7,8-dihydroxyflavone protects human renal proximal tubular cells from hypoxia injury via inhibiting endoplasmic reticulum stress

Abstract

Objective To observe the effects of 7,8-dihydroxyflavone (7,8-DHF) on hypoxia induced endoplasmic reticulum stress (ERS) in human proximal tubular epithelial cells (HK-2). Methods The mRNA level of ERS associated biomarkers was evaluated by RT-PCR assay in cell hypoxia damaged model. And HK-2 cells were pretreated with different concentrations of 7,8-DHF through CCK-8 assay; meanwhile CCAAT/enhancer-binding protein homologous protein (CHOP), Cyr61, Akt and p-Akt were determined by western blotting assay. Moreover, HK-2 cells were pretreated by LY294002, a kind of PI3K/Akt inhibitor, to inhibit the PI3K/Akt signaling, and its effects on protein level induced by 7,8-DHF was detected. HK-2 cells was then over-expressed Cyr61 and exposed to hypoxia Apoptosis rate and CHOP expression were determined. Results Compared to hypoxia group (P<0.01), Hypoxia for 12h was effective in inducing ERS (P<0.01), while pretreatment with 7,8-DHF (100 μmol/L) increased cell proliferation significantly. The protein expressions of Cyr61 and p-Akt in H+7,8-DHF group were higher, but the level of CHOP was decreased (P<0.05). With LY294002 pretreated, the expression of Cyr61, p-Akt was down-regulated (all P<0.05) while the expression of CHOP was up-regulated (P<0.05). In comparison to empty plasmid group, when cells were transfected with over-expression of Cyr61 plasmid and exposed to hypoxia, the number of apoptotic tubular cells was decreased (P<0.01). And over-expression of Cyr61 significantly reduced CHOP expression compared with the empty plasmid group (P<0.01). Conclusion Pretreatment of 7,8-DHF could protect cells from hypoxia injury and inhibit ERS, which may involve the Akt-Cyr61 signaling pathway. Key words: Renal tubular necrosis, acute; Hypoxia, cell; 7,8-Dihydroxyflavone; Cysteine-rich protein 61; Endoplasmic reticulum stress; Protein kinases