Acute Crit Care.  2018 Feb;33(1):34-41. 10.4266/acc.2017.00577.

The Effects of Flecainide Acetate on Inflammatory-Immune Response in Lipopolysaccharide-Stimulated Neutrophils and on Mortality in Septic Rats

• Chung SY
• Kim J
• Bae HB
• Tin TD
• Ju W
• Kwak SH

• KMID: 2436305
• DOI: http://doi.org/10.4266/acc.2017.00577

Abstract

BACKGROUND
Flecainide acetate is a drug used primarily for cardiac arrhythmia. Some studies also imply that flecainide acetate has the potential to regulate inflammatory-immune responses; however, its mechanism of action is contended. We determined the effects of flecainide acetate on lipopolysaccharide (LPS)-stimulated human neutrophils in vitro and on mortality in a septic rat model.
METHODS
Neutrophils from human blood were cultured with varying concentrations of flecainide acetate (1 μM, 10 μM, or 100 μM) with or without LPS (100 ng/ml). To assess neutrophil activation, the protein levels of tumor necrosis factor-alpha (TNF-α) and interleukin (IL)-6 and IL-8 were measured after a 4-hour culture period. To assess the intracellular signaling pathways, the levels of phosphorylation of p38 mitogen-activated protein kinase (p38), extracellular signal-regulated kinase (ERK) 1/2, and c-Jun N-terminal kinase (JNK) were measured after a 30-minute culture period, and the nuclear translocation of nuclear factor (NF)-κB was measured after a 1-hour culture period. Additionally, the survival rate was investigated in a rat sepsis model.
RESULTS
Flecainide acetate down-regulated the activation of proinflammatory cytokines, including TNF-α and IL-6 and IL-8, and intracellular signaling pathways including ERK 1/2 and NF-κB. Flecainide acetate also improved the survival rate in the rat sepsis model.
CONCLUSIONS
Collectively, these findings indicate that flecainide acetate can improve survival in a rat sepsis model by attenuating LPS-induced neutrophil responses. We therefore suggest that flecainide acetate plays an important role in modulating inflammatory-immune responses.

Keyword

lipopolysaccharides; neutrophils; rats; sepsis; sodium channel blockers

MeSH Terms

Animals
Arrhythmias, Cardiac
Cytokines
Flecainide*
Humans
In Vitro Techniques
Interleukin-6
Interleukin-8
Interleukins
JNK Mitogen-Activated Protein Kinases
Lipopolysaccharides
Models, Animal
Mortality*
Neutrophil Activation
Neutrophils*
Phosphorylation
Phosphotransferases
Protein Kinases
Rats*
Sepsis
Sodium Channel Blockers
Survival Rate
Tumor Necrosis Factor-alpha
Cytokines
Flecainide
Interleukin-6
Interleukin-8
Interleukins
JNK Mitogen-Activated Protein Kinases
Lipopolysaccharides
Phosphotransferases
Protein Kinases
Sodium Channel Blockers
Tumor Necrosis Factor-alpha

Figure

• 

  Figure 1. The effects of flecainide acetate on proinflammatory cytokine (A: IL-6, B: IL-8, C: TNF-α) expression in LPS-stimulated neutrophils. Neutrophils (5 × 106/ml) from human blood were incubated for 4 hours with or without flecainide acetate (1, 10, or 100 μM) and with or without LPS (100 ng/ml) with the control group (CON) receiving no treatment. The protein levels were obtained using enzyme-linked immunosorbent assays. A representative experiment, in which each condition was examined in triplicate, is shown. Two additional experiments provided similar results. The data are presented as mean ± standard error for each experimental group. IL: interleukin; LPS: lipopolysaccharide; TNF-α: tumor necrosis factor-alpha. aP < 0.05, vs. CON; bP < 0.05, vs. LPS.

• 

  Figure 2. The effects of flecainide acetate on mitogen-activated protein (A: ERK, B: p38, C: JNK) pathway activation in LPS-stimulated neutrophils. Neutrophils (5 × 106/ml) from human blood were incubated for 30 minutes with or without flecainide acetate (100 μM) and with or without LPS (100 ng/ml) with the control group (CON) receiving no treatment. The histogram shows mean ± standard error from three experiments. ERK: extracellular signal-regulated kinase; LPS: lipopolysaccharide; p38: p38 mitogen-activated protein kinase; JNK: Jun N-terminal kinase. aP < 0.05, vs. CON; bP < 0.05, vs. LPS.

• 

  Figure 3. The effects of flecainide acetate on the nuclear translocation of nuclear factor (NF)-κB in lipopolysaccharide (LPS)-stimulated neutrophils. Neutrophils (5 × 106/ml) from human blood incubated for 1 hour with 100 ng/ml of LPS (LPS) showed more nuclear translocation of NF-κB than neutrophils incubated for 1 hour without LPS (Control) or with 100 μM flecainide acetate (Flecainide). Coincubation of neutrophils with 100 μM flecainide acetate and 100 ng/ml LPS (LPS + flecainide) resulted in less nuclear translocation of NF-κB compared to LPS alone. Three replicate experiments provided similar results.

• 

  Figure 4. The effects of flecainide acetate on mortality in a lipopolysaccharide-induced rat sepsis models. The mortality rates were calculated 24 hours after the intraperitoneal (IP) injection of saline or lipopolysaccharide. Mortality was over 40% in the S-E (positive control) group, whereas mortality was markedly reduced to less than 20% in the F0.1-E (low-dose flecainide acetate) group. All rats in the F0.2-E (high-dose flecainide acetate) group survived. Each value represents mean ± standard error from 10 rats. S-S: subcutaneous (SC) infusion of saline and IP injection saline (n = 10); F-S: SC infusion of flecainide acetate (0.2 mg/kg/hr) and IP injection of saline (n = 10); S-E: SC infusion of saline and IP injection of Escherichia coli endotoxin (25 mg/kg, n = 10); F0.1-E: SC infusion of flecainide acetate (0.1 mg/kg/hr) and IP injection of endotoxin (n = 10); F0.2-E: SC infusion of flecainide acetate (0.2 mg/kg/hr) and IP injection of endotoxin (n = 10). aP < 0.05, vs. the S-S negative-control group; bP < 0.05, vs. the S-E positive-control group.

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