Leonurine Alleviates Ferroptosis In Cisplatin-induced Acute Kidney Injury By Activating The Nrf2 Signalling Pathway

Purpose:Cisplatin-induced acute renal injury（CI-AKI）is one of the most common complications in chemotherapy patients,severely limiting the clinical use of cisplatin（CDDP）,but its pathogenesis has not been fully elucidated.Recent studies have shown that ferroptosis is an important form of cell death in AKI.Nrf2,as one of the most important antioxidant nuclear tran SCription factor,has been shown to regulate ferroptosis by regulating oxidative stress and lipid peroxidation,but its role in ferroptosis in CI-AKI has not been clarified.Leonurine hydrochloride（LH）is a Nrf2activator found in motherwort and its effect of ferroptosis in CI-AKI is unclear.This paper aims to study the protective effect and mechanism of Nrf2 pathway and its activator LH on CI-AKI.Method:In this experiment,CI-AKI model of female C57BL/6 mice（6-8 weeks old）in vivo and RSL3 model of human renal kidney-2 cells（HK-2）in vitro were constructed to explore the role and mechanism of Nrf2 pathway and its activator LH in alleviating CI-AKI.1.Animal experiment:After feeding for one week and waiting for adapting to the environment,the mice were fasted for 18 hours and injected with CDDP（20 mg/kg）intraperitoneally to induce CI-AKI model.Meanwhile,the mice were administered LH（50,100 mg/kg）by gavage at-1,24,and 48 hours after the injection of cisplatin.Finally,we weighed and killed the mice at the 72nd hour after the induction of cisplatin,then collected the kidneys and blood of the mice for the subsequent experiments.2.Cell experiment:Stimulating HK-2 cells with RSL3（0.1,0.2,0.3,0.4,0.5μM）,Erastin（10,20,30,40,50μM）or CDDP（25μM）to induce cell model,and add LH（25,50,and 100 m M）,DFO（10μM）or Fer-1（5μM）pretreatment 1h before adding RSL3/Erastin/CDDP for treatment.Then dying or collecting cells 24h after adding RSL3/Erastin/CDDP and carry out the subsequent experiments.Result:1.Animal experiment（1）Compared with WT mice,Nrf2-KO mice are more susceptible to CI-AKI,which is manifested by aggravation of renal tissue damage,increase of Fe content,increase of MDA and decrease of GSH.（2）Compared with WT mice,ferroptosis related proteins FTH-1,FTL and TFR were significantly increased,GPX4 and x CT protein levels were significantly decreased,and the m RNA levels of GPX4 and x CT were also decreased in Nrf2-KO mice due to the inhibition of Nrf2 signal pathway.（3）LH reduced the indicators of renal injury,alleviated renal pathological changes,inhibited inflammation（F4/80）and reduced the markers of renal tubular injury in CI-AKI mice.（4）LH alleviated iron deposition and inhibited the expression of iron metabolism related proteins in CI-AKI mice.（5）LH inhibited MDA,increased GSH and SOD,activated antioxidant related proteins as well as alleviated mitochondrial damage in CI-AKI mice.（6）LH activated the expression of Nrf2,HO-1 and NQO1 proteins in CI-AKI mice.（7）Compared with WT mice,The therapeutic effect of LH was significantly inhibited in Nrf2-KO mice model.（8）Compared with WT mice,LH could not activate the protein levels of Nrf2 and its downstream HO-1 and NQO1 in Nrf2-KO mice.（9）Compared with WT mice,There were more iron deposition and protein expressions of iron metabolism in Nrf2-KO mice model.Meanwhile,LH could not inhibit these changes in Nrf2-KO mice.（10）Compared with WT mice,The kidney tissues produced more MDA,consumed more GSH,decreased more protein levels of GPX4 as well as x CT,and had more severe mitochondrial damage in Nrf2-KO mice model.Meanwhile,LH could not inhibit these changes in Nrf2-KO mice.2.Cell experiment（1）RSL3 or eristin can induce ferroptosis in HK-2 cells in a dose-dependent manner.LH can alleviate the death of HK-2 cells induced by RSL3 or Erastin and had the similar effect as ferroptosis inhibitors（DFO and Fer-1）.（2）LH alleviated the production of iron deposition induced by RSL3 and inhibitd the elevation of FTH-1,FTL and TFR in HK-2 cells.（3）LH alleviated the decrease of GSH level and the increase of MDA level in HK-2 cells induced by RSL3,as well as inhibited the lipid peroxidation.Western-blot analysis showed that LH activated Nrf2,HO-1,NQO1,GPX4 and x CT protein expressions in HK-2 cells.（4）After pretreatment with Nrf2-si RNA for 72 hours,the protective effect of LH on the survival rate of HK-2 cells stimulated by RSL3 and the anti lipid peroxidation were disappeared.（5）After pretreatment with Nrf2-si RNA for 72 hours,the activation of LH on the expression of Nrf2,HO-1,NQO1,GPX4 and XCT proteins in HK-2 cells stimulated by RSL3 was inhibited,and the inhibition of LH on the Fe²⁺level and the expressions of FTH-1,FTL and TFR proteins in HK-2 cells were also eliminated.Conclusion:1.The renal tissues in Nrf2-KO mice is more prone to ferroptosis and susceptible to CI-AKI.2.LH alleviated ferroptosis in CI-AKI by activating Nrf2 signal pathway.3.As an Nrf2 activator and ferroptosis inhibitor,LH has the clinical potential to treat CI-AKI.