Effects of osthole on renal tubular epithelial cell injury and Nrf2/HO-1 signal pathway induced by high glucose, hypoxia and reoxygenation

Objective To explore the effects of osthole on renal tubular epithelial cell damage and Nrf2/HO-1 signal pathway induced by high glucose, hypoxia and reoxygenation. Methods Human renal tubular epithelial cells HK-2 were cultured in high-glucose medium and subjected to hypoxiareoxygenation treatment to establish a cellular model. CCK-8 experiment was utilized for detecting the effects of 0, 10, 20, 40, 80, 160 μg/ml osthole on its growth and screen the optimal concentration. HK-2 cells were divided into control, model, osthole(80 μg/ml), ML385(Nrf2 inhibitor, 20 nmol/ml) and osthole(80 μg/ml) +ML385(20 nmol/ml) groups. Except for control group, the other groups were induced for establishing cellular models. After high glucose, hypoxia and reoxygenation, CCK-8 experiment was utilized for detecting cellular viability in each group. Kits were employed for measuring the levels of reactive oxygen species(reactive oxygen species, ROS), glutathione peroxidase(GSH-Px), malondialdehyde(MDA), lactate dehydrogenase(LDH), tumor necrosis factor-α(TNF-α), interleukin-6(IL-6) and interleukin-1β(IL-1β);Immunofluorescence for detecting the expression of Nrf2/HO-1 pathway protein;Western blot for detecting the expression of Nrf2/HO-1 pathway protein. Results Compared with control group, HK-2 cellular viability(100±0 vs 43.47±6.14), cellular GSH-Px level(9.53±1.64 vs 3.25±0.32) and cellular Nrf2 protein(1.78±0.30 vs 0.89±0.12) were markedly down-regulated in model group(all P<0.05);the levels of ROS(1.13±0.17 vs 6.52±1.03) and TNF-α spiked markedly (all P<0.05). Compared with model and osthole + ML385 groups,HK-2 cellular viability (43.47±6.14,44.83±7.06 vs 87.76±9.42),cellular GSH-Px level(3.25±0.32,3.27±0.38 vs 9.07±1.78) and cellular Nrf2 protein(0.89±0.12, 0.90±0.18 vs 1.74±0.26) rose in osthole group (P<0.05);ROS(6.52±1.03, 6.47±0.96 vs 1.59±0.21) and TNF-α declined(both P<0.05); HK-2 cellular viability(43.47±6.14, 44.83±7.06 vs 24.80±3.79), cellular GSH-Px level(3.25±0.32, 3.27±0.38 vs 0.88±0.15) and cellular Nrf2 protein(0.89±0.12, 0.90±0.18 vs 0.30±0.05) decreased in ML385 group(all P<0.05);ROS(6.52±1.03, 6.47±0.96 vs 9.76±2.02) and TNF-α increased(all P<0.05). Conclusion Osthole can up-regulate the expression of Nrf2/HO-1 signaling pathway protein, suppress ROS and inflammatory factors production induced by high glucose, hypoxia and reoxygenation, thereby relieving oxidative stress and inflammation and lessening the damage of renal tubular epithelial cells.