Preliminary Study Of Fer-1 Alleviating Hepatic Ischemic Reperfusion Injury In Mice Through DHODH-Mediated Non-Classical Ferroptosis Pathway

Objective: To investigate the role of ferroptosis in hepatic ischemia reperfusion injury(HIRI)by constructing a mice model of HIRI,and to preliminarily study the regulatory mechanism of ferroptosis in HIRI,as well as the mechanism of Fer-1 inhibition of ferroptosis.Methods: Twenty-four 8-week-old male C57BL/6 mice,SPF grade,weighting 23.5-25 g,were randomly divided into three groups: Sham group(n=8),I/R group(n=8),Fer-1+I/R group(n=8).200 ul of normal saline to Sham group,I/R group and Fer-1(5mg/kg)to Fer-1+I/R group,was injected intraperitoneally 1 hour before surgery dissect and supply the liver pedicles of the left liver and middle liver.I/R group and Fer-1+I/R group were blocked blood flow for 30 minutes,meanwhile Sham group didn’t,and samples were taken at 6 hours after recanalization.Serum alanine aminotransferase(ALT)and aspartate aminotransferase(AST)levels were measured by a microplate reader to assess the degree of liver function impairment.Hematoxylin/eosin(HE)stained sections and electron microscopic sections were made from liver tissues.Quantitative real-time reverse transcription polymerase chain reaction(RT-qPCR)was used to detect the expression levels of ferroptosis-specific genes long-chain acylCo A synthetase 4(ACSL4)and Prostaglandin endoperoxide synthase 2(ptgs2)mRNA in liver tissue.The levels of Glutathione(GSH)and malondialdehyde(MDA)were measured to assess the antioxidant and lipid peroxidation metabolism in liver tissue.The expression levels of ferroptosis-related proteins glutathione peroxidase 4(GPX4)and dihydroorotate dehydrogenase(DHODH)were analyzed by Westernblotting.Results: Compared with Sham group,the levels of AST,ALT,ACSL4 mRNA,ptgs2 mRNA,MDA were significantly increased(p<0.05)in I/R group,the levels of GSH was significantly reduced(p<0.05),and there was no significant statistical difference in the expression of GPX4 and DHODH(p>0.05)Compared with I/R group,the levels of AST,ALT,ACSL4 mRNA,ptgs2 mRNA,MDA,GPX4 were significantly reduced(p<0.05)after Fer-1 pretreatment,the expression of DHODH was significantly reduced(p<0.05),the levels of GSH was no significant statistical difference(p>0.05).HE staining showed that the structural destruction and inflammatory response of liver tissue in the Fer-1pretreatment group were alleviated compared with the I/R group,indicating that Fer-1 pretreatment could significantly reduce the abnormal liver function induced by liver ischemia-reperfusion in mice.Electron microscopy showed that in the I/R group,Mitochondrial shrinkage rounded and smaller,reduction or vanishing of mitochondrial cristae significantly,and rupture of some mitochondrial membranes,which are ferroptosis characteristics.Conclusion: Pretreatment of Fer-1 can alleviates HIRI in mice by upregulating the DHODH-mediated non-classical ferroptosis defense rather than GSH/GPX4 axis,and DHODH may be a new target to treat the ischemia-reperfusion injury.