| Parkinson's disease (PD) is a progressive neurodegenerative disorder associated with the loss of dopaminergic neurons originating in the substantia nigra pars compacta (SNc) and terminating in the striatum (Str). The exact pathogenesis of PD has not been revealed now. Accumulating evidence supports that among the proposed mechanisms of dopaminergic degeneration, oxidative stress plays an important role.Ghrelin has been identified as an endogenous ligand for the growth hormone secretagogue receptor (GHS-R) by Kojima in 1999. It is a 28-amino-acid peptide, and ghrelin exerts its biological effects via its fully functional receptor, GHS-Rla. GHS-Rla is a number of G-protein coupled receptor with seven transmembrane-spanning domains. It is mentioned that GHS-Rla is constitutively active, resulting in signaling even in the absence of the ligand. This opens new avenues for the treatment of PD. Several lines of evidence suggest that the effect of ghrelin is not only restricted to feeding behaviour and energy homeostasis, but also concerns anti-oxidative and anti-inflammatory effects, anti-apoptosis, regulation of sleep-wake states and memory, et al. In our previous study, we have observed the neuroprotective effects of ghrelin on dopaminergic neurons against MPTP-induced neurotoxicity by binding with GHS-R1a. However, the exact mechanism is not clear. In order to explore the possible mechanisms, in this study, we investigated the protective effects of ghrelin on MPP+-treated MES23.5 cells which express GHS-R1a and the possible mechanisms using semi-quantitive reverse transcriptase-polymerase chain reaction (RT-PCR), western-blots. Meanwhile, using high performance liquid chromatography-electrochemical detection (HPLC-ECD), RT-PCR and immunohistochemistry, we also detected the protective effect of over-expressed GHS-Rla on MPTP-treated mice. The results were as follows:1. The levels of malondialdehyde (MDA) in MPP+-treated MES23.5 cells were significantly higher than that of the control (P< 0.01). Ghrelin pretreatment could significantly attenuate the MPP+-induced increase in MDA levels (P< 0.01).2. MPP+ treatment resulted in the reduction of catalase (CAT) in both mRNA and protein levels and Cu/Zn-superoxide dismutase (Cu/Zn-SOD) protein levels after 24 h compared with the control (P<0.01), whereas pretreatment with ghrelin enhanced their expression (P<0.01).3. The Bax/Bcl-2 ratio and the active form of caspase-3 protein levels increased in MPP+-treated cells compared with that of the control, while ghrelin pretreatment attenuated these changes.4. Western blots analysis showed the transcription factor nuclear factor E2-related factor 2 (Nrf2) in MES23.5 cells was up-regulated after ghrelin pretreatment in a time-dependent manner from 3 h to 24 h.5. MPP+ induced a significant nuclear factor-κB (NF-κB) nuclear translocation, which was inhibited by ghrelin pretreatment.6. pAdGHS-R1a yielded a large fragment (near 30 kb), plus a smaller fragment of 4.5 kb, indicating the successful construction of recombinant adenovirus expression vector. GHS-R1a was expressed efficiently in 293 cells after infection.7. GHS-R1a protein level was higher than that of control in the SN of mice after injecting the adenovirus encoding GHS-R1a gene.GHS-R1a over-expressed in the SN of mice could significantly ameliorate the reductions of DA and its metabolites (P<0.05), as well as the increase of striatal DA turnover rate induced by MPTP (P<0.05).8. GHS-R1a over-expressed in the SN of mice antagonized MPTP-induced decrease in the number of tyrosine hydroxlase (TH) immunoreactive neurons, and inhibited the reduction of TH mRNA expression induced by MPTP (P<0.05).In conclusion, we demonstrated that the neuroprotective effect of ghrelin on MPP+-treated MES23.5 cells is due to its anti-oxidation properties as well as the regulation of NF-κB and Nrf2. On the other hand, we showed that over-expressed of GHS-R1a in the SN of mice could protect dopaminergic neurons from MPTP-induced neurotoxicity.
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