Effect of linoleic acid on lipopolysaccharide-induced release of inflammatory factors in macrophages of mice

Abstract

Objective To evaluate the effect of linoleic acid on lipopolysaccharide (LPS)-induced release of inflammatory factors in the macrophages of mice. Methods The peritoneal macrophages obtained from C57BL/C mice were seeded in 24-well plates at a density of 4×105 cells/well and in 6-well plates at a density of 2×106 cells/well.The cells were incubated and attached to the wall overnight in a 5% CO2 incubator in humidity at 37 ℃.The experiment was performed in 2 parts.PartⅠ The cells in 24-well plates were randomly divided into 5 groups (n=8 each) using a random number table: control group (group C); LPS group; 3 different concentrations of linoleic acid groups (LA1-3 groups). The sterile anhydrous alcohol 1 μl was added in group LPS, 0.1, 0.5 and 1.0 mol/ml linoleic acid 1 μl were added in LA1-3 groups, respectively, and 30 min later 100 μg/ml LPS 1 μl was added in LPS and LA1-3 groups.The culture medium was collected at 6 h after LPS administration to measure the concentrations of tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) in the supernatant by enzyme-linked immunosorbent assay.PartⅡ The cells in 6-well plates were randomly divided into 3 groups (n=6 each) using a random number table: control group (group C); LPS group; 0.5 mol/ml linoleic acid group (group LA). The sterile anhydrous alcohol 1 μl was added in group LPS, 0.5 mol/ml linoleic acid 1 μl was added in group LA, and 30 min later 100 μg/ml LPS 1 μl was added in LPS and LA groups.At 1 h after administration of LPS, the expression of Toll-like receptor 4 (TLR4) was determined by flow cytometry, and the expression of phosphorylated nuclear factor kappa B (NF-κB) p65 (p-NF-κB p65), phosphorylated extracellular signal-regulated protein kinase (p-ERK) and phosphorylated p38 mitogen-activated protein kinase (p-p38 MAPK) was determined by Western blot. Results PartⅠ Compared with group C, TNF-α and IL-6 concentrations in the supernatant were significantly increased in LPS and LA1-3 groups (P<0.05). Compared with group LPS, TNF-α and IL-6 concentrations in the supernatant were significantly decreased in LA1-3 groups (P<0.05). Compared with group LA1, TNF-α and IL-6 concentrations in the supernatant were significantly decreased in LA2 and LA3 groups (P<0.05). Compared with group LA2, TNF-α and IL-6 concentrations in the supernatant were significantly decreased in group LA3 (P<0.05). PartⅡ Compared with group C, the expression of TLR4, p-NF-κB p65, p-ERK and p-p38 MAPK in macrophages was significantly up-regulated in LPS and LA groups (P<0.05). Compared with group LPS, the expression of TLR4, p-NF-κB p65, p-ERK and p-p38 MAPK in macrophages was significantly down-regulated in group LA (P<0.05). Conclusion Linoleic acid can inhibit LPS-induced release of inflammatory factors in the macrophages of mice, and the mechanism may be related to the inhibition of TLR4 signaling pathway activation. Key words: Linoleic acid; Lipopolysaccharides; Macrophages; Cytokines