Effects of vaccarin on amikacin-induced damage to renal tubular epithelial cells / 安徽医科大学学报

ABSTRACT

Objective @#To establish an in vitro renal injury model of amikacin (AKN) and investigate the protective effect and mechanism of vaccarin (VA) in the AKN-induced in vitro renal injury model .@*Methods @#Human renal tubular epithelial cells (HK-2) were cultured in vitro and incubated with different drugs of AKN or/and VA to de- termine the optimal drug concentration based on cell viability tested by MTT. The changes in intracellular oxidative stress were assessed using the dihydroethidium ( DHE) probe and malondialdehyde ( MDA) /glutathione ( GSH) assay kits at different time points . Total RNA was extracted , and RT-qPCR was performed to detect the changes in the gene expression of kidney injury molecule-1 ( KIM-1) and neutropil gelatinase-associated lipocalin ( NGAL) . Western blot analysis was performed to detect the levels of ferroptosis-related markers solute carrier family 7 member 11 (SLC7A11) and glutathione peroxidase 4 (GPX4) in HK-2 cell lysis .@*Results @#High concentrations of AKN significantly decreased the viability of HK-2 cells in vitro , with a half maximal inhibitory concentration (IC50) of (5 . 74 ±0. 47) mmol/L. VA at concentrations of 25 - 100 μmol/L increased the viability of AKN-stimulated HK- 2 cells (P < 0. 05) . After treatment with AKN (4 mmol/L) , the mRNA expression levels of KIM-1 and NGAL were significantly higher than those of the negative control (NC) group ( P < 0. 001) . VA (50 μmol/L) significantly reduced the mRNA expression levels of KIM-1 (P < 0. 01) and NGAL (P < 0. 05) . The intensity of DHE staining increased after 3 hours of AKN treatment , but the difference was not statistically significant. However , the intensity of DHE staining was significantly higher in the 6 - 24 hours group compared to the 0 - hour group (P < 0. 01) . Furthermore , MDA levels significantly increased , while GSH levels significantly decreased after 6 - 24 hours of AKN treatment , with statistically significant differences (P < 0. 05) . After 6 - 24 hours of AKN stimula- tion , the ferroptosis-related proteins SLC7A11 and GPX4 both significantly decreased (P < 0. 001) . Co-incubation with VA for 24 hours effectively reversed the changes in DHE staining , MDA and GSH levels , as well as the chan- ges of SLC7A11 and GPX4 protein levels (P < 0. 001) .@*Conclusion @#In this study , an in vitro renal injury model was established by stimulating HK-2 cells with high concentrations of AKN , and it was found that VA might allevi- ate the damage to renal tubular cells caused by AKN via inhibiting oxidative stress related ferroptosis .