Epigallocatechin Gallate Attenuates Gentamicin-induced Nephrotoxicity By Suppressing Apoptosis And Ferroptosis

Background: Gentamicin(GEN)is an aminoglycoside antibiotic that is clinically effective against infections caused by Gram-negative bacteria.Although GEN plays an important role in the treatment of a variety of bacterial infections,it has severe nephrotoxicity and ototoxicity during use,and there is currently no approved effective treatment for nephrotoxicity.In recent years,many studies have found that there are various cell death modes in the process of kidney injury caused by GEN,such as cell necroptosis,cell apoptosis,etc.,but the dominant mode of death is still uncertain.A large number of studies on the mechanism of GEN toxicity have found that intracellular oxidation/antioxidant imbalance,calcium ion interference,calcium activated protease activation and so on are closely related to GEN toxicity.In this study,epigallocatechin gallate(EG),a potent antioxidant component in green tea,was used to attenuate the nephrotoxicity caused by GEN.The main mechanism of EG inhibiting kidney injury caused by GEN was also discussed to provide guarantee for the safe clinical use of GEN.Methods: In vivo experiments,SD rats were selected as model objects and were divided into four groups for modeling using GEN and EG: normal control group;EG group;GEN group;in the GEN+EG group,the serum creatinine and urea nitrogen levels of each group were first detected,and the kidney function of SD rats in each group was evaluated.Secondly,the kidney tissue sections of SD rats in each group were pathologically stained.At the same time,the kidney injury degree and ultrastructural changes of SD rats in each group were observed by projection electron microscopy to determine the specific cell death caused by kidney injury and the protective effect of EG on it.Finally,the levels of antioxidant glutathione(GSH),oxidative malondialdehyde(MDA),apoptosis and iron death marker protein in kidney tissues of SD rats in each group were detected.In vitro experiments,normal rat kidney cells 52E(NRK-52E)and human proximal renal tubular epithelial cells(HK-2)were modeled using GEN.The changes of cell vitality and the levels of oxidation/antioxidant substances in each group were detected.NRK-52E cells were stimulated with GEN to observe the changes of reactive oxygen species(ROS)levels over time.The changes of related indexes in each group were detected at protein level.At the same time,small interfering RNA was used to knock down the level of nuclear factor of antioxidant protein erythroid 2associated factor 2(Nrf2)to detect changes in the level of oxidative/antioxidant substances and the level of iron death marker protein of apoptosis.Molecular docking thermal shift technique was used to study the specific mechanism of EG inhibiting kidney injury caused by GEN.Finally,in vitro minimum inhibitory concentration(MIC)test and inhibitory ring test were used to verify whether EG had an effect on the antibacterial action of GEN.Result: The changes of ROS content in NRK-52E cells after GEN stimulation were detected by dihydroacetidium(DHE)staining in vitro.The results showed that the accumulation of ROS in the cells was induced at the early stage of GEN stimulation and remained at a high level from 1.5H to 24 H.In vitro and in vivo studies found that GEN reduced levels of the antioxidant GSH,and that EG administration reversed this effect.EG can increase the level of MDA caused by GEN.The levels of apoptosis marker Caspase3/C-caspase3,iron death marker glutathione peroxidase 4(GPX4)and SLC7A11 were determined,and it was found that EG could reverse apoptosis and iron death induced by GEN.At the same time,the levels of antioxidant protein Nrf2 and heme oxygenase-1(HO-1)were detected,and it was found that EG could activate Nrf2/HO-1 and reverse the decrease of antioxidant protein Nrf2/HO-1 caused by GEN.However,after knocking down Nrf2 level with small interfering RNA,EG lost its protective effect against kidney injury caused by GEN.Molecular docking and thermal shift results confirmed that EG could competitively bind with Nrf2 to Kelch-like ech associated protein 1(KEAP1)and activate Nrf2 protein.Finally,in vitro minimum inhibitory concentration(MIC)test and inhibitory ring test showed that EG had no effect on the antibacterial effect of GEN.Conclusion: In this study,we demonstrated that ROS levels in cells increased rapidly after GEN stimulation and quickly stabilized at abnormal and stable levels.The imbalance of oxidation and antioxidant levels plays a key role in kidney injury caused by GEN,and the use of antioxidant EG can protect kidney injury caused by GEN by inhibiting apoptosis and iron death.At the same time,it has been proved that the protective effect of EG is through the activation of Nrf2/HO-1 pathway.EG has no effect on the antibacterial effect of GEN.