The Role Of MiR-124/Ferroportin1 Signaling Pathway In Neuronal Cell Death In Intracerebral Hemorrhage Model Mice

Background:Intracerebral hemorrhage(ICH)is bleeding within in the brain tissues without obvious external cause,and the incidence of ICH increases with age.The accumulation of iron is shown after ICH,and the incidence of iron accumulation increases with age.Excessive accumulation of iron results in ferroptosis and neuronal cell death.However,the mechanisms of ferroptosis in ICH have not been well clarified.Ferroportin1(Fpn)is involved in maintaining iron homeostasis by transferring excessive intracellular iron out of cells.Therefore,dysregulation of Fpn plays a critical role in regulating iron metabolism in ICH.However,the exact role,the potential mechanisms and its signaling pathway in ICH neuronal cell death and neurological deficits remain unclear and need further exploration.Objective:To investigate the role and mechanism of iron transporter Fpn and its upstream molecule miR-124 in elderly ICH model mice,and whether targeting this pathway can reduce neuronal cell death and improve ICH.Methods:The ICH model mice were constructed by mouse autologous blood injection,detecting the changes of Fpn protein expression by Western blotting(WB)and Fpn m RNA expression by Real-time quantitative polymerase chain reaction(q PCR).The caudate nucleus of Fpn-floxed mice was injected with cre virus to knock down Fpn,and the adeno-associated virus(AAV)of Fpn was injected into the caudate nucleus of mice to overexpress Fpn.Neurological function of ICH mice was assessed by Neurological Deficit Scores(NDS),forelimb placing test,corner turn test and rotarod test.Hematoma was observed by hematoxylin-eosin staining(HE),apoptosis was observed by TUNEL staining and iron accumalation in ICH was observed by Prussian blue reaction staining(Perls staining).The changes of apoptosis and ferroptosis molecules were detected by WB and q PCR.Based on the prediction of website tools,the potential micro RNAs(miRNAs)which possibly regulated Fpn were detected.Combining with luciferase reporter assay,miRNA was detected to regulate Fpn.The clinical symptoms of ICH patients were collected,and the correlation analysis was carried out with the corresponding serum miR-124 levels.The ICH mice were injected with miR-124 antagomir,and the Fpn knockout mice were injected with miR-124 antagomir,then functional changes of nervous system were assessed,apoptosis and ferroptosis were detected.Results:Fpn was detected significantly upregulated in brain tissues of aged ICH mice and elderly ICH patients.Loss of Fpn significantly worsened hematoma volume,apoptosis,ferrroptosis and neurological dysfunction.ICH mice overexpressing Fpn showed significant improvement in these symptoms.The miR-124 was determined to regulate the expression of Fpn after ICH.The level of miR-124 was decreased in the brain tissues and serum of elderly ICH patients.The neurological function of elderly ICH patients was correlated with their serum miR-124 levels.The use of miR-124antagomir enhanced the expression of Fpn and attenuated hemorrhage,apoptosis,ferroptosis and neurological dysfunction.However,the above improvements were not observed in Fpn knockout mice that miR-124 antagomir was administered.Conclusion:The miR-124/Fpn signaling pathway plays a key role in aged ICH model mice.Fpn was significantly upregulated in brain tissues of aged ICH mice and elderly ICH patients.miR-124 could downregulate the expression of Fpn.The level of miR-124 was decreased in the brain tissues and serum of ICH patients,and the neurological dysfunction of ICH patients was correlated with their serum miR-124level.The upregulation of Fpn or the inhibition of miR-124 alleviated hematoma,apoptosis and ferroptosis,and neurological dysfunction in aged ICH model mice.