| Background:Pakinson's disease(PD)is the second leading neurodegenerative disease,and its main hallmark is the the lesion of dopaminergic(DA)neurons in the substantia nigra(SN)and following DA secretion decreased.Several brain-gut peptides have been reported to be related with the function of the central dopaminergic nervous system.Nesfatin-1 is a new brain-gut peptide discovered by Japanese scholar Shinsuke Oh-I in 2006.The main role of nesfatin-1 is to inhibit food taking and regulate energy metabolism.Our previous studies found that nesfatin-1 can antagonize rotenone-induced MES 23.5 cell damage.In both cell and animal experiments,nesfatin-1 can antagonize 1-methyl-4-phenylpyridine ion(MPP~+)-induced MES 23.5 dopaminergic cell lesion and MPTP-induced substantia nigra dopamine neurons lesion in mice.It is related with the function of nesfatin-1 that protected mitochondria and antagonized apoptosis via the C-Raf-ERK signaling pathway.Aim:The above research proves that nesfatin-1 has neuroprotective effects on nigra dopaminergic neurons.It has been reported that the nesfatin-1 level in the blood in patients with PD was lower than that of the control.Would lack of nesfatin-1 in the central nervous system induce the lesion in the substantia nigra-striatum system?Methods:To investigate the role of nesfatin-1 in the normal physiological function of the midbrain striatum dopamine system,nesfatin-1 antibody was injected to the lateral ventricle,which may induce the decrease of nesfatin-1 content in the intraventricular brain,to observe whether the morphology and function of striatum system will be damaged.8-week-old C57BL/6 male mice were selected to conduct lateral ventricle catheterization,and then randomly divided into 4 groups:(1)Control group:injected with normal saline;(2)Anti-nesfatin-1 group:injected with 0.12 mg/m L nesfatin-1 antibody;(3)non-immune anti-mouse IgG antibody:injected with mouse monoclonal IgG1?antibody,MAB 201;(4) MC4R receptor inhibitor group:injected with MC4R receptor inhibitor,SHU 9119.Mice were injected with drugs in lateral ventricle for 14 days.Pole test was used to detect the balance adjustment ability and limb coordination ability of mice;immunofluorescence staining was used to detect the change of the number of TH~+cells in the SN;horseradish catalase DAB method was used to detect the content of nerve fibers in TH~+cells in the str;transmission electron microscopy was used to detect changes in the number of mitochondria,the length of the long axis,and the morphology of the neuron cell nucleus.High performance liquid chromatography(HPLC)technology was used to detect the changes in the content of DA and its metabolites dihydroxyphenylacetic acid(DOPAC)and homovanillic acid(HVA)in the str;Enzyme-linked immunosorbent assay(ELISA)was used to detect the content of brain-derived neurotrophic factor(BDNF)in SN homogenate.Results:1.The weight of mice in each group was compared before and 14 days after the injection of the drug in the lateral ventricle of the mice.The results showed that there was no significant change in the body weight of the mice before and after the drug injection in four groups(p>0.05).2.After 14 days treatment,the pole test was used to detect the changes in balance adjustment ability and limb coordination ability of each group of mice,control group,Anti-nesfatin-1 group,non-immune anti-mouse IgG antibody group,MC4R receptor inhibitor group,the average time from the top to the bottom of the rod was 4.9s,5.2s,5.6s,and 5.2s,respectively.The results showed that there was no significant change in the exercise time of the mice in each group(p>0.05).3.After 14 days treatment,the number of TH+cells in the SN in the Anti-nesfatin-1group decreased by 23%and 30%compared with the control group and non-immune anti-mouse IgG antibody group(p<0.05),the above results show that injection of nesfatin-1antibody into the lateral ventricle can significantly reduce the number of SN dopaminergic neurons in mice.4.After 14 days treatment,TH+immunohistochemical staining of the str was used to observe whether the nerve terminal of the str changed.Immunohistochemical staining results showed that after 14 days injection of 0.12 mg/m L Anti-nesfatin-1 in the lateral ventricle of mice,the number of TH+nerve terminal was significantly reduced when compared with the control group.When the content of nesfatin-1 in the ventricle is reduced,the number of neurons in the substantia nigra DA neurons projected into the striatum is significantly reduced.5.After 14 days treatment,HPLC results showed that the DA content in the str of the Anti-nesfatin-1 group was decreased by 28%,22%and 29%,respectively,when compared with the control group,non-immune anti-mouse IgG antibody group and MC4R receptor inhibitor group,and the differences were statistically significant(p<0.05).The HVA content in the str of the Anti-nesfatin-1 group was decreased by 27%,22%and 19%when compared with the control group,non-immune anti-mouse IgG antibody group and MC4R receptor inhibitor group,and the differences were statistically significant(p<0.05).In term of DOPAC content in the striatum,there was no statistical difference between control group,non-immune anti-mouse IgG antibody group,MC4R receptor inhibitor group and Anti-nesfatin-1 group(p>0.05).When the content of nesfatin-1 in the ventricle is reduced,the content of DA and its metabolite HVA in the str of mice are significantly reduced.6.After 14 days treatment,the mitochondrial number,the length of the long axis of the dopaminergic neurons and morphology of neuronal nucleus in the SN in the control group and the Anti-nesfatin-1 group were observed by transmission electron microscope.The results showed that the number of mitochondria in the Anti-nesfatin-1 group decreased by54%compared with the control group,and the length of the long axis of the mitochondria decreased by 9%,and the difference was statistically significant(p<0.05).In contrast,in the Anti-nesfatin-1 group,the nuclei in the dopaminergic nuclear shrink significantly.When the level of nesfatin-1 in the ventricle decreases,mitochondria in DA neurons in mice would be damaged.7.After 14 days treatment,compared with the control group and non-immune anti-mouse IgG antibody group,the caspase-3 content in the substantia nigra of the Anti-nesfatin-1 group increased by 57%and 102%,respectively,it was statistically significant(p<0.05),when the content of nesfatin-1 in the ventricle decreases,the content of apoptosis factor caspase-3 in the SN of mice would increase,which further induces apoptosis.8.After 14 days treatment,the p-ERK/GAPDH level in Anti-nesfatin-1 group increased by about 44%compared with the control group,and increased by about 34%compared with the non-immune anti-mouse IgG antibody group,it was statistically significant(p<0.05),when the content of nesfatin-1 in the ventricle decreases,the content of p-ERK in the substantia nigra of mice would increase,and the cell pathway will be initiated to trigger the apoptosis effect.9.After 14 days treatment,the ELISA results of the substantia nigra showed that the content of BDNF in the Anti-nesfatin-1 group increased by 89%compared with the control group and increased by 41%compared with the non-immune anti-mouse IgG antibody,it was increased by 89%compared with the MC4R receptor inhibitor group,and the differences were statistically significant(p<0.05).When the content of nesfatin-1 in the ventricle decreases,the content of neurotrophic factor BDNF in the substantia nigra of mice would increase,which will cause a compensation response to cell damage.Conclusion:In summary,consistent with our previous published studies that external administration of nesfatin-1 protects dopaminergic neuron against MPP~+and MPTP induced cytoxicity,when dose Anti-nesfatin-1 antibody to the lateral ventricle of the mice,the content of nesfatin-1 was reduced,which can induced the dopamine system lesion in the nigrostriatal of mice,suggesting that nesfatin-1 plays an important role in maintaining the normal physiological function of the nigrostriatal system,and it is suggested that nesfatin-1 can be used as an early intervention drug for PD. |
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