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

Abstract

Objective 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 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). Results Compared 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). Conclusion The mechanism by which hydrogen inhibits LPS-induced inflammatory factor release from macrophages is related to the activation of Nrf2/ARE signaling pathway in mice. Key words: Nuclear factor-E2 related factor 2; Response elements; Signal transduction; Hydrogen; Lipopolysaccharides; Macrophages; Cytokines