富氢液对内毒素诱导RAW264．7细胞炎性因子释放的影响

Effects of hydrogen-rich medium on lipopolysaccharide-induced release of inflammatory cytoldnes from RAW264.7 macrophages

目的探讨富氢液对内毒素诱导RAW264．7细胞炎性因子释放的影响。方法小鼠巨噬细胞系RAW264．7，培养于含10％胎牛血清的DMEM培养基，接种于6孔细胞培养板，3ml/孔，细胞密度2×106／ml。采用随机数字表法，将其随机分为4组（n=24）：正常对照组（A组）、富氢液组（B组）、脂多糖（LPS）组（C组）和LPS＋富氢液组（D组）。A组不做任何处理；C组和D组加入1μg/ml，LPS，同时B组和D组用0．6mmol／L富氢液孵育。分别于孵育6、12、24（和48h时取6孔，收集细胞培养上清液，测定TNF-a、IL-1β和IL-10的浓度；分别于孵育6、12时取6孔，收集细胞沉淀，提取蛋白，采用Westernblot法测定血红素氧合酶．1（HO．1）表达。结果与A组比较，C组细胞培养上清液TNF—α、IL-1β和IL-10浓度升高，细胞HO-1表达上调（P〈0．05）；与C组比较，D组细胞培养上清液TNF-α和IL-1β浓度降低，IL-α浓度升高，细胞HO-1表达上调（P〈0．05）。结论富氢液可抑制RAW264．7细胞释放促炎细胞因子，促进抗炎细胞因子释放，其机制与上调HO-1表达有关。

Objective To investigate the effects of hydrogen-rich medium on lipopolysaccharide （LPS）- induced release of inflammatory cytokines from mufine macrophage RAW264.7 cells. Methods The RAW264.7 cells were cultured in DMEM culture medium containing 10% fetal bovine serum. The cells were seeded in 6-well plates （3 ml/hole） with a density of 2 × 106/ml and randomly divided into 4 groups （ n = 24 each） ： control group （group A）, hydrogen-rich medium group （group B）, LPS group （group C） and LPS ＋ hydrogen-rich medium group （group D）. Group A received no treatment. LPS 1 μg/ml was added to the medium in groups C and D, while the cells were incubated with 0.6 mmol/L hydrogen-rich medium simultaneously in groups B and D. Six wells in each group were chosen at 6, 12, 24 and 48 h of incubation, and the supernatant was collected to detect the concentrations of TNF-α, IL-1β and IL-10. Six wells in each group were chosen at 6 and 12 h of incubation for de- termination of heme oxygenase-1 （HO-1） expression in the cells by Western blot. Results Compared with group A, the concentrations of TNF-α, IL-β and IL-10 in the supernatant were significantly increased, and the expres- sion of HO-1 was up-regulated in group C （ P 〈 0.05）. Compared with group C, the concentrations of TNF-α and IL-1β in the supernatant were significantly decreased, the concentration of IL-10 in the supernatant was significantly increased, and the expression of HO-1 was up-regulated in group D （ P 〈 0.05）. Conclusion Hydrogen-rich me- dium can inhibit the release of pro-inflammatory cytokines and promote the release of anti-inflammatory cytokines from RAW264.7 cells via up-regnlating HO-1 expression.