Effect Of 6-OHDA On Iron Metabolism Of BV2 Microglia

Parkinson's disease(PD)is a common age-related neurodegenerative disorder characterized by a progressive loss of dopaminergic neurons in the substantia nigra pars compacta(SNpc).The main clinical features of PD include bradykinesia,rigidity,rest tremor and postural instability.Although heredity,environment,oxidative stress and other factors are all responsible for the process of PD,the exact etiology and pathogenesis have not been completely revealed yet.Iron,as an essential nutrient for human beings,is widely involved in physiological and biochemical functions.Previous studies had confirmed that the aggregation of iron which might be due to the abnormal expression of divalent metal transporter 1(DMT1)and ferroportin1(FPN1),played a central role in the pathogenesis of PD.Iron-regulatory proteins(IRPs)can translationally regulate iron transport proteins by binding to iron-responsive elements(IREs)present in the untranslated regions(UTR)of their respective m RNAs.When IRPs bind to the IRE of the 5'-UTR of FPN1,FPN1 m RNA can not be translated;IRPs bind to IREs located in the 3'-UTR of DMT1 m RNA,thereby stabilizing its expression.Hepcidin is an iron-regulated hormone found in recent years.It can inhibit the transcription and translation of FPN1 gene,promote the internalization and degradation of FPN1,reduce FPN1,and play an important role in maintaining the balance of iron homeostasis.Recently,the study on iron deposition are primarily focused on dopaminergic neurons,actually glia cells also play a key role in iron homeostasis.In the midbrain SNpc of PD patients,there exists numerous activated microglia which have a large amount of iron deposition.However,the relationship between microglial activation and iron accumulation has not been elucidated.Previous studies in our laboratory had shown that 6-hydroxydopamine(6-OHDA)activated iron regulatory proteins 1(IRP1)which induced the increased expression of DMT1 and the decreased expression of FPN1,further leading to the accumulation of iron and the toxic injury of dopaminergic neurons.Moreover,the expression of DMT1 and FPN1 were found to be both increased and the ability of iron transport was enhanced in astrocytes treated with 6-OHDA,which could prevent the iron deposition on this occasion.Then,what role will microglia play in the iron metabolism and selective iron deposition in the SNpc of PD? In this study,we aimed to explore the effect of BV2 microglia cell lines treated with 6-OHDA on the iron metabolism using enzyme-linked immunosorbent assay(ELISA),real-time PCR,flow cytometry,western blots and other methods.The results were as follows:1.The protein level of DMT1 was significantly increased in BV2 microglia cell lines treated with 10 ?mol/L 6-OHDA for 24 h compared with the control(P< 0.01,n=6).There was no obvious change in the protein level of FPN1 compared with the control.2.The protein level of IRP1 was remarkably increased in BV2 microglia cell lines treated with 10 ?mol/L 6-OHDA for 24 h compared with the control(P< 0.01,n=6).3.Increased ferrous iron influx was observed in BV2 microglia cell lines treated with10 ?mol/L 6-OHDA compared with the control(P< 0.01,n=6).The ability of ferrous iron efflux had no obvious change.4.The release of hepcidin was significantly reduced in BV2 microglia cell lines treated with 10 ?mol/L 6-OHDA for 24 h compared with the control(P< 0.01,n=6).5.The m RNA levels of tumor necrosis factor-?(TNF-?)and interleukin-1?(IL-1?)was dramatically increased in BV2 microglia cell lines treated with 10 ?mol/L 6-OHDA for24 h compared with the control(P< 0.01,n=6).6.The protein level of DMT1 was decreased and the protein level of FPN1 was increased in BV2 microglia cell lines treated with 100 ?mol/L FAC for 24 h compared with the control(P< 0.01,n=6).7.The protein level of IRP1 was decreased in BV2 microglia cell lines treated with 100?mol/L FAC for 24 h compared with the control(P< 0.001,n=6).8.The protein level of DMT1 was increased and the protein level of FPN1 was decreased in BV2 microglia cell lines treated with 100 ?mol/L DFO for 24 h compared with the control(P< 0.01,n=6).9.The protein level of IRP1 was increased in BV2 microglia cell lines treated with 100?mol/L DFO for 24 h compared with the control(P< 0.01,n=6).Our results indicated that 6-OHDA induced an increased expression of DMT1 and iron import into BV2 microglia,which might be associated with up-regulation of IRP1.6-OHDA had no significant effect on the expression of FPN1,and iron release from BV2 cells,which might be the result of IRP1 up-regulation and hepcidin down-regulation.Furthermore,6-OHDA activated microglia and the release of inflammatory cytokines were enhanced.Iron overload suppressed IRP1 expression,thus down-regulating DMT1 and up-reguating FPN1.On the contray,iron deficiency activated IRP1,leading to increased expression of DMT1,and decreased expression of FPN1.Therefore,6-OHDA can regulate the expression of DMT1 and FPN1 by activating IRP1 and inhibiting hepcidin,thus leading to iron accumulation in microglia.Furthermore,6-OHDA can activate microglia,which lead to increased release of inflammatory factors,causing damage to DA neurons.