ABSTRACT
Objective To evaluate the effect of hydrogen preconditioning during cold ischemia phase on the activity of nuclear factor erythroid 2-related factor 2 ( Nrf2) in rat pulmonary microvascular en-dothelial cells ( PMVECs) subjected to hypoxia-reoxygenation ( H/R) . Methods PMVECs were isolated from clean-grade male Sprague-Dawley rats, aged 2-3 weeks, using the tissue block adherence method and divided into 4 groups ( n=25 each) using a random number table method: control group ( group C) , H/R group, oxygen group ( O group) and hydrogen group ( H group) . Cells were incubated for 4 h with 4℃ low potassium dextransolution ( LPD) pre-equilibrated with 95％ oxygen and 5％ carbondioxide to simulate the cold ischemia phase. LPD pre-balanced with 95％ oxygen and 5％ carbon dioxide was replaced with LPD, and then cells were incubated for 1 h at room temperature to simulate the lung transplantation period. LPD was rapidly replaced with 37℃ M199 complete culture solution, and cells were incubated in the mixture of 40％ oxygen-5％ carbondioxide-55％ nitrogen to simulate the reperfusion period. In O and H groups, the cells were exposed to 40％ oxygen-60％ nitrogen and 3％ hydrogen-40％ oxygen-57％ nitrogen during the cold ischemia period, respectively, and the gas mixture was replaced every 20 min. The cell culture fluid was collected 4 h later for determination of interleukin ( IL )-6, IL-10 and tumor necrosis factor-alpha ( TNF-α) concentrations ( by enzyme-linked immunosorbent assay) and malondialdehyde ( MDA) concen-trations ( by thiobarbituric acid method) . The cytoplasm and nucleoproteins were extracted for measurement of Nrf2 expression ( by Western blot) and cell apoptosis ( by flow cytometry and TUNEL assay) . The cell apoptosis rate was calculated. Results Compared with C group, the IL-6, TNF-α and MDA levels were significantly increased, the IL-10 level was decreased, the apoptosis rate was increased, and the expres-sion of Nrf2 in nucleus was up-regulated in H/R group ( P＞0. 05) . Compared with H/R group, the IL-6, TNF-α and MDA levels were significantly decreased, the IL-10 level was increased, the apoptosis rate was decreased, and the Nrf2 expression in cytoplasm was down-regulated in O and H groups (P＜0. 05), the Nrf2 expression was significantly up-regulated in H group ( P＜0. 05) , and no significant change was found in the expression of Nrf2 in nucleus in O group ( P＞0. 05) . Compared with O group, the IL-6, TNF-αand MDA levels were significantly decreased, the IL-10 level was increased, the apoptosis rate was decreased, the expression of Nrf2 in nucleus was up-regulated, and the expression of Nrf2 protein in cytoplasm was down-regulated in H group ( P＜0. 05 ) . Conclusion The mechanism by which hydrogen preconditioning during cold ischemia phase reduces H/R injury to rat PMVECs is related to activating Nrf2 and thus inhibi-ting oxidative stress.