Effect And Mechanism Of Long-non-coding RNA Lcn2-204 On Myocardial Injury Iron Overload And Ferroptosis In Septic Mice

Objective To observe the change of iron overload and ferroptosis in myocardial injury induced by cecum ligation and perforation(CLP)method in septic mice,and to identify differentially expressed LncRNAs and mRNAs in CLP.To investigate the basic characteristics of LncRNA Lcn2-204 and to explore whether LncRNA Lcn2-204 can be involved in regulating iron metabolism in sepsis in mice and be involved in the process of ferroptosis occurrence.Methods1.Adult male C57BL/6 mice were randomized equally into sham-operated group,CLP group,CLP+ dimethyl sulfoxide(DMSO,lysis medium)group,CLP+ iron chelator dexrazoxane hydrochloride group(DXZ,mice were injected intraperitoneally with 50mg/kg DXZ 2 h before CLP),CLP+ ferroptosis-specific inhibitor ferrostatin-1 group(Fer-1,mice were injected intraperitoneally with 5 mg/kg Fer-1 2 h before CLP),acute iron overloaded group(Iron-dextran,mice were injected intraperitoneally with 1000mg/kg Iron-dextran).2.Adult male C57BL/6J mice were firstly randomized equally into sham and CLP group,which were established to identify differentially expressed long-stranded non-coding RNAs(LncRNAs)and messenger RNAs(mRNAs)by microarray.A coding-noncoding gene co-expression(CNC)network was constructed to reveal the association between the highest upregulated LncRNA-Lcn2-204 and mRNAs,and the potential mechanisms of differentially expressed mRNAs were analyzed by bioinformatics methods.A recombinant adeno-associated virus targeting myocardium with low expression of LncRNA Lcn2-204(sh RNA-Lcn2-204)and its empty vector(NCRNA)were constructed,and the virus was injected into the tail vein 21 days before CLP surgery and Iron-dextran injection.Mice were randomly divided into Sham + NCRNA group,Sham + sh RNALcn2-204 group,CLP + NCRNA group,CLP + sh RNA-Lcn2-204 group,Iron-dextran +NCRNA group,and Iron-dextran + sh RNA-Lcn2-204 group.Mice in the above grouping at 24 h after CLP surgery or 6 h after Iron-dextran injection.The changes of myocardial function in mice were detected by echocardiography.Myocardial injury in the mice was observed with H&E staining,and the changes of myocardial ultrastructure and mitochondria were observed using transmission electron on electron microscopy(TEM)Changes in reactive oxygen species(ROS)levels in myocardial tissue observed by dihydroethidium(DHE)fluorescence staining.TUNEL fluorescence staining to assessed the positive rate of myocardial cell death.The changes of serum creatine kinase isoenzymes(CK-MB),lactate dehydrogenase(LDH),lipocalin-2(Lcn2)levels and myocardial interleukin 6(IL-6),malondialdehyde(MDA),superoxide dismutase(SOD),lipin 1(LPIN1)levels were detected.Real-time fluorescence quantitative PCR verified microarray analysis results,measured LncRNA Lcn2-204 and Lcn2 mRNA expression levels.The protein expressions of myocardial transferrin receptor1(TFR1),ferroprotein 1(FPN1),ferritin,recombinant solute carrier family 7,member 11(SLC7A11,x CT),ferroptosis suppressor protein 1(FSP1),glutathione peroxidase(GPX4),Lcn2 protein expression levels were determined with Western blotting.Changes of the myocardial mitochondrial dihydroorotate-dehydrogenase(DHODH)protein expression levels in the sham,CLP,CLP+DXZ and CLP+Fer-1 groups were also examined.ResultsCompared with the Sham group,in the CLP group,CLP+DMSO group and Iron-dextran group,cardiac output(CO),left ventricular ejection fraction(LVEF),left ventricular fractional shortening(LVFS),and stroke volume(SV)were significantly decreased(P <0.01).Myocardial ROS levels were significantly increased(P < 0.001).Serum CK-MB,LDH,Lcn2 levels and myocardial IL-6 and MDA levels were significantly increased(P< 0.001-0.01),myocardial SOD and LPIN1 levels were significantly decreased(P < 0.001-0.01).Myocardial iron metabolism-related protein TFR1 protein expression was elevated(P < 0.001～0.01),FPN1(P < 0.001-0.01),ferritin(P < 0.01),and ferroptosisrelated protein expression x CT were decreased(P < 0.001-0.05),FSP1(P < 0.05),GPX4 protein expression were decreased(P < 0.001-0.01),Lcn2 protein expression was increased(P < 0.001-0.05).The rate of positive cells in TUNEL staining was increased in the CLP and CLP+DMSO groups(P < 0.001),and was not statistically significant in the Iron-dextran group.H&E staining observed irregular arrangement and partial degeneration of myocardial fibers with inflammatory cell infiltration,interstitial edema,blurred transverse lines,and erythrocyte exudation in the CLP and CLP+DMSO groups of mice,and Iron-dextran group had broken myocardial fibers and edema,but the overall morphology remained intact,with iron deposition in the interstitial and less inflammatory cell infiltration.Transmission electron microscopy observed that some mitochondrial cristae in CLP and CLP+DMSO groups were reduced,the outer membrane was ruptured,some mitochondria became smaller,and the membrane density was increased,while the Iron-dextran group had a more disordered arrangement of myocardial fibers,but some mitochondria had a dense structure,no obvious mitochondrial damage.Compared with the CLP group,in the CLP+DXZ and CLP+Fer-1 groups,CO,SV,LVEF%,and LVFS% increased(P < 0.001-0.01),neater myofilament arrangement and no infiltration of inflammatory cells,more complete myocardial fiber arrangement and less mitochondrial damage,decreased ROS levels(P < 0.001).TUNEL staining positive cell rate was significantly reduced(P < 0.001).Serum CK-MB,LDH,Lcn2 levels and myocardial IL-6 and MDA levels were significantly reduced(P < 0.001-0.01),Myocardial tissue TFR1,Lcn2 protein expression were reduced(P < 0.01～0.05),FPN1(P < 0.01),Ferritin(P < 0.05),x CT(P < 0.001-0.01),FSP1(P < 0.001-0.01),GPX4(P <0.01-0.05)protein expression were elevated,mitochondrial DHODH expression was elevated in the CLP group(P < 0.001).LncRNA Lcn2-204 expression was decreased(P< 0.01),Lcn2 mRNA levels were decreased(P < 0.001-0.01).SOD levels in the CLP+Fer-1 group significantly increased in the CLP+Fer-1 group(P < 0.05),and no significant changes in SOD levels were observed in the CLP+DXZ group.Compared with the CLP group,none of the CLP+DMSO were statistically different.2.Microarray analysis revealed 520 mRNAs and 552 LncRNAs differentially expressed in the Sham and CLP groups,with LncRNA-Lcn2-204 being the highest differentially expressed upregulated LncRNA with a CNC network consisting of 137 positive and 138 negative interactions.Bioinformatics analysis showed that disordered iron metabolism was associated with myocardial injury in sepsis,and bioinformatics analysis enriched several iron-related entries,and six genes(Scara5,Tfrc,Lcn2,Cp,Clic5,Ank1)were closely associated with iron metabolism.Compared with the CLP + NCRNA group,in the CLP + sh RNA-Lcn2-204 group,the left ventricular CO,SV,LVEF%,and LVFS% were increases of mice(P < 0.001-0.05).Myofilaments were more neatly arranged without infiltration of inflammatory cells.ROS levels were significantly decreased(P < 0.01).TUNEL staining positive cell rate was decreased(P < 0.001).Serum CK-MB,LDH,Lcn2 levels and myocardial IL-6 and MDA levels were decreased(P < 0.001-0.05),and myocardial SOD and LPIN1 levels were increased(P < 0.0-0.05).Myocardial tissue TFR1(P < 0.05)and Lcn2(P < 0.01)protein expression were decreased,and FPN1(P < 0.05),Ferritin(P < 0.01),x CT(P < 0.05),FSP1(P < 0.01),and GPX4(P < 0.05)protein expression were increased.LncRNA Lcn2-204 expression was decreased(P < 0.01),Lcn2 mRNA levels were reduced(P < 0.01).Compared with the Iron-dextran + NCRNA group,in the Iron-dextran + sh RNA-Lcn2-204 mice,CO,SV,LVEF%,and LVFS%(P < 0.001-0.05)had reduced.Myofilaments were more neatly arranged,without infiltration of inflammatory cells,and showed less iron deposition.ROS levels were significantly decreased(P < 0.01).TUNEL staining results were not statistically different,serum CK-MB,LDH,Lcn2 levels and myocardial IL-6 and MDA levels were significantly decreased(P < 0.001-0.01),Myocardial SOD levels were significantly increased(P < 0.01),and LPIN1 was not significantly changed.Myocardial TFR1(P < 0.01)and Lcn2 protein expression(P < 0.01)were decreased,FPN1(P < 0.05),Ferritin(P < 0.001),x CT(P < 0.001),FSP1(P < 0.01),and GPX4(P< 0.01)protein expression were increased.LncRNA Lcn2-204 expression was decreased(P < 0.01),Lcn2 mRNA levels were reduced(P < 0.01).Conclusion1.Iron overload and ferroptosis are present in CLP-induced myocardial injury in septic mice,and inhibition of iron overload and ferroptosis can reduce myocardial injury.2.Increased expression of myocardial LncRNA Lcn2-204 in septicemic mice.3.Knockdown of LncRNA Lcn2-204 protects mice from iron metabolism disorders and ferroptosis caused by myocardial injury in sepsis and may exert a protective effect by decreasing Lcn2.