| Backgrounds and Objectives:Sepsis associated Acute Kidney Injury(SAKI)has a high incidence,high mortality and poor prognosis,there is still a lack of specific treatment.Ferroptosis is a unique form of cell death,but whether there is ferroptosis in SAKI has not been reported.Melatonin has also been reported as an effective inhibitor of ferroptosis,but whether it regulates ferroptosis in SAKI and its specific regulatory mechanism was not clear.In this study,we constructed an animal model of SAKI to explore whether there is ferroptosis in SAKI and the role of ferroptosis in SAKI,and to study the role of melatonin in SAKI through the regulation of iron death and its specific regulatory mechanism.The results of this study will lay a foundation for improving the pathogenesis of SAKI,provide a new experimental basis for the treatment of SAKI with melatonin,and provide new therapeutic ideas and targets for the prevention and treatment of SAKI.MethodsThe animal model of SAKI was established by using of 6 to 10 week-old C57BL/6 mice with the cecal ligation and puncure(CLP)surgery.Western Blot technique was used to detect the protein expression levels of GPX4,ACSL4,HO-1 and Nrf2 in renal tissue;immunohistochemical technique was used to detect the expression levels of 4-HNE and FTH1 in renal tissue;related kits were used to detect the levels of tissue iron,GSH and NADPH+;HE pathological staining was used to observe the level of pathological injury in renal tissue;real-time quantitative fluorescence PCR was used to detect the mRNA levels of PTGS2,KIM-1,Lcn2 and IL-18.The specific morphology of renal tissue was observed by transmission electron microscope;and the levels of serum creatinine and urea nitrogen of mice were detected by ELISA technique.Ferroptosis inhibitor Frrostatin-1,HO-1 inhibitor ZnPP and Nrf2 inhibitor ML385,were used to explore whether ferroptosis exists in SAKI,whether melatonin regulates ferroptosis in SAKI and its specific regulatory mechanism.Results1.Ferroptosis is involved in the pathogenesis of SAKI.The related indexes of iron death and the expression of key enzymes of iron death in renal tissue after sepsis were detected,and the characteristic changes of iron death in renal cells after sepsis were directly observed by transmission electron microscope.2.Inhibition of ferroptosis can reduce SAKI.The use of ferroptosis inhibitor Fer-1 significantly reduced ferroptosis and reproved SAKI3.Melatonin improves SAKI by inhibiting Ferroptosis.3.1、Melatonin improved SAKI.The use of melatonin significantly reduced the renal pathological damage of SAKI.3.2、Melatonin suppresses ferroptosis in SAKI.The use of melatonin significantly suppresses ferroptosis in SAKI.3.3、Melatonin attenuates SAKI by inhibiting ferroptosis.Inhibitor of GPX4 and an agonist of ferroptosis RSL3 significantly inhibited the effect of melatonin on suppresses ferroptosis and reduces SAKI.4.MEL inhibiting ferroptosis to improves SAKI by up regulates Nrf2-HO-1.4.1、MEL regulates ferroptosis through upregulates HO-1.Administration of melatonin could partial recovery the protein expression of HO-1 in SAKI.The inhibitor of HO-1 ZnPP inhibited the expression of HO-1 protein in SAKI and inhibited the effects of melatonin on suppresses ferroptosis and improves SAKI.4.2、MEL up regulates HO-1 to suppresses ferroptosis by up regulates Nrf2.Administration of melatonin could increase the protein expression of Nrf2 in SAKI.Nrf2 inhibitor ML385 inhibited the expression of Nrf2 and HO-1 protein expression,and inhibited the effects of melatonin on suppresses ferroptosis and improves SAKI.ConclusionsThis study confirmed that ferroptosis is an important pathological mechanism to promote the development of SAKI,inhibition of ferroptosis can reduce SAKI;Melatonin can improve SAKI by inhibiting ferroptosis,melatonin inhibition of ferroptosis depends on up regulating Nrf2/HO-1. |
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