Effect of hydrogen-rich saline on Akt/Nrf2 signaling pathway during hypoxia/reoxygenation injury to human renal tubular epithelial cells / 中华麻醉学杂志

ABSTRACT

Objective:To evaluate the effect of hydrogen-rich saline on serine threonine protein kinase (Akt) /nuclear factor E2-related factor 2 (Nrf2) signaling pathway during hypoxia/reoxygenation (H/R) injury to human renal tubular epithelial cells.Methods:Human renal tubular epithelial cell line were seeded in 96-well plates at a density of 1.5×10 4 cells/ml (200 μl/well) or in 6-well plates at a density of 2×10 5 cells/ml (2 ml/well) were divided into 5 groups( n=30 each) using a random number table method: control group (group C), hydrogen-rich group (group H), group H/R, H/R plus hydrogen-rich saline group (group H/R+ H) and H/R plus hydrogen-rich saline plus Akt inhibitor uprosertib group (group H/R+ H+ U) .In group C, the cells were incubated for 28 h in an incubator filled with normoxia at 37 ℃ (5%CO 2-21%O 2-74%N 2). In group H, cells were added to the medium containing 0.6 mmol/L hydrogen-rich saline, and then incubated for 28 h in an incubator filled with normoxia at 37 ℃.In group H/R, the cells were incubated in an anaerobic chamber (37 ℃, 5%CO 2-1%O 2-94 %N 2) for 24 h, and then incubated for 4 h in an incubator filled with normoxia at 37 ℃.In group H/R+ H, the cells were incubated in an anaerobic chamber for 24 h, and then incubated for 4 h in an incubator containing 0.6 mmol/L filled with normoxia at 37 ℃.In group H/R+ H+ U, the cells were incubated for 1 h in the culture medium containing uprosertib 10 μmol/L (final concentration) and the other treatments were similar to those previously described in group H/R+ H. After treatment in each group, the cell viability was measured by MTT assay, cell apoptosis was measured using flow cytometry, superoxide dismutase (SOD) activity was measured using xanthine oxidase method), malondialdehyde (MDA) content was detected by thiobarbituric acid method, the expression of Akt, phosphorylated Akt (p-Akt), total Nrf2, nuclear Nrf2 and activated caspase-3 was detected by Western blot, and the expression of Nrf2 mRNA was detected by Real-time PCR. Results:Compared with group C, the cell viability and activity of SOD were significantly decreased, the apoptosis rate and content of MDA were increased, and the expression of p-Akt, nuclear Nrf2, total Nrf2, activated caspase-3 protein and Nrf2 mRNA was up-regulated in group H/R and group H/R+ H ( P<0.05). Compared with group H/R, the cell viability and activity of SOD were significantly increased, the apoptosis rate and content of MDA were decreased, the expression of p-Akt, nuclear Nrf2, total Nrf2 and Nrf2 mRNA was up-regulated and expression of activated caspase-3 protein was down-regulated in group H/R+ H ( P<0.05). Compared with group H/R+ H, the cell viability and activity of SOD was significantly decreased, the apoptosis rate and content of MDA were increased, the expression of p-Akt, nuclear Nrf2, total Nrf2 protein and Nrf2m RNA was down-regulated, and the expression of activated caspase-3 protein was up-regulated in group H/R+ H+ U ( P<0.05). Conclusion:The mechanism by which hydrogen-rich saline attenuates H/R injury to human renal tubular epithelial cells is related to improving activation of Akt/Nrf2 signaling pathway, decreasing oxidative stress response and inhibiting cell apoptosis.