The Protective Effects And Mechanisms Of Daphnetin On Acute Kidney Injury

Acute kidney injury(AKI)is a clinical syndrome with high mortality caused by multiple etiologies,and it is also one of the most common critical illnesses in clinical practice.Its medical expenses are very staggering.At present,effective prevention and treatment measures are still lacking.It is for the above reasons that AKI has received widespread attention,and it has become a public health problem that needs to be faced and urgently solved by the whole world.A large number of literature have reported that drug-induced kidney injury(DKI)is one of the important causes of AKI,especially that antibiotics and anti-tumor drugs have serious side effects on the kidney.In addition,cisplatin(CDDP)and gentamicin(GM)induced AKI accounted for a large proportion in clinical drug-induced kidney injury.Therefore,clinical researchers often use the side effects of these two drugs to establish animal models for kidney injury related research.AKI not only endangers human health,but also causes harm to the livestock industry.Bacterial infection in the digestive tract of livestock and poultry is one of the major problems in the breeding industry.The prevalence of diseases caused by intestinal bacterial infections such as salmonellosis and Escherichia coli infection has become increasingly complicated and increasingly difficult to control.And the emergence of new epidemic and pathogenic characteristics,not only will cause serious intestinal diseases,but also cause death and great economic losses.Aminoglycoside antibiotics have strong antibacterial activity and broad antibacterial spectrum,which have been widely used in livestock and poultry to treat Gram-negative bacterial infections,such as Escherichia coli and Salmonella infection.GM is widely used and very effective aminoglycoside antibiotic.It is usually used to treat gram-negative infection in livestock and poultry,but causes serious complication such as nephrotoxicity is important confining factors for the clinical application.With this background,the objective of this study was to investigate the protective effects,molecular mechanisms and prevention strategy of kidney injury by constructing CDDP and GM-induced AKI animal models,and,this study is particularly important from the perspective of human medicine and veterinary.Numerous studies have confirmed that Nrf2 signaling pathway is a regulator of mitochondrial dysfunction,oxidative stress and inflammation,and is a potential therapeutic target of AKI.The pharmacological activation of Nrf2 may provide a new treatment for the clinical prevention and treatment of AKI.In recent years,as a major Nrf2 activator,natural products counteract oxidative stress by modulating the Nrf2/ARE pathway,which has become an important research direction for the prevention and treatment of various diseases home and abroad.Daphnetin is a natural coumarin derivative,and it may be a clinically effective agent for the prevention and treatment of certain diseases as an activator of Nrf2.However,the protective effect of Daphnetin against CDDP/GM-induced nephrotoxicity and the molecular mechanisms have not previously been investigaged.The present study explored the protective effects and mechanism of Daphnetin against CDDP-induced nephrotoxicity in HK2 cells and AKI model.In HK2 cells,CDDP was selected to induce oxidative stress,apoptosis and inflammation in order to investigate the anti-oxidative,anti-apoptotic and anti-inflammatory properties and mechanisms of Daphnetin,as well as the interaction of antioxidant,anti-apoptotic and anti-inflammatory mechanisms.Furthermore,the effect of Daphnetin on the anticancer activity of CDDP was subsequently assessed in human ovarian carcinoma cell lines A2780,OVCAR-8 and SKOV3 and the NSCLC cell lines A549 and H1299,respectively,to determine the effect of ruthenium on CDDP anticancer activity.In this process,we applied the methods of CCK8 assays,Hoehcst/PI staining,flow cytometry(FCM),Western blot.In mice,CDDP was selected to induce AKI to investigate the protective effects of Daphnetin on CDDP-induced oxidative,apoptosis and inflammatory kidney injuries and the underlying mechanisms.Furthermore,the knockout mice were selected to explore the relationship between antioxidant,antiapoptotic and anti-inflammatory mechanisms of Daphnetin in AKI.It was also examined whether Daphnetin could affect CDDP-induced tumoricidal activity using a B16 melanoma tumor mouse model.In this process,we applied the methods of HE staining,TUNEL staining and Western blot.The present study also explored the protective effects and mechanism of Daphnetin against GM-induced nephrotoxicity in m TEC cells and AKI model.In m TEC cells,GM was selected to induce oxidative stress and apoptosis in order to investigate the anti-oxidative and anti-apoptotic properties and mechanisms of Daphnetin.In this process,we applied the methods of CCK8 assays,Hoehcst/PI staining,Western blot.In mice,GM was selected to induce AKI to investigate the protective effects of Daphnetin on GM-induced oxidative and apoptosis kidney injuries and the underlying mechanisms.In this process,we applied the methods of HE staining and Western blot.The major research results are as follows:1.In HK2 cells,Daphnetin had potential antioxidant and anti-apoptosis activities,which was reflected in the inhibitory effects on CDDP-induced cytotoxicity,apoptosis and ROS production.Furthermore,Daphnetin not only up-regulated SIRT1/SIRT6/Nrf2/ARE pathway to mediate antioxidant activity,but also suppressed CDDP-activated MAPK,NF-κB and p53 pathways to mediate anti-inflammatory and anti-apoptosis activities.2.In m TEC cells,Daphnetin had potential antioxidant and anti-apoptosis activities,which was reflected in the inhibitory effects on GM-induced cytotoxicity,apoptosis and ROS production.Furthermore,Daphnetin up-regulated SIRT3/Nrf2/ARE pathway to mediate antioxidant activity.3.In C57BL/6J mice,Daphnetin significantly ameliorated CDDP-induced weight loss,elevated renal index,blood BUN and CRE,swelling and histopathological injury in kidney tissue.Daphnetin also decreased the MPO and MDA production,SOD and GSH consumption in kidney tissue.Furthermore,Daphnetin up-regulated SIRT1/SIRT6/Nrf2/ARE pathway and inhibited CDDPactivated MAPK,NF-κB and p53 pathways in CDDP-induced AKI.4.In CDDP-induced AKI,the protection of Daphnetin was fairly weak in Nrf2-/-mice compared with WT mice,which was reflected in impaired inhibition of CDDP-induced weight loss,elevated renal index,blood BUN and CRE,swelling,histopathological injury and MAPK,NF-κB,p53 pathways activation in kidney tissue,indicating its antioxidant,anti-apoptosis,anti-inflammatory and protective effects in CDDP-induced AKI was dependent on Nrf2.5.Daphnetin did not attenuate the tumoricidal effect of CDDP in human ovarian carcinoma cell lines A2780,OVCAR-8 and SKOV3 cells and nonsmall cell lung cancer cell lines A549 and H1299,but rather enhanced it.And in B16 melanoma tumor model of C57BL/6J mice,Daphnetin not only improved CDDP-induced kidney damage,but also increased anticancer effects on CDDP in a syngeneic tumor model.6.In ICR mice,Daphnetin significantly ameliorated GM-induced elevated blood BUN and CRE,swelling,histopathological injury in kidney tissue.Daphnetin also decreased the MPO and MDA production,SOD and GSH consumption in kidney tissue.Furthermore,Daphnetin up-regulated SIRT3/Nrf2/ARE pathway and inhibited CDDP-activated p53 pathways in GM-induced AKI.In conclusion,Daphnetin confers protection against CDDP/GM-induced AKI in mice by regulating oxidative stress,inflammation,mitochondrial dysfunction and apoptosis,which might come from the up-regulation of SIRT1/SIRT3/SIRT6 mediated-Nrf2/ARE pathway activation.Therefore,Daphnetin is expected to be applied to the prevention and treatment of various drug side effects in humans and animals,thereby changing or improving the body’s disease resistance,protecting human health and reducing the economic loss of livestock and poultry farming.