Mechanism Of Farrerol Attenuates Cisplatin-induced Acute Kidney Injury By Regulating Nrf2 Signaling Pathway

Purpose:CDDP,as the prefered chemotherapy drug,can treat bladder cancer,testicular cancer and many other tumours.And CDDP also has many side effects such as bone marrow suppression,nephrotoxicity and ototoxicity.After being absorbed,90%of CDDP is excreted by the kidneys,so it is easy to accumulate in the kidney’s proximal tubule cells and cause nephrotoxicity.Although CDDP is usually administered in small and divided doses,combined with comprehensive treatment methods such as hydration,mannitol and antioxidants,about one-third of patients receiving CDDP treatment will have renal dysfunction.Far is a natural molecular product with antioxidant effects,but its protective effects and potential mechanisms on CDDP-induced AKI are unknown.The aim of this study was to investigate the protective effect of Far on CDDP-induced nephrotoxicity and its mechanism.Methods:In this experiment,the CDDP-associated C57BL/6 mice model and MTEC cells’model were used to detect the protective role and mechasim of Far on CDDP-treated AKI.1.Animal experiment:Starvation for 12 hours,mice were then treated with Far（20 mg/kg）at-1h,24h,and 48h after intraperitoneal injection of CDDP（20mg/kg）,respectively.Subsequently,weighing and excuting the mice,and collectting blood and renal at 72 hours for renal function assessment.（1）Investigated the impacts of Far on CDDP-stimulated mice,including body’s and kidneys’weight,renal pathological changes,KIM1,NGAL,SCr and BUN.（2）Detectd the regulation of Far on MDA’s,MPO’s,SOD’s and GSH’s production in the CDDP-treated kidney tissues.（3）Explored Far’s influence on the protein’s levels of Keap1,NOX4,Nrf2,HO-1 and NQO1 in CDDP-induced mice kidney tissues by Western Blot.（4）Analyzed the influence of Far on the relationship between MAPK,NF-κB,NLRP3,P53,Bcl2,Caspase-3 and Bax in mice via Western BBlot.（5）Explored the impacts of Far on CDDP-associated changes in body’s and kidneys’weight,BUN,SCr,KIM1,and NGAL in Nrf2^-/-mice.（6）Detected MDA’s,MPO’s,SOD’s and GSH’s levels in the kidney tissues of Nrf2^-/-mice dealt with CDDP.（7）Studied the Far’s influences on the protein’s levels of Nrf2/AREs signaling pathway in the Nrf2^-/-mice’renal tissues via Western Blot.2.Cell experiment:Incubated MTEC cells with different doses of Far（5,10 and 20μM）,then treated them with CDDP（20μM）for 18 hours after 1 hour.And the cells were collected for the following studies.（1）qRT-PCR was utilized to explore the influence of different doses of Far on MTEC cells’Nrf2 m RNA level.（2）CCK-8 was applied to measure the cytoprotection of different concentrations of Far on CDDP-induced MTEC cytotoxicity（3）After pretreatment with Nrf2 inhibitor for 1 hour,CCK-8 was applied to detect the Far’s influence on CDDP-related MTEC cells.（4）ROS assay was utilized to monitored the impact of Far on CDDP-induced ROS levels in MTEC cells.（5）After pretreatment with Nrf2 inhibitor for 1 hour,the effect of Far on CDDP-induced ROS levels in MTEC cells was detected.（6）Utilizing Western Blot to consider the Far’s function on the levels of Keap1,NOX4,Nrf2,HO-1 and NQO1 protein in MTEC cells.Results:1.Animal experiment:（1）Far can improve the the loss of weight,the increase of kidney index,BUN and SCr,the enhancement of KIM1 and NGAL,and the kidney pathological damage induced by CDDP.（2）Far can effectively reduc MPO’s and MDA’s production and GSH’s and SOD’s consumption in the CDDP-treated kidney tissues.（3）Far can upregulate Nrf2’s,HO-1’s and NQO1’s protein and alleviate Keap1’s and NOX4’s level,and decline NOX4’s and Keap1’s protein expression in the kidney tissues stimulated by CDDP.（4）Far can effectively decrease MAPK’s phosphorylation,p-NF-κB’s and NLRP3’s activation,and p-p53’s,Bax’s and Caspase-3’s expression in the CDDP-related AKI mice model.（5）Nrf2^-/-mice have greater weight loss and increased KIM1,NGAL,SCr and BUN than C57BL/6 wild-type mice.Nevertheless,the above phenomenon was not significantly improved after Far treatment.（6）In the Nrf2^-/-mice model,the generation of MDA and MPO and the loss of SOD and GSH are more significant than those of C57BL/6 wild-type mice,while Far treatment cannot ameliorate the generation of MPO and MDA and the depletion of GSH and SOD.（7）In the Nrf2^-/-mice model,Far cannot upregulate the Nrf2/AREs signaling pathway protein,and alleviate NOX4’s level.2.Cell experiment:（1）Far upregulated the m RNA of Nrf2 in a dose-dependent way in MTEC cells.（2）CCK-8 results indicated that Far was able to attenuate CDDP-stimulated MTEC cytotoxicity in a concentration-dependent manner.Furthermore,the cytoprotection of Far on CDDP-related MTEC cytotoxicity was effectively reversed after Nrf2 inhibitor pretreatment.（3）MTEC cells produced high levels of ROS after CDDP treatment,but Far markedly alleviated ROS’s production in a dose-dependent way.Additionally,after pre-incubation with Nrf2 inhibitor for 1h,Far effectively reversed the elimination of ROS in MTEC cells.（4）Far regulated the Nrf/AREs oxidation pathway in a dose-dependent way in MTEC cells.Conclusion:1.Far inhibited CDDP-induced ROS production in MTEC cells,thereby improving CDDP-induced cytotoxicity.2.Far attenuated the oxidative stress induced by CDDP by regulating the Nrf2/AREs signaling pathway.3.Far downregulated the expression of p-NF-κB/NLRP3 and p-p53/Bax/Caspase3by phosphorylating the MAPK signalling pathway,through restraining CDDP-related inflammation and apoptosis.