory mediators.
CONCLUSION
These results support the possibility of pretreatment with bortezomib as a new therapeutic target for the treatment of overwhelming inflammation, which is a characteristic of severe sepsis.

Keyword

Bortezomib; proteasome inhibitor; lipopolysaccharide; inflammatory cytokines; cecal ligation and puncture

MeSH Terms

Animals
Boronic Acids/administration & dosage/pharmacology/*therapeutic use
Cecum/*pathology
Cell Adhesion Molecules/metabolism
Cell Line
Cell Proliferation/drug effects
Cell Survival/drug effects
Chymotrypsin/metabolism
Cytokines/*metabolism
Disease Models, Animal
Inflammation Mediators/*metabolism
Ligation
Lipopolysaccharides/pharmacology
Lung/drug effects/metabolism/pathology
Male
Mice, Inbred C57BL
Nitric Oxide/metabolism
Proteasome Inhibitors/pharmacology
Punctures
Pyrazines/administration & dosage/pharmacology/*therapeutic use
Sepsis/*drug therapy
Boronic Acids
Cell Adhesion Molecules
Chymotrypsin
Cytokines
Inflammation Mediators
Lipopolysaccharides
Proteasome Inhibitors
Pyrazines
Nitric Oxide

Figure

• 

  Fig. 1 The concentrations of viable RAW 264.7 cells measured using the CCK-8 assay to evaluate the effect of pre-incubation with bortezomib 1 hr before LPS treatment on the cell proliferation. On experiment day 3, LPS at 50 ng/mL (A) and 100 ng/mL (B) was administered to the growing RAW 264.7 cell lines of 8×103 cells/mL at 1 hr after the application of bortezomib with various concentrations from 0 to 100 nM. Arrows (↓) indicate the reference values in samples that had received only LPS application with 50 and 100 ng/mL, without pre-incubation with bortezomib. *Statistical significance with p-values less than 0.05 when compared with reference values (LPS alone, arrows). CCK-8, Cell Counting Kit-8; LPS, lipopolysaccharide; OD, optical density.

• 

  Fig. 2 The chymotrypsin-like activity of proteasomes measured by the bioluminescent proteasome assay. RAW 264.7 cells (arrow) that were not treated with bortezomib and lipopolysaccharide (100 ng/mL) were used as the reference control to determine statistical significance (*p<0.01). †Means statistically significant with a p-value less than 0.01 when compared with bortezomib at a 25 nM concentration (asterisk). The results are reported as mean±standard error of the mean of three independent experiments in triplicate. RLU, relative luminescent units; LPS, lipopolysaccharide; Bor, bortezomib.

• 

  Fig. 3 The expression levels of inflammatory cytokines and intercellular adhesion molecules based on the administration of bortezomib and LPS at various concentrations. (A) The RT-PCR products expressed by agarose gel electrophoresis. (B-F) The relative expression rate of RT-PCR products by internal control using mouse GAPDH in each cytokine and ICAM-1. These semi-quantitation was performed using the Multi Guage Version 3.0 program. *Statistical significance with p-values less than 0.01. **Statistical significance with p-values less than 0.001. †,‡Reference values for statistical comparison. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; TNF-α, tumor necrosis factor-alpha; IFN-α, interferon-gamma; IL, interleukin; ICAM, intercellular adhesion molecule; LPS, lipopolysaccharide.

• 

  Fig. 4 The measurement of nitric oxide concentrations using the diazotization assay (Griess method) in order to analyze the effect of bortezomib on nitric oxide production after LPS stimulation. On the third day of RAW 264.7 cell growth, the supernatant of the cell line growth media received bortezomib (0 or 50 nM) and then LPS (0 or 100 ng/mL) with a 1 hr interval. These samples were used for the nitric oxide detection assay 16 and 24 hr after administration of bortezomib and/or LPS to the cell lines. *p-values less than 0.001 compared with the LPS-only treatment group for the 16 and 24 hr time periods. LPS, lipopolysaccharide; Bor, bortezomib.

• 

  Fig. 5 Comparison of 7-day mortalities after CLP surgery by the Kaplan-Meier survival curve. Bortezomib and normal saline (at 1 mL) were always injected intraperitoneally. CLP, cecal ligation and puncture; NS, normal saline; Bor, bortezomib.

• 

  Fig. 6 Histologic findings of lung tissue after H&E staining (×200). All lung tissues were harvested from the mice that were alive at 7 days after the surgery. (A) Normal mice (negative control). (B) Normal saline 1 mL injection IP at 1 hr before sham surgery (negative control). (C) Normal saline 1 mL injection IP at 1 hr before CLP surgery (positive control). (D) Bortezomib 0.01 mg/kg injection IP at 1 hr before CLP surgery. (E) Bortezomib 0.1 mg/kg injection, IP at 1 hr before CLP surgery. (F) Bortezomib 0.01 mg/kg injection, IP at 24 hr after CLP surgery. IP, intraperitoneal; CLP, cecal ligation and puncture.

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