| Parkinson’s disease (PD) is a common neurodegenerative disorder characterized symptomatically by resting tremor, rigidity, and bradykinesia. Neuropathological hallmarks of the disease include the degeneration and loss of dopaminergic neurons in the substantia nigra (SN) and the subsequent dopamine (DA) depletion in the striatum. The exact pathogenesis of PD has not been revealed yet. Multiple factors might be involved, such as heredity, environmental factors, oxidative stress, excitatory toxin, autoimmunity, iron accumulation and cell apoptosis. Both microglia activation and abnormal iron deposit in SN are involved in the pathogenesis of PD. However, the relationship between microglia activation and iron accumulation was not fully elucidated.Lactoferrin (Lf) is an iron-binding protein belonging to the transferrin family, which is produced by the exocrine glands and is widely distributed in all body fluids such as tears,saliva,milk and neutrophilic leukocytes. Lactoferrin receptor (LfR) has been shown to be present at the surface of different tissues and cell types, participating in Lf internalization by a receptor-mediated pathway. Lf can bind and transfer Fe3+ions, playing a key role in iron metabolism and transport. Lf also has a wide variety of biological functions, for example, antioxidant activities, anti-carcinogenic and anti-inflammatory properties, many of which do not appear to be connected with its iron binding ability. There are mainly two forms of Lf:iron-free(apo-lactoferrin,apo-Lf) and iron-saturated (holo-lactoferrin, holo-Lf). In the brain, Lf is only synthesized and released by activated microglia, and Lf mRNA levels were increased in a1-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP) mouse model of PD. LfR is mainly expressed in neurons. Immunohistochemical studies of PD patients revealed an increase of LfR on SNpc neurons and microvessels. These findings indicate a relevance of Lf/LfR in SN iron accumulation and the subsequent degeneration of dopaminergic neurons in PD.In this study, using enzyme-linked immunoabsorbent assay, real-time PCR, flow cytometry, immunofluorescence, western blots, HPLC, laser confocal scanning microscopy and other methods, we investigated how iron levels affected the Lf release in activated microglia, observed the effects of Lf on primary cultured ventral mesencephalon (VM) neurons against1-methyl-4-phenylpyridinium ion (MPP+) and MPTP-induced PD mice models. The results were as follows: 1.Lf release was significantly increased in primary cultured microglia with100μmol/L MPP+or800ng/ml lipopolysaccharide (LPS) treatment for24h (P<0.05), and this effect was enhanced by pretreatment of100μmol/L ferric ammonium citrate (FAC)(P<0.05).2.Lf mRNA expression was increased significantly in100μmol/L MPP+or800ng/ml LPS treated microglia, and this effect was enhanced with100μmol/L FAC pretreatment, compared with MPP+or800ng/ml LPS treated group (P<0.05).3.100μmol/L MPP+treatment resulted in a reduction of the mitochondrial transmembrane potential (△Ψm)(P<0.05) in primary cultured VM neurons.100ng/ml apo-Lf or100ng/ml holo-Lf pretreatment for4h could antagonize MPP+-induced dissipation of△Ψm (P<0.05).4.LfR protein level was up-regulated in primary cultured VM neurons with100ng/ml apo-Lf or100ng/ml holo-Lf treatment for24h (P<0.05).5.Blocking LfR for1h significantly blocked the protective effect of Lf on△Ψm against MPP+(P<0.05).6.100μmol/L MPP+treatment resulted in a reduction of Superoxide Dismutase (SOD) protein expression in primary cultured VM neurons.100ng/ml apo-Lf or100ng/ml holo-Lf pretreatment could antagonize MPP+-induced decrease of SOD (P<0.05).7.100μmol/L MPP+treatment resulted in a significant decrease of Bcl-2protein level in primary cultured VM neurons.100ng/ml apo-Lf or100ng/ml holo-Lf pretreatment could antagonize MPP+-induced decrease of Bcl-2and increase the ratio of Bcl-2/Bax (P<0.05).8.100ng/ml apo-Lf but not holo-Lf could reduce the level of labile iron pool (LIP) in primary cultured VM neurons.9. MPTP significantly decreased the numbers of tyrosine hydroxylase-immunoreactive (TH-IR) neurons in C57BL/6mouse. Pretreatment with apo-Lf could reverse the decrease of the TH neurons induced by MPTP (P<0.05).10. Pretreatment of apo-Lf could protect against the decrease of DA content in the striatum induced by MPTP (P<0.05).The above results suggest that activated microglia can synthesize and release abundant Lf. This process is further enhanced by iron load. In VM neurons, Lf exerts neuroprotective effects against MPP+-induced neurotoxicity by a mechanism, believed to enhance the△Ψm, improve the activity of SOD, increase the expression of anti-apoptotic protein Bcl-2and apo-Lf play the role of iron chelator. In MPTP-induced PD mice model, the nigral TH-IR neurons and striatal DA content were significantly decreased. Apo-Lf pretreatment could reduce the toxic effects of MPTP. The present study provides powerful evidence on etiology and promising strategies on prevention and therapy of PD. |
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