Effect of low-dose hydrocortisone and inhaled nitric oxide on inflammatory mediators and local pulmonary metalloproteinases activity in LPS-induced sepsis in piglets

Abstract

Hospital mortality in sepsis varies between 30-45%. It has been shown that administration of inhaled nitric oxide (iNO) and intravenous corticosteroid in a porcine endotoxemia model attenuated the systemic inflammatory response. We explored the anti-inflammatory effect of a double-treatment strategy (iNO + low-dose steroid) on the lungs in a long-term porcine endotoxic shock model. As metalloproteinases (MMPs) are involved in the initiation of multiple organ dysfunction in septic shock, we evaluated the influence of this combination therapy on MMP2 and MMP9 activity and proIL-1β maturation. A shock-like condition was established in 23 animals by continuous infusion of E. coli lipopolysaccharide (LPS) for 10 h. Then the animals were observed for 10 h. Twelve pigs received iNO and hydrocortisone (iNO treatment started 3 h after the initial LPS infusion and continued until the end of the experiment). Eleven pigs were controls. Pigs treated with iNO and hydrocortisone displayed less inflammatory infiltrates in the lungs than the controls and a lower level of IL-1β. The proMMP2 was significantly decreased in the iNO and hydrocortisone group. The amount of an active MMP9 (~ 60 kDa) was decreased in the iNO and hydrocortisone group. Total gelatinolytic activity was lower in the iNO and hydrocortisone group. Reduced MMP activity was accompanied by a 2.5-fold decrease of the active IL-1β form (17 kDa) in the pulmonary tissue of iNO combined with hydrocortisone exposed pigs. We demonstrated that in a porcine endotoxemia model the NO inhalation combined with intravenous hydrocortisone led to the attenuation of the inflammatory cascade induced by bacterial LPS. The decrease in pulmonary MMPs activities was accompanied by reduced proIL-1β processing.

Figure 1

Study protocol. The experiment was run for 20 h. Shock was induced by continuous intravenous (IV) infusion of lipopolysaccharide (LPS). After anesthesia and instrumentation an initial dose of 2.5 µg/kg/h was administered for 1.5 h, then the dose was changed to 0.5 µg/kg/h for the next 8.5 h (a total of 10 h of LPS infusion), followed by a further 10 h of observation. Animals were randomized into two groups: the control group (standard treatment) and the iNO + HCT group (standard treatment + inhaled NO and IV hydrocortisone).

Figure 2

Pigs treated with the combination of iNO and IV hydrocortisone (HCT) displayed significantly less inflammatory infiltrates in lungs than control animals. Representative photomicrograph of lung sections from the control group and iNO + HCT treatment group, stained by the H&E method. Pulmonary congestion (black arrows) in the control group (A) and iNO + HCT (B) group; note hemosiderin granules (white arrow); globular hyaline microthrombus (black arrows) in the control group (C) and iNO + HCT (D) group; fluid in the alveolar spaces (black arrows) in the control group (E) and iNO + HCT (F) group; inflammatory infiltration (black arrows) in the control group (G) and iNO + HCT (H) group. (A,B) Original magnification × 1250; (C,D) original magnification × 800; (E,F) original magnification × 400; (G,H) original magnification × 100.

Figure 3

The level of IL-1β is significantly lower in the group treated with iNO and IV hydrocortisone (iNO + HCT) than in the control group. Main proinflammatory mediators in pulmonary homogenates in a porcine endotoxemia model. Values are presented as means ± SEM. Statistical significance was determined by the Mann–Whitney U test. *P < 0.05 vs. control group. HCT hydrocortisone, iNO inhaled nitric oxide, IL-1β interleukin-1β, TNF-α tumor necrosis factor α, IL-8 interleukin-8, IL-6 interleukin-6.

Figure 4

MMP activities are reduced in the pulmonary tissue of iNO and IV hydrocortisone (iNO + HCT) exposed pigs. Gelatinolytic activity in pulmonary homogenates. (A) representative gelatin zymograms of pulmonary homogenates from the iNO + HCT group and the control group; densytometric analysis of MMP zymographic activity was performed using pulmonary homogenates from the pigs in the control group and from the iNO + HCT group. The optical density value for the pig with the lowest activity from the control group was set to 1, and then the results for all other animals were calculated accordingly. (B) An active form of MMP9 (60 kDa) and proMMP2 (72 kDa); (C) The total gelatinolytic activity quantified using a biotin–gelatin assay in pulmonary homogenates from the iNO + HCT and control groups. The measured mean absorbance A₄₅₀ value for a pig with the lowest gelatinolytic activity was considered as 100%; results for all other animals were expressed as a percentage of this activity. Values are presented as means ± SEM. The Mann–Whitney U test was used for statistical analysis. *p < 0.05; **p < 0.005; ***p < 0.0005 vs. control group.

Figure 5

The active IL-1β form (17 kDa) decreases in the pulmonary tissue of iNO and IV hydrocortisone (iNO + HCT) exposed pigs. Densitometric analysis of the mature IL-1β band (17 kDa) in pulmonary homogenates from the control and iNO + HCT groups. The optical density value for the same pig as used in the analysis of gelatinolytic activity (from the control group) was set to 1, and then the results for all other animals were calculated accordingly. Representative Western blots using pulmonary homogenates from a pig from the iNO + HCT group and the control group show 33 kDa proIL-1β band and a mature (active) 17 kDa IL-1β band. GAPDH was used as a loading control for protein normalization. Values are presented as means ± SEM. The Mann–Whitney U test was used for statistical analysis. *p < 0.05 vs. control group.