MiR-106b-5p/ACSL4 Axis Mediates Ferroptosis In Pulmonary Ischemia-reperfusion Injury

Objective:Pulmonary ischemia-reperfusion(IR)injury is a common perioperative complication which occurs in patients in a variety of clinical conditions.Its occurrence can lead to acute lung injury,and increase early postoperative mortality.Therefore,it’s of great importance to study the pathologies of lung IR for further potential therapies.Ferroptosis is an iron-dependent cell death driven by lipid peroxidation,which was different from other modes of regulated cell death biochemically,genetically and morpholohically.The hallmarks of ferroptosis include iron overload,lipid peroxidation accumulation,loss of antioxidant defense,degradation of GPX4 protein,as well as smaller mitochondria with increased membrane density or ruptured membrane.Ferroptosis has been confirmed to be involved in the IR injury of numerous organs.However,the role of ferroptosis in pulmonary IR injury remains unclear.The oxidation of polyunsaturated fatty acids play a central role in ferroptosis,which requires the activation of lipid synthesis and metabolic pathway.Acyl-Co A synthetase long-chain family member 4(ACSL4)catalyzes arachidonic acid(AA)into AA-Co A for further production of oxidized phosphatidylethanolamine to trigger ferroptotic death.ACSL4 is a key regulator of ferroptosis sensitivity,and we speculated that it might mediate the ferroptotic damage induced by IR.It has been reported that mi R-106b-5p is involved in the metabolism of oxidized phospholipids and fatty acids.According to the results of bioinformatics prediction and in vivo experiments,it is suggested that mi R-106b-5p may regulate ACSL4 and modulate ferroptosis pathway during pulmonary IR.The aims of this study are: 1)to examine the hallmarks of ferroptosis and investigate the role of ferroptosis in lung IR injury;3)to explore the role of ACSL4 in ferroptosis induced by lung IR;4)to examine that mi R-106b-5p regulates ferroptosis in lung epithelial cells by targeting ACSL4.Part I: Ferroptosis was involved in lung ischemia-reperfusion injury in miceMethods:We assigned the mice randomly into: Control group,Sham group,IR 1 h group and IR 2 h group.Mice in the IR 1-and 2-hours groups received ischemia by left pulmonary hilum clamping for 1-h,followed by reperfusion for 1 or 2 h with clamp removed,respectively.Mice were sacrificed at the end of the procedure,with lungs perfused with phosphate buffer saline(PBS)and the lung lobes harvested for iron staining,iron content measurement,western blotting,malondialdehyde(MDA),glutathione(GSH),and transmission electron microscopy(TEM)evaluation.According to the results from first part,the mice were further randomly assigned into: Sham group,ferroptosis inhibitor liproxstatin-1(Lip-1)+Sham group,IR group(receiving ischemia for 1-h,followed by reperfusion for 2 h)and Lip-1+ IR group.In the Lip-1 treatment groups,10 mg/kg of Lip-1 was administered via i.p.1 hour before mechanical ventilation or lung ischemia.Mice were sacrificed at the end of the procedure,with lungs perfused with PBS and the lung lobes harvested for MDA,GSH and lactate dehydrogenase(LDH)activity measurements,expression levels of GPX4 and ACSL4,TEM evaluation,H&E staining,We/dry weight(W/D)ratio,oxygenation indexes and pro-inflammatory cytokines(IL-1β,IL-6,and TNF-α)measurements.A549 cells were also divided into: normoxia group,(2)Lip-1 + normoxia group,(3)hypoxia/regeneration(HR)group,and(4)Lip-1 + HR group.Cells after certain treatment were collected for corresponding detection: cell viability,LDH activity,lipid peroxidation measurement and GPX4 expression level.Results:Iron staining and tissue iron measurement both showed that iron content in mouse lung were increased during ischemia-reperfusion and were significantly elevated when reperfusion is up to 2 h.The expression levels of ferroptosis related proteins such as ACSL4 and glutathioneperioxidase 4(GPX4)were also dramatically different between Sham group and IR group.ACSL4 expression level were increased from 1 hour of reperfusion,while GPX4 expression was significantly decreased until 2 h of reperfusion.Pulmonary IR treatment not only markedly increased the MDA level but also decreased the level of total GSH.TEM images showed that shrunken mitochondria in the random field were more prominent at 2 hours of reperfusion.The MDA levels in lung tissue after IR declined after Lip-1 treatment.Reduced T-GSH content and elevated LDH activity after lung IR were also prevented after Lip-1 pretreatment.Western blotting showed that the IR-induced suppression of GPX4 expression in lung tissue was restored back after Lip-1 treatment.However,no changes were found in ACSL4 expression.Consistently,fewer small mitochondria were observed when ferroptosis was inhibited by with Lip-1.H&E staining indicated less severe lung injury in mice receiving Lip-1 treatment than those in the IR group.The deterioration of W/D ratio and gas exchange function after IR were also prevented by Lip-1 administration.IR enhanced the levels of pro-inflammatory cytokines in mouse lung tissue,which were also decreased by Lip-1 treatment.In vitro,cell viability and cell damage of A549 lung epithelial cells caused by HR was partially prevented by preincubation with Lip-1.HR-induced lipid ROS elevation was significantly decreased after Lip-1 treatment.In addition,Lip-1 also upregulated the concentrations of GPX4 expression levels in cells after HR.Part II: ACSL4 inhibition mitigates ferroptotic damage during lung ischemia-reperfusion injury in vivo and in vitroMethods:We assigned the mice randomly into: Sham group,ACSL4 inhibitor rosiglitazone(ROSI)+Sham group,IR group and ROSI+IR group.In the ROSI treatment groups,0.5 mg/kg of ROSI was administered via i.p.1 hour before mechanical ventilation or lung ischemia.Mice were sacrificed at the end of the procedure,with lungs perfused with PBS and the lung lobes harvested for the following measurements: MDA content,GSH level and LDH activity,GPX4 and ACSL4 expression levels,TEM evaluation,H&E staining,W/D ratio,oxygenation indexes and pro-inflammatory cytokines(IL-1β,IL-6,and TNF-α)levels.A549 cells were also divided into: si-NC+normoxia group,si-ACSL4+ normoxia group,si-NC+HR group,and si-ACSL4+HR group.Cells after certain treatment were collected for corresponding detection: ACSL4 protein level,cell viability,LDH activity,lipid peroxidation measurement and GPX4 expression level.Results:ROSI reduced the MDA production and LDH activity in IR-injured lung tissues.The variation of T-GSH after IR was partially prevented by ROSI treatment.ROSI also abrogated the declined levels of GPX4 in lung tissues after IR.There was no significantly difference between ROSI+IR group and IR group in the expression level of ACSL4.Consistently,fewer ferroptosis-like mitochondria were observed after using ROSI.Additionally,ROSI treatment also improved pulmonary function by reducing histological injury measured by H&E,W/D ratio,and increased Pa O2 after lung IR.A dramatical increase in the IL-1β,IL-6,and TNF-α levels in IR mice were efficiently diminished after ROSI treatment.In vitro,the ACSL4 expression was effectively decreased after si-ACSL4 transfection.Decrease in cell viability of A549 cells after HR was partially blocked by ACSL4 knockdown.Conversely,the absence of ACSL4 decreased LDH activity in cell culture supernatant.ACSL4 loss decreased lipid ROS in A549 cells,relative to those in Control cells after HR.GPX4 expression level was also significantly increased in the ACSL4-knockout cells.Part III: mi R-106b-5p regulates ferroptosis in lung epithelial cells by targeting ACSL4 under hypoxia-regenerationMethods:(1)The expression difference of mi R-106b-5p between Sham group and IR group was detected in vivo.(2)In vitro,to investigate the effect of mi R-106b-5p,the cells were divided into: NC+normoxia group,mi R-106b-5p mimic+normoxia group,NC+HR group,and mi R-106b-5p mimic+HR group.(3)For mi R-106b-5p inhibition experiments,cells were divided into: inhibitor NC+normoxia group,mi R-106b-5p inhibitor+ normoxia group,inhibitor NC+HR group,and mi R-106b-5p inhibitor+HR group.To verify whether ACSL4 mediated the protective effect of mi R-106b-5p on ferroptosis induced by HR,cells were divided into: NC+vector+HR group,mi R-106b-5p mimic+vector+HR group,(3)NC+pc ACSL4+HR group,and mi R-106b-5p mimic+pc ACSL4+HR group.Detection of cell viability,the lipid ROS level and the protein expression of ACSL4,as well as luciferase reporter experiment were performed.Results:The expression of mi R-106b-5p were reduced in the IR group,relative to Sham group.In vitro,mi R-106b-5p downregulated ACSL4 protein,while inhibition of mi R-106b-5p upregulated ACSL4.Consistently,luciferase reporter assay confirmed the regulatory mechanism of mi R-106b-5p on ACSL4.In addition,mi R-106b-5p increased cell viability and decreased lipid ROS level after HR and vice versa,and the protective effect of mi R-106b-5p was partially restored by overexpression of ACSL4.Conclusions:1.These results indicated that ferroptotic damage contributed to pulmonary IR injury in a murine model at 2 hours of reperfusion.2.Inhibition of ferroptosis plays a protective role in lung IR both in vivo and in vitro models.3.Inhibition of ACSL4 mitigated ferroptosis during lung IR.Consistently,knockdown of ACSL4 could decrease ferroptosis sensitivity in lung epithelial cells under HR condition.4.Overexpression of mi R-106b-5p protected lung epithelial cells ferroptosis through targeting ACSL4 under HR condition.