The Role Of Over-expressed DMT1 In Iron Accumulation In MES23.5 Cells And The Mechanism Of The Protective Effect Of Ginsenoside Rg1

Parkinson's disease(PD) is a progressive neurodegenerative disorder characterized by selectively dopaminergic neurons loss in the substantia nigra pars compacta(SNpc) and the depletion of dopamine(DA) in the striatum.Recently many literatures have confirmed that iron plays a key role in Parkinson's disease.However,the underlying mechanisms of iron accumulation remain unclear.The identification of new iron transporters provides an approach to investigate cellular mechanism of iron accumulation.DMT1,also known as Nramp2(nature resistance associated macrophage protein 2), is newly identified iron transporter.DMT1 is involved in the brain iron metabolism and highly expressed in the neurons of the substantial nigra in PD,which correlates with the iron abnormally deposited in the same area.Our previous studies showed increased DMT1 expression and elevated iron levels in the SNpc of PD mouse model.This suggested the up-regulation of divalent metal transporter 1(DMT1) might be a contributing factor to iron accumulation.In the present study,we aim to verify the hypothesis that increased DMT1 expression caused the iron accumulation.We chose MES23.5 cells as the experimental neuronal model.Using molecular biology,immunofluorescence,laser confocal scanning microscopy,flow cytometry,HPLC and other methods,we investigated the role of over expressed DMT1 in iron accumulation in MES23.5 cells.The upregulation of DMT1 provided new pharmacologic target to treat the disease. Ginsenoside Rg1 is one of the main components of ginseng,which has a variety of biological functions including but not limited to anti-oxidative,estrogen-like activity. The present study was carried out to elucidate the effect of the antioxidant drug ginsenoside Rg1 on levels of expression of the non-heme iron transporter,divalent metal transporter-1(DMT1),and iron levels in MES23.5 dopaminergic cells after 6-hydroxydopamine(6-OHDA) treatment.The main findings are as follows:Ⅰ.The role of over expressed DMT1 in iron accumulation in MES23.5 cells. 1.DMT1+IRE and DMT1-IRE were expressed on MES23.5 cells.2.Human DMT1+IRE and DMT1-IRE genes were amplified by RT-PCR.Recombinant adenovirus AdDMT1+IRE and AdDMT1-IRE were obtained after packaging and amplification in HEK293 cells.3.Increased DMT1 expression was confirmed by western blots.Data showed that DMT1+IRE protein levels increased significantly in AdDMT1+IRE infected cells compared to AdGFP infected cells(P＜0.01).DMT1-IRE protein levels increased significantly in AdDMT1-IRE infected cells compared to AdGFP infected cells (P＜0.01).4.Cell viability reduced when treated with 100μmol/L ferrous iron in AdDMT 1 +IRE or AdDMT1-IRE infected MES23.5 cells(P＜0.05).However,no significant change was observed in AdGFP infected cells.5.Intracellular iron levels,hydroxyl free radicals and lipid peroxidation increased in AdDMT1+IRE or AdDMT1-IRE infected MES23.5 cells after iron incubation compared to cells infected with AdGFP(P＜0.01).6.When cells were treated with ferrous iron,AdDMT1+IRE or AdDMT1-IRE infected cells showed more activated-caspase-3(P＜0.01),apparent ladder patterns, fragmentation of chromatin and hypercondensed(brightly stained) nuclei(P＜0.05).7.Dopamine contens in AdDMT1+IRE or AdDMT1-IRE infected cells were both decreased after iron treatment compared to cells infected with AdGFP(P＜0.05).8.pcDNA3.1-DMT1+IRE and pcDNA3.1-DMT1-IRE expression vectors were successfully constructed.There was a significant decrease in the fluorescence intensity in pcDNA3.1-DMT1+IRE or pcDNA3.1-DMT1-IRE transfected cells compared to the contról when perfused with 1 mmol/L ferrous iron(Two-way ANOVA,F=25.995,P＜0.01,pcDNA3.1-DMT1+IRE vs.control;P＜0.01, pcDNA3.1 -DMT1-IRE vs.control). 9.The production of ROS and changes of mitochondrial transmembrane potential (△Ψm) were detected by Flow Cytometer.Overexpression of DMT1(DMTI+IRE or DMTI-IRE) increased ROS production and decreased mitochondrial transmembrane potential(△Ψm) compared to the control after iron treatment (P＜0.05).Ⅱ.The role of ginsenoside Rg1 in 6-OHDA-induced iron accumulation in MES23.5 cells1.We examined DMT1+IRE expression levels by western blots in MES23.5 cells. DMT1+IRE protein in 6-OHDA treated MES23.5 cells was up-regulated compared to the control,while pretreated with Rg1 or vitamin E could attenuated this upregulation of DMT1 +IRE(P＜0.05).2.Quantitative PCR was conducted to measure DMTI+IRE mRNA levels.There was an increase of DMT1+IRE mRNA in 6-OHDA treated cells and a decrease of DMT1+IRE mRNA in Rg1 or vitamin E pretreated cells(P＜0.05).3.There was a significant decrease in the fluorescence intensity in 6-OHDA treated cells compared to the control when perfused with 1 mmol/L ferrous iron.The fluorescence intensity restored to the control levels when pretreated with Rg1 or vitamin E(Two-way ANOVA,F=18.102,P＜0.01,cells treated with 6-OHDA vs. control,P＜0.01,cells pretreated with Rg1 or vitamin E vs.cells treated with 6-OHDA).4.IRP1 and IRP2 were upregulated in protein levels after 6-OHDA treatment,when pretreated with Rg1 or vitamin E,the two protein levels restored to the levels of the control(P＜0.05).5.Both IRP1 and IRP2 were upregulated in mRNA levels after 6-OHDA treatment, while when pretreated with Rg1 or vitamin E,the mRNA levels of both IRP1 and IRP2 restored to the levels of control(P＜0.05).6.Increased iron influx by 6-OHDA(10μmol/L) treatment resulted in a reduction of the mitochondrial transmembrane potential after iron treatment.While Rg1 could attenuate the reduction of mitochondrial transmembrane potential.In conclusion,these results suggested that DMT1+IRE and DMT1-IRE were expressed on MES23.5 cells.Increased two forms of DMT1 expression in MES23.5 cells caused the increased intracellular iron accumulation.Elevated cellular iron induced oxidative stress,increased caspase-3 activity and ultimately apoptosis.This supports the function of increased DMT1 expression in iron accumulation and the notion that increased DMT1 results in oxidative stress and apoptosis,processes known to occur in PD.We also reported that Rg1 could attenuate the upregulation of DMT1+IRE and decrease the cellular iron accumulation.And this effect may contribute to the capacity of Rg1 to attenuate the expression of IRP1 and IRP2.