| Parkinson's disease (PD) is a progressive neurodegenerative disorder associated withthe loss of dopaminergic neurons originating in the substantia nigra pars compacta (SNc)and terminating in the striatum (Str). This disorder is characterized symptomatically bybradykinesia, resting tremor and rigidity. The exact pathogenesis of PD has not beenrevealed yet. However, lots of researches suggests that multiple factors might be involved,such as heredity, environment, oxidative stress, excitotoxin, autoimmunity, ironaccumulation and cell apoptosis. Recent studies on the mechanism of the loss ofdopaminergic neurons support that apoptosis plays an important role in PD pathogenesis.Concentration of chromatin and apoptotic bodies could be observed in dopaminergicneurons by electron microscopy in the SN of PD patients. Agents that inhibit apoptosiscould prevent, or at least partly delay the progress of PD.Ghrelin, a novel 28-amino-acid brain-gut peptide, has been identified as anendogenous ligand for the growth hormone (GH) secretagogue receptor (GHS-R) byKojima in 1999. It is produced and secreted mainly from the stomach, and acts on thepituitary and hypothalamus to stimulate growth hormone (GH) release, adiposity, andappetite. Recent studies have reported that ghrelin stimulates proliferation and inhibitsapoptosis in several cell types, such as 3T3-L1 adipocytes and osteoblastic MC3T3-E1cells etc. D-lys~3-GHRP-6, the antagonist against GHS-R 1a, can eliminate the anti-apoptotic effect of ghrelin on 3T3-L1 adipocytes and primarily cultured hypothalamusneurons.In the present experiment, we investigated whether ghrelin could protectdopaminergic neurons. Using 1-methy-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)induced mice model of PD, we detected the effects of ghrelin on the contents of dopamine(DA) and its metabolites in the Str by high performance liquid chromatography-electrochemical detection (HPLC-ECD), and the mRNA and protein expression of TH inthe SN respectively by semi-quantitive reverse transcriptase- polymerase chain reaction(RT-PCR) and immunohistochemistry. Furtherly, to explore the probable mechanism ofghrelin's neuroprotective effect, we observed the colocalization of its full functionalreceptor—GHS-R 1a and TH in the SN by double immunofluorescent staining. Using RT-PCR and immunohistochemistry, we also detected the changes of Bcl-2, Bax and caspase-3 expression in the SN. Finally, we investigated the effect of D-Lys~3-GHRP-6—theantagonist of GHS-R 1a, on the indexes above.The results were as follows: 1. TH and GHS-R 1a colocalized in the SNpc of C57BL/6 mice.2. MPTP decreased the contents of DA, 3,4-dihydrophenyl acetic acid (DOPAC) andhomovanillic acid (HVA) in the Str (P＜0.05), and increased the striatal DA turnoverrate (P＜0.05). Pretreated with ghrelin (200 ng, 400 ng) significantly ameliorated thereductions of DA and its metabolites (P＜0.05), as well as the increase of striatal DAturnover rate induced by MPTP (P＜0.05). Ghrelin alone did not affect either striatalDA turnover rate or the contents of DA and its metabolisms (P＞0.05). D-Lys~3-GHRP-6 completely abolished the effects of ghrelin.3. MPTP reduced the number of TH immunoreactive neurons and TH mRNAexpression in the SN (P＜0.05). Pretreatment with 100 ng, 200 ng and 400 ng ghrelininhibited the reductions induced by MPTP (P＜0.05). Ghrelin alone did not changeTH mRNA expression and TH immunoreactive cell numbers in the SN (P＞0.05). Theneuroprotective effect of ghrelin could be thoroughly blocked by D-Lys~3-GHRP-6.4. Compared with the normal control mice, MPTP group exhibited a significant increaseof caspase-3 immunoreactive cell numbers in the SNpc (P＜0.05). Ghrelin inhibitedthe activity of caspase-3 dose-dependently. 50 ng and 100 ng ghrelin could not reducethe number of caspase-3 immunoreactive cells (P＞0.05). However, 200 ng and400 ng ghrelin showed a remarkable inhibition of caspase-3 activity compared withMPTP group (P＜0.05), which was stronger than lower doses. Ghrelin alone did notaffect caspase-3 immunoreactive cell numbers in the SNpc of nomal mice (P＞0.05).D-Lys~3-GHRP-6 could block ghrelin's neuroprotective effect against MPTP.5. The mRNA and protein expression of Bcl-2 in the SN decreased after injections ofMPTP (P＜0.05). 100 ng, 200 ng and 400 ng ghrelin increased SN Bcl-2immunoreactive cell numbers and mRNA expression of PD mice (P＜0.05). And theincreases of Bcl-2 mRNA and protein were propotional with the doses of ghrelin.Ghrelin alone did not change Bcl-2 mRNA expression and Bcl-2 immunoreactive cellnumbers in the SNpc (P＞0.05). The up-regulation of Bcl-2 expression in MPTP miceinduced by ghrelin was inhibited by D-Lys~3-GHRP-6.6. MPTP induced a remarkable increase of Bax mRNA expression in the SN (P＜0.01).All doses of ghrelin (50 ng, 100 ng, 200 ng and 400 ng) could down-regulate BaxmRNA expression in the SN of MPTP mice (P＜0.05). The inhibitive effect paralleledthe dose of ghrelin. The expression of Bax mRNA of normal mice was not affectedby ghrelin (P＞0.05). D-Lys~3-GHRP-6 thoroughly blocked the effect of ghrelin onBax mRNA expression.7. The ratio of SN Bcl-2/Bax increased in the Ghrelin group (P＜0.05). MPTP induced areduction of Bcl-2/Bax (P＜0.05). Pretreated with 50 ng and 100 ng ghrelin inhibited the reduction of Bcl-2/Bax induced by MPTP (P＜0.05). 200 ng and 400 ng ghrelineven reversed this reduction (P＜0.05). Blocking GHS-R 1a with D-Lys~3- GHRP-6abolished the effect of ghrelin.In conclusion, ghrelin inhibited the reduction of TH mRNA and protein expressionin the SN induced by MPTP, as well as the contents of DA and its metabolists in the Str.The probable mechanism of this protective effect might be anti-apoptosis by enhancingBcl-2 expression, reducing Bax expression and inhibiting caspase-3 activity. Theneuroprotective effect was mediated by GHS-R 1a. Our data provided new evidences forthe cell protective effect of ghrelin, and offered a new way possible to prevent and treatPD. |
