| Parkinson's disease(PD) is a progressive neurodegenerative disorder associated with the loss of dopaminergic neurons originating in the substantia nigra pars compacta(SNpc) and terminating in the striatum(Str).The exact pathogenesis of PD has not been revealed yet.Multiple factors might be involved,such as heredity,environmental factors,oxidative stress,excitotoxin,autoimmunity,iron accumulation and cell apoptosis.Recent studies showed that concentration of chromatin and apoptotic bodies could be observed in dopaminergic neurons of the SN of PD patients,which supported apoptosis played an important role in PD.Ghrelin,an endogenous peptide first discovered in 1999,is a 28-amino-acid protein with Ser3 modified by a fatty acid,primarily n-octanoic acid.Although ghrelin is essentially secreted by the stomach and duodenum,it could also be secreted by the brain cells,such as cells in the pituitary and hypothalamus.It has been identified as a solely endogenous ligand for the growth hormone secretagogue receptor(GHS-R),whose function is exerting bioactivity through acting on this receptor.Besides the pituitary and hypothalamus,GHS-R has been widely detected in other brain regions,such as the arcuate nucleus,SN,ventral tegmental area,and etc,suggesting ghrelin might have some unknown biological effects in these areas.Recent studies have reported that ghrelin could inhibit apoptosis in several cell lines via its fully functional receptor,GHS-R 1a.In the previous study,we have observed the neuroprotective effects of ghrelin on dopaminergic neurons in vivo in MPTP-treated Parkinson's disease mice.In order to illustrate the underlying mechanisms,in the present study,using immunofluorescence,western blotting, cell viability assay,flow cytometry and the other molecular biology methods,we conducted our experiment in vitro in MPP+-treated MES23.5 cells.The results were as follows:1.GHS-R 1a was abundantly expressed in MES23.5 cells.2.Cell viability was measured after 0-1000μmol/L MPP+ treatment for 24 hrs.A significant reduction of cell viability was observed when treated with 10 -1000μmol/L MPP+.Similar results were observed in measuring the leakage of the cytosolic LDH using LDH assay.200μmol/L MPP+ was chosen to do the further experiments.There was a significant increase in cell viability with ghrelin pretreatment(10-7-10-13 mol/L) compared to the cells with solely MPP+ treatment.The percentage of viable cells in MPP+ group decreased to 68.37%compared to the control,however,it increased to 89.57%,87.16%,93.67%,88.45%,86.88%and 82.46%with 10-7 mol/L,10-8 mol/L,10-9 mol/L,10-10 mol/L,10-11 mol/L and 10-12 mol/L ghrelin pretreatment,respectively.LDH assay further confirmed that ghrelin(10-9 mol/L) pretreatment for 20 min could significantly inhibit LDH leakage outside the cells induced by MPP+.Ghrelin(10-9 mol/L) was chosen to do the following experiments.3.In MPP+(200μmol/L) treated group,nuclei of MES23.5 cells appeared hyper-condensed. However,pretreatment with ghrelin(10-9 mol/L) could significantly abolish MPP+-induced nuclear morphological changes There was a significant reduction of△ψM in MPP+-treated cells.However,a restoration of△ψM was observed in cells with ghrelin (10-9 mol/L) pretreatment.Cells treated with MPP+ showed increased ROS production compared to the control.However,cells pretreated with ghrelin showed a less ROS production.Consistent with the above observation,activation of the effector caspase was also observed.Cells incubated with MPP+ resulted in an increase in the activated caspase-3,and this effect could be significantly abolished by ghrelin(10-90 mol/L pretreatment.4.Ghrelin induced the phosphorylation of extracellular signal regulated protein kinase (EKR) in a time-dependent manner rapidly.ERK1/2 inhibitor MAPK inhibitor PD98059 (50μmol/L) could significantly block Ghrelin-induced phosphorylation.It could also significantly antagonized the protective effects of Ghrelin against MPP+,as indicated by cell viability assay,intracellular△ψM and ROS formation.5,Protein kinase C(PKC) inhibitor GF109203 X(5μmol/L) and the tyrosine kinase (PTK) inhibitor tyrphostin-23(10μmol/L) significantly attenuate Ghrelin-induced phosphorylation of ERK;and they could also significantly antagonized the protective effects of Ghrelin against MPP+.6,In MES23.5 cells overexpressed with GHS-R1a,low dose of ghrelin(10-13 mol/L) that had no protective effect against MPP+,showed the observable protective effects.7,When pSilencer-GHS-R1a expression vectors were transfected into MES23.5 cells that induced GHS-R1a low expression,the neuroprotective effects of Ghrelin were abolished.The present study first showed that GHS-R1a was abundantly expressed on MES23.5 cells,which hinted that ghrelin might exert some effect on this kind of cell.As expected,ghrelin could significantly antagonize MPP+-induced neurotoxicity in MES23.5 cells.Ghrelin exerted this effect through restoration of mitochondria transmembrane potential,inhibition of ROS production and caspase-3 activation.The anti-apoptotic effects of Ghrelin might be mediated via a GHS-R1a-tyrosine kinase/PKC-MAPK-dependent signalling pathway.These suggested that ghrelin might function as a neuroprotective agent and be a novel therapeutic strategy for the treatment of PD.
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