Effect of hydrogen preconditioning during cold ischemia phase on activity of Nrf2 in rat pulmonary microvascular endothelial cells subjected to hypoxia-reoxygenation

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 endothelial 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) concentrations (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 expression 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). Compared with O group, the IL-6, TNF-α and 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 inhibiting oxidative stress. Key words: Hydrogen; Cold ischemia; Microvessels; Endothelial cells; Lung; NF-E2-related factor 2