Neuroprotective Effect And Mechanisms Of Lactoferrin On MPTP Induced Mice Model Of Parkinson’s Disease

Parkinson’s disease（PD）is the second most common neurodegenerative disease after Alzheimer’s disease.Its pathological features are dopaminergic neurons denaturing death in the substantia nigra（SN）,significant reduction of dopamine（DA）content in striatum（Str）and Lewy body in residual neurons of substantia nigra（SN）.The main clinical manifestations of PD include motor symptoms characterized by resting tremor,bradykinesia,myotonia and posture balance and non-motor symptoms such as autonomic dysfunction,sensory disturbance,sleep disorder and mental cognitive impairment.According to Chinese Parkinson’s Disease Treatment Guide（Third Edition）,the prevalence of PD in Chinese people over 60 years old is about 1.7%,and the current number of patients is about 2.7 million,experts in WHO predict that the number of PD patients in China is expected to reach 5 million by 2030.Therefore,from the perspective of sociology and economics,PD has become a major social problem that affects the life quality in population and seriously hinders the sustainable development of the economy.The pathogenesis of PD is influenced by environmental and genetic factors,but its specific pathogenesis is not completely clear,although it is known to be associated with inflammation,oxidative stress,apoptosis and impaired mitochondrial function.Therefore,it is necessary to further explore the pathogenesis of PD and new prevention target for the molecular mechanism of the disease in order to reverse or prevent the progression of the disease.Lactoferrin（Lf）is a non-heme iron-binding glycoprotein belonging to the transferrin family,composed of a single polypeptide chain folded into two symmetrical lobes,both of which show a high degree of homology and reversibly bind Fe³⁺iron.The molecular weight of Lf is 80kDa.Lf enters the cell membrane via a lactoferrin receptor（LfR）-mediated pathway and can enter the brain via the brain blood barrier（BBB）.Lf exists mainly in two forms:apo-lactoferrin（apo-Lf）and iron-saturated-lactoferrin（holo-Lf）.The affinity of Lf for iron is more than 300 times that of other transferrins,and the affinity in an acidic environment is significantly increased.Lf plays an important role in a variety of biological functions including iron metabolism,immune regulation,anti-apoptosis and antioxidants.Lf is synthesized and released by activated microglia in the brain,and apo-Lf is the major form of microglia release.Since Dexter DT et al first found elevated levels of iron in neuronal melanocytes of the substantia nigra pars compacta（SNpc）of PD patients,a large number of studies have found an increase in iron levels in PD patients,MPTP and 6-OHDA-induced PD animal models,which suggested that brain iron metabolism dysfunction played a key role in the pathogenesis of PD.Many studies found that Lf and LfR expression increased in the SN neurons of PD patients and MPTP-induced PD animal models.Our previous studies found that Lf were synthesized and released from activated microglia,and iron overload further enhanced this process.In ventral mesencephalon（VM）neurons,apo-Lf and holo-Lf increased copper-zinc superoxide dismutase（Cu/Zn-SOD）and B-cell lymphoma-2（Bcl-2）by protecting mitochondria to exert its neuroprotective effect against MPP⁺.This suggested that Lf could protect neurotoxin-treated dopaminergic neurons,which may be involved in the antioxidant and anti-apoptotic activities of apo-Lf and holo-Lf.This study aims to further observe whether two different forms of Lf have neuroprotective effects in vivo and explore their central and peripheral mechanisms,so as to provide experimental basis for finding new prevention target for PD.Male C57BL/6N mice aged 9-10 weeks and weighed 20-30g were allocated into ten groups according to random number table.The groups were as followed:Control,MPTP,MPTP+15mg/kg holo-Lf,MPTP+10mg/kg holo-Lf,MPTP+5mg/kg holo-Lf,MPTP+10mg/kg apo-Lf,MPTP+5mg/kg apo-Lf,15mg/kg holo-Lf,10mg/kg holo-Lf,and 10mg/kg apo-Lf.Doses of holo-Lf and apo-Lf were chosen according to our preliminary experiments.Immunohistochemistry,Immunofluorescence,Western Blot,HPLC,Prussian blue staining,ELISA,behavior observation,ultraviolet spectroscopy and blood routine examination were used to detect SN LfR expression;tyrosine hydroxylase immunoreactivity（TH-ir）changes in SN;DA and its metabolites dihydroxy-phenyl acetic acid（DOPAC）and homovanillic acid（HVA）content changes in Str;iron staining positive neurons in SN;the change of the mice body movement coordination;content changes of monoamine oxidase-B（MAO-B）in brain and peripheral blood;the expression changes of DMT1 and FPN1 in SN,and the levels of Bcl-2,Bax,cleaved Caspase-3,Cu/Zn-SOD,CD86;the changes of iron staining positive neurons in liver and spleen;serum iron,serum ferritin,total iron binding force（TIBC）and the changes of RBC and hemoglobin related indicators,so as to investigate the neuroprotective effect and its mechanisms of Lf on MPTP induced mice model of PD.Results are as follows:1.Neuroprotective effect of Lf on MPTP induced mice model of PD（1）Lf intragastric administration caused an increase in LfR expression in SN.Compared with the control group,LfR expression increased in groups treated with MPTP and pretreated with Lf.Intragastric administration of Lf without MPTP also showed increased in LfR expression in the SN compared with the control group.（P<0.05,n=6）.（2）Lf pretreatment reduced MPTP-induced TH-ir neuron loss in SN.MPTP caused significant TH-ir neuron loss in SN compared to the control group.High doses pretreatment with Lf（15mg/kg holo-Lf and 10mg/kg apo-Lf）rescued TH-ir eurons lost due to MPTP treatment（P<0.05,n=7）.No difference was observed etween solo holo-Lf or apo-Lf treatment groups and the control group（P>0.05,n=7）.（3）Lf pretreatment reduced the decrease of DA,DOPAC,HVA and the increase of DA metabolic rate in Str induced by MPTP.Compared to the control group,MPTP treatment significantly decreased DA,DOPAC and HVA levels,and increased the metabolic rate of DA in Str.High doses Lf（15mg/kg holo-Lf and 10mg/kg apo-Lf） pretreatment had an antagonistic effect on MPTP toxicity,restoring the levels of DA and normalized DA metabolic rate（P<0.05,n=6）.No difference was observed between solo holo-Lf or apo-Lf treatment groups and the control group（P>0.05,n=6）.（4）Lf pretreatment improved limb movement disorder induced by MPTP.The pole test showed MPTP-treated mice exhibited a pronounced prolongation of time on the pole compared to the control group.Lf pretreatment obviously relieved thephenomenon（P<0.05,n=7）.There were no differences between mice treated with Lf sololy and the control group（P>0.05,n=7）.2.Central mechanism of the neuroprotective effect of Lf on MPTP induced mice model of PD.（1）Lf pretreatment reduced the number of iron positive staining cells in SN induced by MPTP.Compared to the control group,MPTP treatment caused a significant increased numbers iron-positive staining cells in SN,and all doses of Lf pretreatment substantially attenuated MPTP-induced elevated iron positive stained cells（P<0.05,n=7）.Iron positive cell numbers were unchanged between Lf treated mice and the control group（P>0.05,n=7）.（2）Lf pretreatment down-regulated increased expression of DMT1 and decreased expression of FPN1 induced by MPTP in SN.Compared to the control group,MPTP induced an increase in DMT1 expression and a decrease in FPN1 expression in SN.High doses Lf（15mg/kg holo-Lf and 10mg/kg apo-Lf）pretreatment attenuated or reversed the increased expression of DMT1 and the reduced expression of FPN1 resulting from MPTP toxicity（P<0.05,n=7）.The level of DMT1 and FPN1 were unchanged between solo holo-Lf or apo-Lf groups and the control group（P>0.05,n=7）.（3）Lf pretreatment antagonized apoptosis related protein Bcl-2,Bax,Bcl-2/Bax ratio and cleaved Caspase-3 expression induced by MPTP in SN.MPTP resulted in decreased Bcl-2 protein expression and Bcl-2/Bax ratio,and an increased cleaved Caspase-3 expression in SN,compared to control group.These changes in protein levels were antagonized by high dose Lf（15mg/kg holo-Lf and 10mg/kg apo-Lf）pretreatment（P<0.05,n=6）.The protein level of Bax was not altered between treatment groups（P>0.05,n=6）.Mice administrated intragastrically with holo-Lf or apo-Lf sololy did not show protein levels alterations compared to control group（P>0.05,n=6）.（4）Lf pretreatment increased Cu/Zn-SOD protein expression decrease induced by MPTP in SN.MPTP treatment reduced expression of Cu/Zn-SOD protein in SN compared to control group.High dose Lf（15mg/kg holo-Lf and 10mg/kg apo-Lf） pretreatment antagonized the reduced expression of Cu/Zn-SOD protein induced by MPTP（P<0.05,n=7）.No difference in Cu/Zn-SOD protein expression was observed among only holo-Lf or apo-Lf given groups and the control group（P>0.05,n=7）.（5）Lf pretreatment antagonized MPTP-induced increase in CD86 protein expression in SN.Compared to control group,MPTP could increase the expression of CD86 protein in SN,and 15mg/kg holo-lf pretreatment could reduce the increased expression of CD86 induced by MPTP（P<0.05,n=8）.No changes in CD86 protein expression were observed in the group treated with 15mg/kg holo-Lf sololy（P>0.05,n=8）.3.Peripheric mechanism of the neuroprotective effect of Lf on MPTP induced mice model of PD.（1）Lf pretreatment and/or MPTP treatment did not affect iron staining in the liver.Prussian blue combined with DAB showed no statistically significant difference in the number of iron staining positive cells among different groups（P>0.05,n=6）.（2）Lf pretreatment antagonized the decrease of spleen weight in mice induced by MPTP.MPTP treatment resulted in a lower spleen weight compared to control group,while high and moderate doses of Lf（15mg/kg holo-Lf,10mg/kg holo-Lf and 10mg/kg apo-Lf）pretreatment reversed the reduction in spleen weight（P<0.05, n=6）.Compared to control group,there was no change in spleen weight treated with the holo-Lf or apo-Lf group sololy（P>0.05,n=6）.（3）Lf pretreatment reduced MPTP induced spleen iron decreased.Compared to control group,MPTP decreased iron level in mice spleen tissue,high and moderate doses of Lf（15mg/kg holo-Lf,10mg/kg holo-Lf and 10mg/kg apo-Lf）pretreatment reversed the decreased iron level and normalized iron storage in spleen（P<0.05,n=6）.The iron level was unchanged in solo holo-Lf or apo-Lf groups compared to control group（P>0.05,n=6）（4）Lf pretreatment corrected disturbance of iron metabolism induced by MPTP in peripheral blood.Compared to control group,MPTP treatment significantly increased serum iron and ferritin levels and decreased serum TIBC,high doses of Lf（15mg/kg holo-Lf and 10mg/kg apo-Lf）pretreatment corrected the increased peripheral serum iron,ferritin and the decreased TIBC induced by MPTP（P<0.05,n=5）.No significant differences in iron status were found between the holo-Lf or apo-Lf groups and the control group（P>0.05,n=5）.（5）Blood routine tests showed plasma measures of hemolysis and anemia including RBC,HGB,HTC,MCV,MCH,and MCHC,there were no differences among all ten groups（P>0.05,n=5）.In summary,these results indicate that Lf has a neuroprotective effect on MPTP-induced PD model mice,and its mechanism may be related to the reduction of brain iron,anti-oxidative stress,anti-apoptosis and anti-microglia-mediated inflammatory response.Apo-Lf and Holo-Lf have the same protection effect in PD.Lf did not affect peripheral iron levels,but was able to correct peripheral iron metabolism disorders in MPTP-induced PD model mice.Therefore,Lf is a safe and reliable neuroprotective agent that can reduce brain iron without affecting peripheral iron.