The Mechanism Of Quercetin Alleviates Cisplatin-induced Acute Kidney Injury In Mice By Inhibiting Ferroptosis

ObjectiveAccording to the clinical stages,AKI can be classified as "uresis","clearance","edema","drowning"in traditional Chinese medicine.Kidney is the main target of toxic effects of various chemicals.Drug-induced renal injury is a common type of acute renal injury(AKI).Cisplatin is a cell non-specific inorganic platinum-based chemotherapy drug,which is widely used in the treatment of solid malignancies.The incidence of cisplatin-induced AKI is 20%-30%.It is an urgent clinical problem to improve the renal injury during its use.At present,it is confirmed that lipid peroxidation occurs in kidney tissue of AKI mouse model and be improved by drugs related to inhibiting ferroptosis.Therefore,ferroptosis may be a key target for the treatment of cisplatin induced AKI,but the mechanism of it needs further research.Quercetin,as a flavanol found in most vegetables and fruits,has strong antioxidant,antiinflammatory properties,lipid oxidation inhibition ability and protective effect on ferroptosis induced cell death.Therefore,this research used animal and cell experiments to validate the protective effect and mechanism of quercetin on cisplatin induced AKI in mouse and cell models.Combined with the ferroptosis inducer RSL3 induced ferroptosis cell model,further explore the pharmacological mechanism of quercetin on AKI,providing therapeutic basis and ideas for alleviating cisplatin induced AKI.Methods1.C57BL/6 male mice were used as the experimental study object in vivo.Cisplatin solution was injected intraperitoneally to simulate the AKI model induced by cisplatin.The experiment was divided into five groups:control group,AKI model group(cisplatin 20mg/kg),low,medium,and high dose quercetin group(quercetin 5mg/kg,25mg/kg,50mg/kg).The renal function of mice was evaluated by measuring the content of urea nitrogen(BUN)and creatinine(CRE)in the serum of mice,and the pathological changes of renal tissue were evaluated by HE staining,Sirius red staining and PSA staining.Through the detection of ROS level,oxidative stress level,iron load and ferroptosis related mRNA expression,the possible mechanism of AKI mice injury after cisplatin administration and the renal protective mechanism of quercetin through regulating ferroptosis were clarified.2.The rat renal tubular epithelial cell NRK-52E was used to construct an in vitro model of cisplatin-induced injury.The experiment was divided into four groups:control group,cisplatin administration model group,and different doses of quercetin administration group(2.5μM,5μM)intervention group.The role of ferroptosis in cell injury after cisplatin administration and the protective effect of quercetin on kidney and its possible mechanism were clarified by detecting cell viability,mitochondrial membrane potential,oxidative stress response,and ferroptosis related gene.3.NRK-52E cells were induced by ferroptosis inducer RSL3 to establish an ferroptosis cell model.The experiment was divided into four groups:control group,RSL3 induced ferroptosis model group,RSL3 induced plus different doses of quercetin(2.5μM,5μM)intervention group.The possible mechanism of quercetin’s protective effect on renal tissue was identified by detecting RSL3-induced cell viability,mitochondrial membrane potential,oxidative stress response,endogenous antioxidant level,lipid peroxidation activity,iron load,and ferroptosis related gene.Results1.The results of in vivo experiment showed that the body weight of mice in each group did not change significantly after administration(P>0.05),and there was no significant difference between the groups(P>0.05).Compared with the blank control group,the BUN and CRE of AKI group mice increased(P<0.05).Compared with the control group mice,the kidney tissue of AKI group mice showed diffuse denudation of renal tubular cells,dilation of tubular cells,formation of intratubular cast,widening of glomerular space and glomerular atrophy in HE staining results.After quercetin treatment,the degree of renal tissue lesion was significantly reduced.Sirius red staining results showed that the kidney tissue of AKI group mice showed fibrosis,and quercetin treatment reduced the occurrence of this situation.The results of PAS staining showed that the area of glomerulus in the kidney tissue of AKI group was significantly increased,and the number of purplish red particles in the mesangial area and renal tubular area increased.The area of glomerulus decreased after quercetin intervention and the preservation of renal tubular expansion,swelling,and cast formation was less than that of AKI group.The mRNA expression of ferroptosis related genes ACSL4,ALOX-5,HO-1,PTGS-2,slc7a11 and NRF2 was detected by q11T-PCR.The results showed that the mRNA levels of ACSL4,ALOX-5,HO-1,PTGS-2,SLC7A11 and NRF2 in the kidney of AKI group mice were higher than those of the control group(P<0.05).The mRNA levels of ACSL4,ALOX-5,HO-1,PTGS-2,SLC7A11 and NRF2 in the kidney of mice in quercetin group were significantly lower than those in AKI group(P<0.05).Compared with the control group,the results of immunofluorescence staining showed that the renal fluorescence intensity of AKI mice decreased,and quercetin treatment could significantly reduce GPX4 depletion.ALOX5 fluorescein staining showed that the fluorescence intensity of renal tubules in AKI group increased,and quercetin treatment could significantly reduce the expression of ALOX5.Prussian blue staining showed that there was no iron precipitation in the kidneys of each group.The results of DHE staining,4-HNE staining and CellROX showed that the content of ROS in kidney tissue of AKI group mice was greatly affected,while quercetin reduced the production of ROS after administration.Compared with the control group,the level of 4-HNE in kidney tissue of AKI group mice was significantly increased,while quercetin significantly decreased the level of 4-HNE in kidney tissue.2.NRK-52E as an in vitro study object,the dose of cisplatin can be higher than 12.5μg/ml according to the MTT test results.The cell viability was less than 50%at 25μg/ml.The results of MTT test and live/dead cell staining showed that quercetin could reduce the cell damage caused by cisplatin after administration(P<0.05).Under the condition of cisplatin administration,the number of living cells and the morphology of cells were significantly improved after the ferroptosis inhibitors FPL62064 and Fer-1 were administered.The results of immunofluorescence staining with GPX4 and ALOX5 antibodies showed that the ALOX5 fluorescence intensity of cells in cisplatin group increased significantly,and the ALOX5 fluorescence intensity decreased after quercetin administration(P<0.05).The GPX4 fluorescence intensity of cells in cisplatin group decreased significantly,and the fluorescence intensity increased after quercetin administration.The results of JC-1 fluorescent probe kit showed that,compared with the control group,the number of JC-1 aggregates of NRK-52E cells in cisplatin group decreased,and the number of JC-1 aggregates increased after quercetin administration.The content of Fe2+,ROS and 4-HNE in the cisplatin group was significantly higher than that in the control group.Quercetin and ferroptosis inhibitors Ferrostatin-1 and FPL62064 could reduce the content of these indicators.3.From the MTT results of the in vitro experiment,the RSL3 concentration is greater than 0.315 μg/ml significantly decreased the cell activity(P<0.05).After administration of quercetin,it can significantly inhibit the effect of RSL3 on the activity of NRK-52E cells and protect cells(P<0.05).The staining results of living/dead cells showed that RSL3 had obvious killing effect on cells(P<0.05).Immunofluorescence staining with GPX4 and ALOX5 showed that the ALOX5 fluorescence intensity of cells significantly increased after RSL3 administration,and decreased after quercetin administration(P<0.05).After administration of RSL3,the fluorescence intensity of GPX4 decreased significantly,and the fluorescence intensity increased after administration of quercetin.The results of JC-1 fluorescent probe kit showed that compared with the control group,the number of JC-1 aggregates in NRK-52E cells decreased after RSL3 modeling,and the number of JC-1 aggregates increased after quercetin administration.The content of ROS and 4-HNE in RSL3 group was significantly higher than that in the control group,while quercetin and ferroptosis inhibitors Ferrostatin-1 and FPL62064 could reduce the content of these indicators.Conclusion1.It can be seen the ferroptosis in the AKI model group with cisplatin administration,and quercetin can reduce the degree of renal tissue damage and improve renal function.2.The AKI model of NRK-52E cells treated with cisplatin has ferroptosis.Quercetin,FPL62064 and Fer-1 can reduce the cell damage induced by cisplatin.3.Quercetin may reduce the cisplatin induced-AKI caused by regulating the expression of ferroptosis related genes ACSL4 and ALOX5 to inhibit ferroptosis through reducing iron content,lipid peroxidation and increasing endogenous antioxidant activity.