Exogenous carbon monoxide attenuates inflammatory responses in the small intestine of septic mice

Abstract

To determine whether the carbon monoxide (CO)-releasing molecules (CORM)-liberated CO suppress inflammatory responses in the small intestine of septic mice. The C57BL/6 mice (male, n = 36; weight 20 ± 2 g) were assigned to four groups in three respective experiments. Sepsis in mice was induced by cecal ligation and puncture (CLP) (24 h). Tricarbonyldichlororuthenium (II) dimer (CORM-2) (8 mg/kg, i.v.) was administrated immediately after induction of CLP. The levels of inflammatory cytokines [interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α)] in tissue homogenates were measured with enzyme-linked immunosorbent assay. The levels of malondialdehyde (MDA) in the tissues were determined. The levels of nitric oxide (NO) in tissue homogenate were measured and the expression levels of intercellular adhesion molecule 1 (ICAM-1) and inducible nitric oxide synthase (iNOS) in the small intestine were also assessed. NO and IL-8 levels in the supernatants were determined after the human adenocarcinoma cell line Caco-2 was stimulated by lipopolysaccharide (LPS) (10 g/mL) for 4 h in vitro. At 24 h after CLP, histological analysis showed that the ileum and jejunum from CLP mice induced severe edema and sloughing of the villous tips, as well as infiltration of inflammatory cells into the mucosa. Semi-quantitative analysis of histological samples of ileum and jejunum showed that granulocyte infiltration in the septic mice was significantly increased compared to that in the sham group. Administration of CORM-2 significantly decreased granulocyte infiltration. At 24 h after CLP, the tissue MDA levels in the mid-ileum and mid-jejunum significantly increased compared to the sham animals (103.68 ± 23.88 nmol/mL vs 39.66 ± 8.23 nmol/mL, 89.66 ± 9.98 nmol/mL vs 32.32 ± 7.43 nmol/mL, P < 0.01). In vitro administration of CORM-2, tissue MDA levels were significantly decreased (50.65 ± 11.46 nmol/mL, 59.32 ± 6.62 nmol/mL, P < 0.05). Meanwhile, the tissue IL-1β and TNF-α levels in the mid-ileum significantly increased compared to the sham animals (6.66 ± 1.09 pg/mL vs 1.67 ± 0.45 pg/mL, 19.34 ± 3.99 pg/mL vs 3.98 ± 0.87 pg/mL, P < 0.01). In vitro administration of CORM-2, tissue IL-1β and TNF-α levels were significantly decreased (3.87 ± 1.08 pg/mL, 10.45 ± 2.48 pg/mL, P < 0.05). The levels of NO in mid-ileum and mid-jejunum tissue homogenate were also decreased (14.69 ± 2.45 nmol/mL vs 24.36 ± 2.97 nmol/mL, 18.47 ± 2.47 nmol/mL vs 27.33 ± 3.87 nmol/mL, P < 0.05). The expression of iNOS and ICAM-1 in the mid-ileum of septic mice at 24 h after CLP induction significantly increased compared to the sham animals. In vitro administration of CORM-2, expression of iNOS and ICAM-1 were significantly decreased. In parallel, the levels of NO and IL-8 in the supernatants of Caco-2 stimulated by LPS was markedly decreased in CORM-2-treated Caco-2 cells (2.22 ± 0.12 nmol/mL vs 6.25 ± 1.69 nmol/mL, 24.97 ± 3.01 pg/mL vs 49.45 ± 5.11 pg/mL, P < 0.05). CORM-released CO attenuates the inflammatory cytokine production (IL-1β and TNF-α), and suppress the oxidative stress in the small intestine during sepsis by interfering with protein expression of ICAM-1 and iNOS.