NLRP3炎症小体在富氢液减轻LPS致小鼠巨噬细胞线粒体损伤中的作用

摘要

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 apoptosisassociated 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-l8 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.%目的 探讨NOD样受体热蛋白结构域相关蛋白3(NLRP3)炎症小体在富氢液减轻LPS致小鼠巨噬细胞线粒体损伤中的作用.方法 常规培养小鼠巨噬细胞株RAW264.7细胞,采用随机数字表法随机分为4组(n=6):对照组(C组)、LPS组、富氢液+LPS组(LPS+H2组)和富氢液+LPS+ATP组(LPS+ATP+H2组).LPS组给予浓度为1μg/ml的LPS孵育30 min.LPS+H2组和LPS+ATP+H2组同时给予浓度为1μg/ml的LPS和浓度为0.6 mmol/L的富氢液孵育30 min,然后LPS+ATP+H2组给予浓度为1 nmol/L的ATP孵育6h.采用JC-1法测定线粒体膜电位(MMP),采用Clark氧电极法测定线粒体呼吸控制率(RCR).采用Western blot法测定细胞NLRP3、caspase-1和凋亡相关点状蛋白(ASC)的表达.采用ELISA法检测上清液IL-1β、IL-18和IL-6的浓度.结果 与C组比较,LPS组MMP和RCR降低,上清液IL-1β、IL-18和IL-6浓度升高,细胞NLRP3、caspase-1和ASC表达上调(P＜0.05);与LPS比较,LPS+H2组MMP和RCR升高,上清液IL-1β、IL-18和IL-6浓度降低,细胞NLRP3、caspase-1和ASC表达下调(P＜0.05);与LPS+H2组比较,LPS+ ATP+H2组MMP和RCR升高,上清液IL-1β、IL-18和IL-6浓度降低,细胞NLRP3、caspase-1和ASC表达下调(P＜0.05).结论 富氢液可通过抑制NLRP3炎症小体激活,减轻LPS致小鼠巨噬细胞线粒体损伤.