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)signifi-cantly 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.