Melatonin pretreatment modulates anti-inflammatory, antioxidant, YKL-40, and matrix metalloproteinases in endotoxemic rat lung tissue

Abstract

We aimed to investigate the effects of melatonin administered before and during endotoxemia on the lung tissue of rats, cytokine, YKL-40, matrix metalloproteinase (MMP) and inhibitor levels, oxidative stress parameters, and energy balance. Sepsis was induced with lipopolysaccharide (LPS), the cell wall molecule of gram negative bacteria. Rats were divided into four groups, Control, LPS (Escherichia coli O127:B8, 20 mg/kg), melatonin (10 mg/kg), and melatonin+LPS (M+LPS). After injections, lung tissues samples were taken for experimental analyses. YKL-40, thiobarbituric acid reactive substances (TBARS), glutathione reductase (GR), glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) enzymes levels were measured, high-energy components were analyzed; tumor necrosis factor-alpha (TNF-α), MMP-2, YKL-40, MMP-9, myeloperoxidase (MPO), tissue inhibitors of matrix metalloproteinase (TIMP)-1, and interleukin (IL)-10 immunoreactivities were investigated. In LPS group, YKL-40, creatine phosphate (both, p < 0.05), SOD, GR, adenosine mono-phophate (AMP), adenosine tri-phosphate (ATP) (for all, p < 0.01) were significantly decreased, while TBARS and adenosine di-phosphate (ADP) levels were increased (p < 0.01, p < 0.05; respectively) compared to other groups. MMP-2 and -9, TIMP-1, TNF-α, IL-10, and MPO immunoreactivity were investigated in LPS group. On the contrary, in M+LPS group, MMP-9, TIMP-1 immunoreactivities were not found and IL-10 and MMP-2 immunoreactivities were found with little involvement. In M+LPS group, YKL-40, GR, AMP, ATP, creatine phosphate (for all, p < 0.05), and SOD (p < 0.01) levels were significantly increased and TBARS levels were decreased (p < 0.05). In our study, we suggest that melatonin exerts a protective and curative effect by reducing the matrix metalloproteinase levels responsible for tissue damage balance, stimulating the release of antioxidant enzymes, regulating cytokines and energy balance during endotoxemia.

Keywords: Melatonin; YKL-40; cytokines; lipopolysaccharide; matrix metalloproteinases; oxidative stress.

Figure 1.

Diagram of experimental procedure.

Figure 2.

AMP, ADP, ATP, creatine, creatine p, ADP/ATP ratio levels in lung tissue were measured with HPLC in all experimental groups; control (n = 6), LPS (n = 8; 10 mg/kg i.p.), melatonin (n = 8; 10 mg/kg × 3 doses i.p.), melatonin+LPS (n = 8; LPS group treated with melatonin). *p < 0.05 (Tukey test); **p < 0.01 (Tukey test) LPS versus other groups, ⁺p < 0.5 (Tukey test) M+LPS versus LPS.

Figure 3.

YKL-40 levels in lung tissue of all experimental groups; control (n = 6), LPS (n = 8; 10 mg/kg i.p.), melatonin (n = 8; 10 mg/kg × 3 doses i.p.), melatonin+LPS (n = 8; LPS group treated with melatonin). *p < 0.05 (Tukey test); ⁺p < 0.5 (Tukey test) M+LPS versus LPS.

Figure 4.

Antioxidant enzymes chain (GR, GSH-Px, SOD) levels in lung tissue of all experimental groups; control (n = 6), LPS (n = 8; 10 mg/kg i.p.), melatonin (n = 8; 10 mg/kg × 3 doses i.p.), melatonin+LPS (n = 8; LPS group treated with melatonin). *p < 0.05 (Tukey test); **p < 0.01 LPS (Tukey test) versus other groups, ⁺p < 0.5 (Tukey test) M+LPS versus LPS.

Figure 5.

Lipid peroxidation (TBARS) in lung tissue of all experimental groups; control (n = 6), LPS (n = 8; 10 mg/kg i.p.), melatonin (n = 8; 10 mg/kg × 3 doses i.p.), melatonin+LPS (n = 8; LPS group treated with melatonin). **p < 0.01 (Tukey test) control versus LPS; ⁺p < 0.5 (Tukey test) M+LPS versus LPS.

Figure 6.

Sections of the lung tissue immunoreactivity from all groups; control (n = 6), LPS (n = 8; 10 mg/kg i.p.), melatonin (n = 8; 10 mg/kg × 3 doses i.p.), melatonin+LPS (n = 8; LPS group treated with melatonin) stained with TNF-α, IL-10, YKL-40, MMP-2, MMP-9, TIMP-1, and MPO staining. For each specimen, one score was assigned according to the percentage of positive cells: < 5% of the cells: 1 point; 6–35% of the cells: 2 points; 36–70% of the cells: 3 points; and > 71% of the cells: 4 points. Another score was assigned according to the intensity of staining, with negative staining equal to 0 point; no involvement, 1 point; mild involvement, 2 points; moderate involvement and intense involvement 3 points. (A color version of this figure is available in the online journal.)

Figure 7.

H-scores of sections of the lung tissue immunoreactivity from all groups. “a” indicates p < 0.05 versus all experimental groups; “b” indicates p < 0.05 versus control group (F = 5.463).

Figure 8.

Sections of the lung tissue from all groups stained with H&E. (a) Control, (b) melatonin, (c) LPS, and (d) M+LPS group, 50 µm scale bar. (A color version of this figure is available in the online journal.)