Nrf2／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

Role of Nrf2/ARE signaling pathway in inhibition of LPS-induced inflammatory factor release from macrophages by hydrogen

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 1 μg/ml；LPS＋H2组加入LPS 1 μg/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 1 μg/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。

ObjectiveTo 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.MethodsRAW264.7 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 1 μg/ml was added, and the culture medium was then replaced with the culture medium containing 0.6 mmol/L hydrogen-rich saline.In group siRNA＋ LPS＋ H2, after Nrf2-siRN was successfully transfected into the cells, the cells were continuously 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 separated 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）.ResultsCompared with group C, 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 HO-1 in cells was significantly up-regulated in LPS and siRNA＋ LPS＋ H2 groups, and the expression of Nrf2 in cells was significantly 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 significantly 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 significantly down-regulated in group LPS＋ H2＋ siRNA（P〈0.05）.ConclusionThe mechanism by which hydrogen inhibits LPS-induced inflammatory factor release from macrophages is related to the activation of Nrf2/ARE signaling pathway in mice.