Treatment with histone deacetylase inhibitor attenuates MAP kinase mediated liver injury in a lethal model of septic shock

Abstract

Background: Despite global efforts to improve the treatment of sepsis, it remains a leading cause of morbidity and mortality in intensive care units. We have previously shown that suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, markedly improves survival in a murine model of lipopolysaccharide (LPS)-induced shock. SAHA has anti-inflammatory properties that have not been fully characterized. The liver plays an important role in the production of acute phase reactants involved in the inflammatory cascade and is also one of the major organs that can become dysfunctional in septic shock. The purpose of this study was to assess the effect of SAHA treatment on MAP kinases and associated inflammatory markers in murine liver after LPS-induced injury.

Methods: C57B1/6J mice were randomly divided into three groups: (A) experimental-given intraperitoneal (i.p.) SAHA (50 mg/kg) in dimethyl sulfoxide (DMSO) vehicle solution (n = 12); (B) control- given vehicle only (n = 12), and; (C) sham-given no treatment (n = 7). Two hours later, experimental and control mice were injected with LPS (20 mg/kg, i.p.) and experimental mice received a second dose of SAHA. Livers were harvested at 3, 24, and 48 h for analysis of inflammatory markers using Western Blot, Polymerase Chain Reaction (PCR), and Enzyme-Linked Immunosorbent Assay (ELISA) techniques.

Results: After 3 h, the livers of animals treated with SAHA showed significantly (P < 0.05) decreased expression of the pro-inflammatory MAP kinases phosphorylated p38, phosphorylated ERK, myeloperoxidase and interleukin-6, and increased levels of the anti-inflammatory interleukin-10 compared with controls. Phospho-p38 expression remained low in the SAHA treated groups at 24 and 48 h.

Conclusion: Administration of SAHA is associated with attenuation of MAPK activation and alteration of inflammatory and anti-inflammatory markers in murine liver after a lethal LPS insult. The suppression of MAPK activity is rapid (within 3 h), and is sustained for up to 48 h post-treatment. These results may in part account for the improvement in survival shown in this model.

FIG. 1

SAHA reduces the expression of phospho-p38. The cytosol fraction from the livers of mice treated with or without SAHA at 3 h after LPS insult was subjected to western blot with anti-phospho-p38, p38, and actin antibodies. Upper panel shows a representative western blot. Protein bands of phospho-p38 were quantified by densitometry and expressed as mean ± SD, n = 3. The asterisk indicates that a value significantly differs from the LPS group (P < 0.004). There was no difference between groups in the expression of non-phosphorylated p38.

FIG. 2

SAHA attenuates p38 activation for up to 48 h. The cytosol fraction from the livers of mice treated with SAHA at 3, 24, and 48 h after LPS insult was subjected to western blot with anti-phospho-p38 and p38 antibodies. Upper panel shows a representative western blot. The levels of phospho-p38 remained low in the SAHA treated group even 48 h after LPS and SAHA injection. There was no significant difference between phospho-p38 expression at 3, 24, or 48 h. Bars represent standard error (SE).

FIG. 3

SAHA reduces the expression of phospho-ERK. The cytosol fraction from the livers of mice treated with (n = 5) or without SAHA (n = 5) at 3 h after LPS insult was subjected to western blot with anti-phospho-ERK and actin antibodies. Seven sham animals were used as comparison and are represented in the graph, though only four are shown in the Western blot. Protein bands of phospho-ERK were quantified by densitometry and expressed as mean ± SD. The asterisk indicates that the SAHA 3 h group significantly differs from the LPS 3h group (P < 0.006).

FIG. 4

SAHA decreases myeloperoxidase activity. Myeloperoxidase activity in liver tissues of mice treated with or without SAHA at 3 h after LPS injection was analyzed by quantifying MPO concentration. Values represent the mean ± SEM (n = 3). The asterisk indicates that a value significantly differs from the LPS group (P < 0.05).

FIG. 5

SAHA reduces the expression of IL-6. Total RNA from the livers of mice treated with or without SAHA at 3 h after LPS insult was subjected to RT-PCR for analysis of IL-6 gene expression with GAPDH as an internal control. Sham serves as animal control. DNA bands of IL-6 were quantified by densitometry and expressed as mean ±SD (n = 3). The asterisk indicates that a value significantly differs from the LPS group (p < 0.023).

FIG. 6

SAHA reduces the expression of IL-10. Total RNA from the livers of mice treated with or without SAHA at 3 h after LPS insult was subjected to RT-PCR for analysis of IL-10 gene expression with GAPDH as an internal control. Sham serves as animal control. DNA bands of IL-10 were quantified by densitometry and expressed as mean ±SD (n = 3). The asterisk indicates that a value significantly differs from the LPS group (P < 0.033).