Role of NLRP3 inflammasome in hydrogen-rich saline-induced reduction of LPS-caused damage to mitochondria in macrophages of mice

Abstract

Objective To investigate the role of nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3(NLRP3)inflammasome in hydrogen-rich saline-induced reduction of lipopolysaccharide(LPS)-caused damage to mitochondria in macrophages of mice. Methods Macrophage line RAW264.7 of mice were routinely cultured and divided into 4 groups(n=6 each)using a random number table method: control group(group C), group LPS, hydrogen-rich saline plus LPS group(group LPS+ H2)and hydrogen-rich saline plus LPS plus ATP group(group LPS+ ATP+ H2). LPS was given at the concentration of 1 μg/ml, and the cells were then incubated for 30 min in group LPS.LPS at the concentration of 1 μg/ml and hydrogen-rich saline at the concentration of 0.6 mmol/L were simultaneously given, and the cells were then incubated for 30 min in LPS+ H2 and LPS+ ATP+ H2 groups.ATP at the concentration of 1 nmol/L was then given, and the cells were incubated for 6 h in group LPS+ ATP+ H2.Mitochondrial membrane potential(MMP)was determined by JC-1 staining, and respiratory control ratio(RCR)was measured using a Clark-type electrode.The expression of NLRP3, caspase-1 and apoptosis-associated speck-like protein containing C-terminal caspase recruitment domain(ASC)was determined by Western blot.The concentrations of INTERLEUKIN-1 BETA(IL-1β), IL-18 and IL-6 in the supernatant were determined by enzyme-linked immunosorbent assay. Results Compared with group C, MMP and RCR were significantly decreased, the concentrations of IL-1β, IL-18 and IL-6 in the supernatant were increased, and the expression of NLRP3, caspase-1 and ASC was up-regulated in group LPS(P<0.05). Compared with group LPS, MMP and RCR were significantly increased, the concentrations of IL-1β, IL-18 and IL-6 in the supernatant were decreased, and the expression of NLRP3, caspase-1 and ASC was down-regulated in group LPS+ H2(P<0.05). Compared with group LPS+ H2, MMP and RCR were significantly increased, the concentrations of IL-1β, IL-18 and IL-6 in the supernatant were decreased, and the expression of NLRP3, caspase-1 and ASC was down-regulated in group LPS+ ATP+ H2(P<0.05). Conclusion Hydrogen-rich saline can reduce LPS-caused damage to mitochondria in macrophages of mice through inhibiting the activation of NLRP3 inflammasome. Key words: Hydrogen; Endotoxins; Macrophages; Mitochondria; NOD like receptor pynn domain containing 3