| Parkinson’s disease（PD）is a progressive neurodegenerative disease which is characterized by the degeneration of dopaminergic（DAergic）neurons in the substania nigra pars compacta and subsequently reduced release of dopamine（DA）in the striatum（Str）.The clinical manifestations are resting tremor,muscle rigidity and bradykinesia.Although the etiology of PD has not been clarified up to now,the environment,gene,aging and oxidative stress are considered as contributing factors to the etiology of PD.In recent years,it has been found that some molecule peptides such as ghrelin can effectively protect the nervous system.Therefore,discovering an endogenous small molecule active substance has become one of the potential therapies for PD.Ghrelin,a newly discovered brain-gut peptide in 1999,is the only endogenous ligand for the growth hormone secretagogue receptor（GHSR）.And it could freely cross the blood-brain barrier.The endogenous ghrelin is involved in food intake and energy metabolism.Our previous study first reported that ghrelin could regulate the electrical activity of DAergic neurons,affect the release of DA,and exert its protective effect on DAergic neurons through anti-oxidation and anti-apoptosis.Furthermore,our recent study found that plasma total ghrelin and active ghrelin in the early PD patients were significantly decreased.There was no typical postprandial inhibitory and pre-meal secretion peak of plasma ghrelin in PD patients.Therefore,the question is,whether continuous dosing ghrelin before the onset of decreased plasma ghrelin could slow down the PD progression.To evaluate the potential roles of pretreatment ghrelin in the early stages of PD,continuous dosing ghrelin and non-sense peptide were administered subcutaneously through Alzet Osmotic Pumps to alpha-synuclein（α-syn）A53T+/+transgenic mice(α-SynA53T+/+)and wide type（WT）mice from the age of 4 w.The mice were divided into P（non-sense peptide）-WT group,P-A53T group,G（ghrelin）-WT group and G-A53T group.The expression of Iba1 and tyrosine hydroxylase（TH）-positive cells were detected by the immunofluorescent technique and the contents of DA in the Str were measured by high performance liquid chromatography（HPLC）.The protein levels of TH,Bcl-2,Bax,superoxide dismutase1（SOD1）and interleukin-6（IL-6）were detected by western blot.We used enzyme-linked immunoabsorbant assay（ELISA）method to detect the plasma total and active ghrelin levels.The results were as follows:1.After the treatment with ghrelin for 4 w,there were no differences in the number of TH-positive neurons and the protein levels of TH in the SN of G-A53T at the age of 3months in comparison with the control（P>0.05）.The content of DA（11.71±1.37 ng/mg）in the Str of G-A53T at the age of 3 months was significantly increased in comparison with the control（P<0.05）.Compared with the control,the number of TH-positive neurons in the SN of P-A53T group mice were decreased by 12%（P<0.05）.Correspondingly,the protein level of TH in the SN were decreased by 23%,and the content of DA in the Str was 5.57±2.48 ng/mg,which were decreased by 51%at the age of 3 months（P<0.01）.Ghrelin administration had no effect on the number of TH-positive neurons,TH protein levels in the SN and the DA contents in the Str in WT mice at the age of 3 months.2.After the treatment with ghrelin for 8 w,in comparison with the control,the number of TH-positive neurons in the SN of G-A53T group mice were increased by 13%and 15%at the age of 3 months and 6 months,respectively（P<0.05）.The protein levels of TH in the SN of G-A53T group mice at the age of 3 months and 6 months were increased by 17%and 42%,respectively（P<0.01）,and the contents of DA in the Str of G-A53T group mice at the age of 3 months and 6 months were 13.31±2.46 ng/mg and 8.61±1.75 ng/mg,which were increased by 162%and 69%,respectively（P<0.01）.The number of TH-positive neurons in the SN of P-A53T group mice at the age of 3 months and 6 months were decreased by 14%and 16%compared with the control（P<0.05）.Correspondingly,the protein levels of TH in the SN were decreased by 16%and 43%at the age of 3 months and6 months,respectively（P<0.01）,and the contents of DA in the Str of P-A53T group mice were 5.06±2.48 ng/mg and 5.09±1.07 ng/mg,at the age of 3 months and 6 months which were decreased by 36%and 33%,respectively（P<0.01,P<0.001）.Ghrelin administration had no effect on WT mice in terms of the number of TH-positive neurons,the protein levels of TH in the SN and the contents of DA in the Str at the age of 3 months and 6 months.3.After the treatment with ghrelin for 4 w,there were no significant differences of plasma total ghrelin of G-A53T（4.82±1.49 ng/mL）group mice at the age of 3 months compared with the control（P>0.05）.The plasma total ghrelin of P-A53T group mice was2.97±0.81 ng/mL,which was decreased by 38%（P<0.05）at the age of 3 months.After the treatment with ghrelin for 8 w,the plasma total ghrelin of G-A53T group mice was 4.52±0.36 ng/mL,which was increased by 20%compared with the control at the age of 3months（P<0.05）.There were no significant differences between G-A53T（4.44±1.34ng/m L）and the control in plasma total ghrelin at the age of 6 months（P>0.05）.Compared with the control,the plasma total ghrelin of P-A53T group were decreased by 36%and 33%at the age of 3 months and 6 months,respectively（P<0.05）.Ghrelin administration had no effect on WT mice in terms of plasma total ghrelin.4.After the treatment with ghrelin for 4 w,plasma active ghrelin of G-A53T group at the age of 3 months was 172.27±38.56 pg/m L,which was increased by 26%compared with the control（P<0.05）.The plasma active ghrelin of P-A53T group 3 months was 167.85±15.39 pg/mL,which was decreased by 22%compared with the control at the age of 3months（P<0.05）.After the treatment with ghrelin for 8 w,the plasma active ghrelin of G-A53T group mice 6 months were 193.87±17.60 pg/mL and 167.92±29.06 pg/m L which were increased by 33%and 14%,respectively,compared with the control at the age of 3months and 6 months（P<0.05）.The plasma active ghrelin of P-A53T group mice at the age of 3 months and 6 months were 139.85±15.03 pg/m L and 122.10±32.35 pg/m L,decreased by 27%and 26%,respectively（P<0.05）.Ghrelin administration had no effect on WT mice in terms of plasma active ghrelin.5.After the treatment with ghrelin for 8 w,the body weight of G-A53T group mice increased over the age of 3-6 months compared with the control（P<0.05）.The body weight of P-A53T group mice decreased over the age of 3-6 months compared with the control（P<0.05）.Ghrelin administration had no effect on WT mice in terms of body weight.6.After the treatment with ghrelin for 4 w,no significant differences were observed in the ratio of Bcl-2/Bax protein levels in the SN among the four groups at the age of 3months（P>0.05）.After the treatment with ghrelin for 8 w,no significant differences were observed in the ratio of Bcl-2/Bax protein levels in the SN among the four groups at the age of 3 months（P>0.05）.At the age of 6 months,in the ratio of Bcl-2/Bax protein levels in the SN of G-A53T group mice were increased by 13%compared with the control（P<0.05）.The ratio of Bcl-2/Bax protein levels in the SN of P-A53T group mice were decreased by 46%at the age of 6 months compared with the control（P<0.05）.Ghrelin administration had no effect on WT mice in terms of the ratio of Bcl-2/Bax protein levels in the SN.7.After the treatment with ghrelin for 4 w,no significant differences were observed in the protein levels of SOD1 in the SN among the four groups at the age of 3 months（P>0.05）.After the treatment with continuous dosing ghrelin for 8 w,no significant differences were observed in the protein levels of SOD1 in the SN among the four groups at the age of 3 months.At the age of 6 months,the protein levels of SOD1 in the SN of G-A53T group mice were increased by 33%compared with the control（P<0.01）.The protein levels of SOD1 in the SN of P-A53T group mice were decreased by 32%at the age of 6months compared with the control（P<0.001）.Ghrelin administration had no effect on WT mice in terms of the protein levels of SOD1 in the SN.8.After the treatment with ghrelin for 4 w,no significant differences were observed in the number of microglia and the protein level of IL-6 in the SN of G-A53T group mice at the age of 3 months compared with the control（P>0.05）.The number of microglia was increased by 29%and the protein level of IL-6 was increased by 28%in the SN of P-A53T group mice at the age of 3 months compared with the control（P<0.05）.After the treatment with ghrelin for 8 w,the number of microglia in the SN of G-A53T group mice were decreased by 22%and 45%at the age of 3 months and 6 months,respectively（P<0.05,P<0.01）.Compared with the control,the number of microglia in the SN of P-A53T group mice were increased by 22%and 58%at the age of 3 months and 6 months,respectively（P<0.05,P<0.01）.Correspondingly,the protein levels of IL-6 in the SN of G-A53T group mice were decreased by 42%and 47%at the age of 3 months and 6 months,respectively（P<0.05）.The protein levels of IL-6 in the SN of P-A53T group mice increased by 37%and 39%at the age of 3 months and 6 months,respectively（P<0.05）.Ghrelin administration has no effect on WT mice in terms of the number of microglia and the protein levels of IL-6 in the SN.To sum up,with the progression ofα-SynA53T+/+mice,the plasma ghrelin decreased,and continuous dosing ghrelin administration could restore the decreased plasma ghrelin to normal levels.Continuous dosing ghrelin administration did not lead to obesity of WT mice.With the recovery of the plasma ghrelin to normal levels,the degeneration of DAergic neurons ofα-SynA53T+/+mice were improved significantly in terms of the increased TH positive cell number and the protein level of TH in the SN.After the treatment with ghrelin for 4 w,ghrelin had no neuroprotective effects on DAergic neurons at the age of 3 months.However,after the treatment with ghrelin for 8 w,ghrelin had neuroprotective effect on the DAergic neurons in the SN at the age of 3 months,and this effect could last for 6 months.And this early intervention of ghrelin may delay the progression ofα-Syn A53T+/+mice by inhibiting apoptosis,decreasing oxidative stress and anti-inflammation.Therefore,ghrelin can improve and the progression of neurodegenerative diseases,which will have important potential clinical value in preventing the onset of PD. |
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