Cytoprotective effects of hypoxia against oxidative stress in human lens epithelial cells by up-regulating Nrf2 expression

Abstract

Abstract AIM: To investigate the protective mechanism that hypoxia may attenuate human lens epithelial cells (LECs) injury caused by oxidative stress. METHODS: Human LECs were cultured in different atmospheric levels of O2 at different 4-Hydroxynonenal (4-HNE) concentrations and detected the viability by CCK-8. The apoptosis, reactive oxygen species (ROS), and senescent level were analyzed in both hypoxia and normoxia cultured LECs by Annexin V/PI staining, fluorescent probe (DCFH-DA), β-galactosidase staining, and BrdU incorporation assay respectively. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assays were performed to quantify the mRNA levels of genes-associated oxidative stress, antioxidant damage, and apoptosis. Protein blotting analyses were performed with antibodies specific to antioxidant and apoptosis-specific proteins. The expression level of endogenous Nrf2 was changed by transient transfection, and the cell viability was studied under different conditions after 4-HNE treatment. RESULTS: The proliferative activity of LECs was inhibited by 4-HNE treatment in both normoxic and hypoxic conditions, but hypoxia can attenuate the injury of 4-HNE to LECs by comparing with normoxia (***P < 0.001). We found that hypoxia-cultured LECs can inhibit the apoptosis, ROS, and senescent level by comparing with normoxia-cultured LECs. The qRT-PCR assays showed that hypoxia promote LECs to up-express HIF-1α and Nrf2 (***P < 0.001) and its downstream target genes NQO1, GSTP and p53 mRNA (**P < 0.01, ***P < 0.001), and down-express caspase3 gene (**P < 0.01, ***P < 0.001). The hypoxia increased the expression of HIF-1α (*P < 0.05, **P < 0.01) and Nrf2 (**P < 0.01, ***P < 0.001) and its downstream target proteins NQO1, GSTP and p53 (*P < 0.05, **P < 0.01, ***P < 0.001), and reduced caspase3 (**P < 0.01, ***P < 0.001) protein expression by western blotting. Alteration of Nrf2 expression can eliminate the protective effect of hypoxia on 4-HNE induced injury. CONCLUSION: The results suggested that the hypoxic condition was inhibited the injury effect of 4-HNE induced oxidative stress by up-expressing antioxidant gene Nrf2 and its downstream genes, including p53, and inhibit LECs apoptosis by reducing caspase3 expression in LECs, so activate the protective mechanism to LECs survival in oxidative stress.