Regulatory role of the TLR4/JNK signaling pathway in sepsis‑induced myocardial dysfunction

Abstract

Sepsis is a life‑threatening organ dysfunction caused by a dysregulated host response to infection, and is a leading cause of mortality worldwide. Myocardial dysfunction is associated with poor prognosis in patients with sepsis and contributes to a high risk of mortality. However, the pathophysiological mechanisms underlying sepsis‑induced myocardial dysfunction are not completely understood. The aim of the present study was to investigate the role of toll‑like receptor 4 (TLR4)/c‑Jun N‑terminal kinase (JNK) signaling in pro‑inflammatory cytokine expression and cardiac dysfunction during lipopolysaccharide (LPS)‑induced sepsis in mice. C57BL/6 mice were pretreated with TAK‑242 or saline for 1 h and then subjected to LPS (12 mg/kg, intraperitoneal) treatment. Cardiac function was assessed using an echocardiogram. The morphological changes of the myocardium were examined by hematoxylin and eosin staining and transmission electron microscopy. The serum protein levels of cardiac troponin I (cTnI) and tumor necrosis factor‑α (TNF‑α) were determined by an enzyme‑linked immunosorbent assay (ELISA). The TLR4 and JNK mRNA levels were analyzed via reverse transcription‑quantitative PCR. TLR4, JNK and phosphorylated‑JNK protein levels were measured by western blotting. In response to LPS, the activation of TLR4 and JNK in the myocardium was upregulated. There were significant increases in the serum levels of TNF‑α and cTnI, as well as histopathological changes in the myocardium and suppressed cardiac function, following LPS stimulation. Inhibition of TLR4 activation using TAK‑242 led to a decrease in the activation of JNK and reduced the protein expression of TNF‑α in plasma, and alleviated histological myocardial injury and improved cardiac function during sepsis in mice. The present data suggested that the TLR4/JNK signaling pathway played a critical role in regulating the production of pro‑inflammatory cytokines and myocardial dysfunction induced by LPS.

Keywords: sepsis; myocardial dysfunction; lipopolysaccharide; animal models; Toll‑like receptor 4; c‑Jun N‑terminal kinase signaling pathway.

Figure 1.

Representative echocardiographic images (M-mode) in different groups. Left, representative echocardiographic images in sham group (top), LPS group (middle) and TAK-242 group (bottom) at 3 h after LPS or saline injection. Middle, representative echocardiographic images in sham group (top), LPS group (middle) and TAK-242 group (bottom) at 12 h after LPS or saline injection. Right, representative echocardiographic images in sham group (top), LPS group (middle) and TAK-242 group (bottom) at 24 h after LPS or saline injection. LPS, lipopolysaccharide.

Figure 2.

Echocardiographic characterization of cardiac function in different groups. Quantitative assessment of dilation and systolic function of heart based on (A) LVEDD, (B) LVEDV, (C) LVESD, (D) LVESV, (E) LVEF and (F) LVFS. Data are expressed as the mean ± SD from six animals in each group. *P<0.05 vs. sham group; ^#P<0.05 vs. LPS group. LVEDD, left ventricular end diastolic diameter; LVESD, left ventricular end systole diameter; LVEDV, left ventricular end diastolic volume; LVESV, left ventricular end systolic volume; LVEF, left ventricle ejection fraction; LVFS, left ventricle fractional shortening; LPS, lipopolysaccharide.

Figure 3.

Histological changes of myocardium in different groups. (A) Representative images of hematoxylin and eosin-stained sections of the mouse heart tissues in different groups. (A-1) The LPS (top) and TAK-242 (bottom) groups. (A-2) The sham group. Magnification, ×400. (B) Representative images of transmission electron micrographs of the mouse heart in different groups. Black arrows indicate myofibrillar disarray, and red arrows indicate mitochondrial swelling and cracking. (B-1) The LPS (top) and TAK-242 (bottom) groups. (B-2) The sham group. Magnification, ×3,000. LPS, lipopolysaccharide.

Figure 4.

Protein levels of cTnI and TNF-α of mouse serum in different groups. Statistical results from ELISA for (A) cTnI and (B) TNF-α. Data are expressed as the mean ± SD from six animals in each group. *P<0.05 vs. sham group; ^#P<0.05 vs. LPS group. cTnI, cardiac troponin I; TNF-α, tumor necrosis factor-α; ELISA, enzyme linked immunosorbent assay; LPS, lipopolysaccharide.

Figure 5.

TLR4 and JNK gene expression in mouse hearts from different groups. The relative expression levels of cardiac (A) TLR4 and (B) JNK mRNA were assessed by reverse transcription-quantitative PCR. Results were normalized to GAPDH. Data are expressed as mean ± SD (each group, n=6). *P<0.05 vs. sham group; ^#P<0.05 vs. LPS group. TLR4, toll-like receptor 4; JNK, c-Jun N-terminal kinase; LPS, lipopolysaccharide.

Figure 6.

TLR4, JNK and p-JNK protein expression in the mouse hearts from different groups. Representative western blotting images showing protein expression of (A-1) TLR4 and (B-1) total and p-JNK in the mouse heart from various groups. Semi-quantification of western blot analysis, (A-2) TLR4, (B-2) p-JNK/JNK and (B-3) expression. Protein expression shown as relative densitometric absorption units (left axis). Data are expressed as the mean ± SD (each group, n=6). *P<0.05 vs. sham group; ^#P<0.05 vs. LPS groups. TLR4, toll-like receptor 4; JNK, c-Jun N-terminal kinase; LPS, lipopolysaccharide; p-, phosphorylated.