| Parkinson's disease (PD) is a progressive neurodegenerative disorder and the increased iron levels in the substantia nigra (SN) might be involved in the loss of dopaminergic neurons and the depletion of dopamine (DA) in the striatum (Str). However, the mechanism of why iron increased in SN is still unknown. Perhaps it is related with the misregulation of some iron transport proteins. Iron accumulation in SN of PD patients might be resulted from either increased iron uptake or decreased iron release. Over past years, iron uptake by cells and the related mechanism have received considerable attention. However, little knowledge is available on how iron is released. Ferroportinl (FP1), hephaestin (HP) and ceruloplasmin (CP) are three iron export proteins, so far. FP1 was a newly discovered transmembrane iron export protein, and it was found to be expressed in all kinds of tissues. CP is a ferroxidase that converts highly toxic ferrous iron (Fe2+) to its non-toxic ferric form (Fe3+). It is abundant in the brain, the liver and other organs, however, it is lack in the gut. HP is the analog of CP and can function in the gut instead of CP. Iron is taken up by cells and then may be retained, or transported across the basolateral surface by FP1. The form of iron that is exported by FP1 might be Fe2+, FP1 may work with HP and CP, Fe2+ is oxidized to Fe3+, Fe3+ bound to transferrin (Tf) for plasma transport. Lack of these proteins results in iron accumulation in the organs. However, the knowledge about brain iron metabolism is preliminary at present, especially about the mechanism of iron export. Using immunofluorescence, molecularbiolgy, electricalchemistry and other technology, we investigated the iron staining and the loss of DA neurons in SN of 6-hydroxydopamine (6-OHDA) lesioned rats; Determined how the three proteins function in the glioma C6 cell line; Compared the changes of expressionsof FP1, CP and HP in 6-OHDA lesioned rats. The results were as follows:1. 1, 3, 5, 7, 14 days after 6-OHDA treatment and also in the PD model rats, the density of the tyrosine hydroxylase immunoreactive (TH-ir) cells in the lesioned SN decreased by 52%, 68%, 81%, 85%, 97% and 99%, respectively.2. 1,3, 5, 7, 14 days following 6-OHDA lesions, iron levels increased compared with normal and unlesioned side (P<0.05). Although iron levels have the upward tendency, however, there were no statistical significance among them (P>0.05). In the PD model rats iron staining increased in the lesioned SN compared with Id group (P<0.05).3. 1, 3, 5, 7, 14 days following 6-OHDA lesions, DA release in Str of lesioned side did not change compared with normal group and unlesioned side (P>0.05). Only on 14 days of postlesion and in PD rats, did the DA release decreased (P<0.05).4. FP1 expressed in the brain of rats. It was distributed mostly in the cortext, hippocampus, SN, and ependymal cells around the third ventricles. FP1 also expressed in all kinds of rat brain cells including astrocyte, microglia, oligodendrocyte and neurons.5. Id after 6-OHDA lesions, the expression of FP1 decreased both in mRNA and protein levels. And in PD model rats, further decreases were investigated in the lesioned SN.6. CP expressed in the brain of rats. It was distributed mostly in the cortext, hippocampus, SN, and ependymal cells around the third ventricles. CP co-localized with FP1 on the same cells.7. Id after 6-OHDA lesions, the expression of CP decreased both in mRNA and proteinlevels compared with the normal and unlesioned side. In PD model rats, evident decreases were investigated in lesioned SN.8. HP expressed in the brain of rats. It was distributed mostly in the cortext, hippocampus, SN, and ependymal cells around the third'ventricles. HP also expressed in all kinds of rat brain cells including astrocyte, microglia, oligodendrocyte and neurons, which indicated that FP1 and HP were co-localized in the brain.9. Id after 6-OHDA lesions, the expression of HP decreased both in mRNA and protein levels compared with the normal and unlesioned side. In PD model rats, evident decreases were investigated in lesioned SN.10. Using Calcein and Laser Confocal Scanning Microscope, we investigated the iron export function of FP1, HP and CP on glioma C6 cell lines. In the time point of the first 5min, there were no differences among the groups of control and antibody treatment. At the second time point, iron export in C6 cells decreased in the groups of FP1, CP, HP and FP/CP/HP blocked, compared with control group. At the third time point, iron export in C6 cells decreased in the groups of FP1, HP and FP/CP/HP blocked, compared with control and CP blocked groups, and the iron export decreased in CP blocked group compared with control group. From the fourth to the fourteenth 5 min time points, there were statistical significances among the groups of control, FP1, CP, HP and FP1/CP/HP blocked when intercomparsion was made, except for the groups between FP1 blocked and FP1/CP/HP blocked. There was much more iron exported out of C6 cells when treated with CP antibody compared with the group of HP blocked.The results suggest that FP1, HP and CP expressed widely in the brain of rats. FP1 and HP also expressed in all types of rat brain cells indicating that they are co-expressed...
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