摘要目的：评价NF?E2相关因子2（ Nrf2）∕抗氧化反应序列原件（ ARE）信号通路在氢抑制LPS致巨噬细胞炎症因子释放中的作用。方法小鼠巨噬细胞株RAW264．7接种于6孔培养板（2×106个∕孔），采用随机数字表法将其分为4组（ n＝24）：对照组（ C组）、LPS组、富氢液＋LPS组（ LPS＋H2组）和Nrf2小干扰RNA（ siRNA）＋ LPS＋富氢液组（ siRNA＋LPS＋H2组）。 LPS组加入LPS 1μg∕ml；LPS＋H2组加入LPS 1μg∕ml，培养基更换为0．6 mmol∕L的富氢液培养基；siRNA＋LPS＋H2组将Nrf2?siRN成功转染至细胞后，继续培养24 h，加入LPS 1μg∕ml，培养基更换为0．6 mmol∕L的富氢液培养基。于孵育24 h时，收集上清液，采用比色法测定LDH活性，采用ELISA法检测TNF?α、IL?1β、高迁移率族蛋白B1（ HMGB1）和IL?6的浓度；收集细胞，采用MTT法测定细胞增殖水平，采用Western blot法检测Nrf2和血红素氧合酶?1（ HO?1）的表达水平。结果与C组比较，LPS组和siRNA＋ LPS＋H2组上清液 LDH活性、TNF?α、IL?1β、IL?6和HMGB1的浓度升高，细胞增殖水平降低，HO?1表达上调， LPS组和LPS＋H2组细胞Nrf2表达上调（P＜0．05）；与LPS组比较，LPS＋H2组上清液LDH活性、TNF?α、IL?1β、IL?6和HMGB1的浓度降低，细胞增殖水平升高，Nrf2和HO?1的表达上调，siRNA＋LPS＋H2组细胞Nrf2和HO?1的表达下调（P＜0．05）；与LPS＋H2比较，LPS＋H2＋siRNA组上清液LDH活性、TNF?α、IL?1β、IL?6和HMGB1的浓度升高，细胞增殖水平降低，Nrf2和HO?1的表达下调（ P＜0．05）。结论氢抑制LPS致巨噬细胞炎症因子释放的机制与激活Nrf2∕ARE信号通路有关。

abstractsObjective To evaluate the role of nuclear factor erythroid 2?related factor 2 ( Nrf2)∕antioxidant response element( ARE) signaling pathway in inhibition of lipopolysaccharide ( LPS)?induced inflammatory factor release from macrophages by hydrogen. Methods RAW264. 7 macrophages of mice were cultured in 6?well plates (2×106 cells∕well) and were divided into 4 groups (n=24 each) using a random number table: control group ( group C); LPS group; hydrogen?rich saline+LPS group ( group LPS+H2); Nrf2 small interference RNA (siRNA)+LPS+hydrogen?rich saline group (siRNA+LPS+H2 group) . LPS 1 μg∕ml was added in group LPS. In group LPS+H2 , LPS 1μg∕ml was added, and the cul?ture medium was then replaced with the culture medium containing 0. 6 mmol∕L hydrogen?rich saline. In&nbsp;group siRNA+LPS+H2 , after Nrf2?siRN was successfully transfected into the cells, the cells were continu?ously incubated for 24 h, and the culture medium was then replaced with the culture medium containing 0.6 mmol∕L hydrogen?rich saline after LPS 1 μg∕ml was added. At 24 h of incubation, the supernatant was sep?arated for determination of the lactic dehydrogenase (LDH) activity (using colorimetric method) and for detection of the concentrations of tumor necrosis factor?alpha ( TNF?α) , interleukin?1 beta ( IL?1β) , high mobility group box?1 (HMGB1) and IL?6 (by ELISA). The cells were collected for measurement of the proliferation of cells ( by methyl thiazolyl tetrazolium assay) and for determination of the expression of Nrf2 and heme oxygenase?1 ( HO?1) in cells ( by Western blot) . Results Compared with group C, the LDH activity and concentrations of TNF?α, IL?1β, IL?6 and HMGB1 in the supernatant were significantly in?creased, the proliferation of cells was significantly decreased, and the expression of HO?1 in cells was sig?nificantly up?regulated in LPS and siRNA+LPS+H2 groups, and the expression of Nrf2 in cells was signifi?cantly up?regulated in LPS and LPS+H2 groups (P<0.05). Compared with group LPS, the LDH activity and concentrations of TNF?α, IL?1β, IL?6 and HMGB1 in the supernatant were significantly decreased, the proliferation of cells was significantly increased, and the expression of Nrf2 and HO?1 in cells was sig?nificantly up?regulated in group LPS+H2 , and the expression of Nrf2 and HO?1 in cells was significantly down?regulated in group siRNA+LPS+H2 ( P<0.05) . Compared with group LPS+H2 , the LDH activity and concentrations of TNF?α, IL?1β, IL?6 and HMGB1 in the supernatant were significantly increased, the proliferation of cells was significantly decreased, and the expression of Nrf2 and HO?1 in cells was signifi?cantly down?regulated in group LPS+H2+siRNA ( P<0.05) . Conclusion The mechanism by which hydro?gen inhibits LPS?induced inflammatory factor release from macrophages is related to the activation of Nrf2∕ARE signaling pathway in mice.