| Aging is the important risk factor in neurodegenerative diseases, including Parkinson’s disease. Neurodegeneration or neurodegenerative diseases is the public health problem that is impact on population health and quality of life. It is also a major problem that is worthy of scientific research. It is imperative that promote the window of the basic and clinical research forward about Parkinson’s disease and other neurodegenerative diseases. To explore the biochemical molecular mechanisms of carbonyl stress and the biochemical molecular mechanisms age-related in striatal aging and Parkinson’s disease, to explore the impact of exercise on the striatal in the early neurodegeneration and its biochemical and molecular regulation mechanism, and to commit to screening the early new targets of neurodegeneration, this paper constructed two parts of model that are striatal aging model and6-OHDA-induced Parkinson’s disease model based on the carbonyl stress of the common biochemical process in aging, and the regular aerobic exercise intervention was carried out in the models. It provides research information for sports science and geriatrics degenerative diseases including the early development process of Parkinson’s disease in a very important theoretical and clinical significance.Methods:(1) In this study,6-month-old,16-month-old and25-month-old specific pathogen free (SPF) male Sprague-Dawley Rat (SD) rats were divided into young (Y-SED), middle-aged (M-SED) and old-aged (O-SED) sedentary control group, and the corresponding Y-EX, M-EX and O-EX in the aerobic exercise runner group. The10-weeks of regular moderate-intensity aerobic exercise intervention was carried out in the aerobic exercise runner group. The open-field test, the horizontal rope test and the adhesive removal test were used for indicating behavioral of motion. Blood biochemical markers of oxidative stress were detected. TUNEL assay for apoptosis, HE staining and electron microscopy were used for observing the striatum stereological indexes. Then the improved avidin-biological enrichment protein carbonyl proteomics and electrospray ionization quadrupole time of flight mass spectrometry (ESI-Q-TOF-MS/MS) were used to separate and identify the protein carbonyl and explore its biochemical and molecular regulation mechanism. The miRNA microarray of miRNA miRCURYTM LNA Array (v.18.0)(Exiqon, Danish) was deteced to excavate the importance of protein carbonyl specific mechanisms of transcription and translation in this project. qRT-PCR, immunohistochemistry and Western Blot was performed to screen and verify the related signal pathways for the biochemical molecular mechanisms.(2) The16-month-old male SD rats were divided into sedentary control Parkinson’s disease group (SED-PD) and aerobic exercise Parkinson’s disease group (EX-PD), the corresponding normal side SED-NOR and EX-NOR were the control. After8-weeks moderate-intensity aerobic exercise,6-OHDA (2.0mg/mL) was injected into the right unilateral striatum with double coordinate two targets for causing Parkinson’s disease. After testing the success of the Parkinson’s disease model, this paper adopt the method of (1) to explore the process of carbonyl stress mechanism in PD and the mechanism of action of exercise intervention.Resu lts:(1):Rats movement behavior, morphology and biochemistry related indexes showed that age-related and degenerative changes was occured in the process of aging. The horizontal rope test results showed that there age-related decline significantly (P<0.05) in sedentary control experimental rats. From the observation of the electron microscope, tomenta in elderly rat striatum arranged obviously confusion and loose, there was less tomenta between nodular or above, and the smoothness of tomenta down. Aoptotic nuclei were appeared and apoptotic rate was100%in aging rat striatum, and it showed age-related trend. Compared with the Y-SED group, apoptotic index increased by288.88%in the M-SED (P<0.05), while apoptotic index increased by392.22%in the O-SED(P<0.05). Compared with the Y-SED group, Beclinl/p-actin ratio rised trend in the M-SED and O-SED group (PO.05), and there were highly correlated with apoptosis.The carbonyl proteins were enriched by the improved avidin-biological enrichment protein carbonyl proteomics and identified by ESI-Q-TOF-MS/MS mass spectrometry. It found that protein carbonyl did not show incremental change with aging. It showed that the proteins were modified by carbonyl selectively. The relative amount of protein was not the decision factors of protein carbonyl levels.The number of carbonyl proteins is78in young sedentary control group, followed by69in old-aged rats and middle-aged was the least52. And oxidative modification sites were identified in protein carbonylation and the highest rate of percent76.47%in middle-aged rats, followed by72.63%in old-aged rats, and69.65%of young minimum. The biological function of the carbonyl proteins was involved in energy metabolism, mitochondrial inner membrane, ATP activity, substantia nigra development, synaptic conductance regulator protein carbonyl axons and other related parts and aging glycosylation pathways. The carbonyl proteins plays an important role in AMPK pathway, CaMK pathway and PI3R/Akt/mTOR pathway and the related network in age-related aging process. The experimental results showed that, compared with the Y-SED and M-SED group, AMPKal/p-actin ratio in O-SED group was upregulated with rising trend (P<0.05). The expression of CaMKⅡα and Vdacl was changed with age-related trends, and compared with the Y-SED, the expression of CaMKⅡα and Vdacl in the M-SED and O-SED was significantly increased (P<0.05). The expression of PI3K, Akt and mTOR was downregulated with aging, and compared with the Y-SED, the expression of PI3K, Akt and mTOR in the O-SED was significantly reduced (P<0.05). The results showed that these three signaling pathways were involved in regulating the relationship in the balance and stability between apoptosis and autophagy during striatum aging, which affects the development of aging process.(2) From the observation of the electron microscope, compared with the Y-SED, M-SED and O-SED groups, the tomenta was arranged with tightly, and there was more numbers and larger volume nodules in rat striatum in the Y-EX,M-EX and O-EX groups. The expression levels of BDNF was improved by regular aerobic exercise in all ages striatum (P<0.05), and it showed age-related trends. Oxidative stress and its related indexes in rat blood and tissue changes was not the same in regular aerobic exercise, and the blood and tissue trends in the corresponding of different presents was a stress stimulus itself. The striatal apoptosis index in each age group was increased respectively with205.56%,57%and68.24%in aerobic exercise (P<0.01). And Beclin1/β-actin ratio was increased significantly (P<0.05), while was increased significantly in M-EX and O-EX group (P<0.01).The carbonyl proteins were identified by ESI-Q-TOF-MS/MS, there were some carbonyl proteins were produced a certain influence on mitochondrial and substantia nigra development related signal transduction in regular aerobic exercise. The expression of calreticulin was improved by regular aerobic exercise which was associated with aging processes, and the heterogeneous nuclear ribonucleoprotein A2/B1carbonyl protein which had important relationships within DNA, and14-3-3which was closely related to the development of the substantia nigra and synaptic etc. The miRNA microarray results showed the expression of miRNA207was upregulated and the expression of miRNA542was downgulated which were involved in neural aging or neurodegenerative that had a close relationship with the stimulation of the nervous system receptor interaction pathways, calcineurin signaling pathway, γ-aminobutyric acid synaptic signaling pathway, glycolysis signaling pathways etc. The results showed that AMPKα1/β-actin ratio were increased significantly in rat striatum in regular aerobic exercise (P<0.05), with showing the age-related trends. Compared with the corresponding sedentary control group, AMPKα1/β-actin in the Y-EX, M-EX and O-EX group was increased by46.18%,62.85%and22.84%(P<0.05). Compared with the corresponding sedentary control group, the expression of CaMKIIa and Vdacl in the aerobic exercise runner group was increased significantly (P<0.05), which the expression upregulation had a very significant in M-EX than M-SED (P<O.01). Compared with M-SED and O-SED, the expression of PI3K, Akt and mTOR was upregulated in the corresponding M-EX and O-EX, which compared with O-SED, the expression of PI3K, Akt and mTOR in O-EX was increased significantly (P<0.01). Come to rule, aerobic exercise was in favor of the biological function and improve striatal neural mechanisms of aging, was inspired by the AMPK pathway, CaMKpathways and PI3K/Akt/mTOR pathway in rat striatum. It may be participated in the regulation of the balance relationship of homeostasis between autophagy and apoptosis.(3) The rat model of Parkinson’s disease was constructed successfully which was induced by6-OHDA, the process did not cause iatrogenic inflammation and a large of area apoptosis or necrosis. It showed that the striatal axons and nerve atrophy of the villi and other morphological changes had a close relationship with Parkinson’s disease from scanning electron microscope. Apoptotic nuclei could be observed by using transmission electron microscopy and TUNEL assay, and the incidence of apoptosis was100%in rat striatum Parkinson’s disease model. With the results of the fifth chapter analyzed, compared with M-SED group, the apoptotic index was increased significantly in SED-NOR group (P<0.01). Compared with the SED-NOR group, the apoptosis index was increased by40.72%in SED-PD group (P<0.01). Western blot analysis showed that, compared with the SED-NOR group, Beclin1/β-actin ratio was decreased in SED-PD group(P<0.05).The carbonyl proteins was identified by ESI-Q-TOF-MS/MS, which had an important role in and involved in mitochondrial inner membrane, energy metabolism, substantia nigra development, synaptic conductance regulator, monovalent cation transporter, microtubules, actin cytoskeleton and other related regulatory proteins in the process of Parkinson’s disease. Compared with the SED-NOR group, the expression of AMPK was increased significantly in SED-PD(P<0.01). Compared with SED-NOR, the expression of CaMKⅡα, CaMKV and Vdacl was significantly upregulated in SED-PD (P<0.01). Compared with SED-NOR, the expression of PI3R, Akt and mTOR was significantly upregulated in SED-PD (P<0.01). The development of Parkinson’s disease was closely related to striatal neurons autophagy disorder which could promote apoptosis. To a certain extent, AMPK pathway down CaMK signaling pathway which could promote the apoptosis, and it played an important role in the regulation Parkinson’s disease.(4) Pre-aerobic exercise improved the resilience in Parkinson damage which6-OHDA-induced in rats corpus striatum, and had a certain impact on ATP binding activity and amide binding related signal transduction protein carbonyl, and the expression of CamⅡα, HSP60, Synl, Map1and Map2protein carbonylation was improved that was related with aging and degenerative diseases. The expression of miRNA3557was upregulated and the expression of miRNA324was downgulated by pre-aerobic exercise, whch had a close relationship with Parkinson regulation on the stimulation of the nervous system receptor interaction pathways, calcineurin signaling pathway, amphetamine into addiction, glycosylphosphatidylinositol(GPI)-anchor biosynthesis, endocytosis, drug metabolism-cytochrome and nucleotide excision repair. Compared with the EX-NOR group, AMPKα1/β-actin ratio was increased in the EX-PD group(P<0.05), but compared with SED-PD group, AMPKα1/β-actin ratio was declined in EX-PD group (P<0.05). Compared with SED-NOR, the expression of CamⅡα, CaMKV Vdacl were significantly reduced in EX-NOR (P<0.01). Compared with SED-PD, the expression of CaMKV and Vdacl was slightly reduced in EX-PD (P>0.05), only the expression of CaMKIIa was slightly increased in EX-PD (P>0.05). Compared with the EX-NOR, the expression of PI3R, Akt and mTOR was also increased significantly in EX-PD (P<0.01). However, compared with the SED-PD, the expression of PI3R and mTOR was slightly raised in EX-PD (P>0.05), then the expression of Akt was significantly reduced (P<0.01). Compared with SED-PD, the expression of Syp and Ywhah was significantly reduced in EX-PD (P<0.05,P<0.01), which UCH-L1upregulation (P<0.01). Come to a conclusion, autophagy and apoptosis were induced in pre-aerobic exercise in rat striatum of Parkinson’s disease with appropriate increasing, the AMPK pathway and CaMK signaling pathway were downregulated, and the PI3R/Akt/mTOR signaling pathway was activated, and PI3R/Akt/mTOR signaling pathway coordinated CaMK regulatory pathway and AMPK signaling pathway network, thereby increasing the expression level of UCH-L1and the index of autophagy jointly in Parkinson’s disease with the occurrence or development of disease.Conclusion:The comprehensive results showed that, in the development of age-related aging, and with Parkinson’s disease occurrence, the carbonyl proteins was modified selectively, the relative amount of protein is not the decision factors of protein carbonyl levels, and the network of AMPK signaling pathway, CaMK signaling pathway and PI3R/Akt/mTOR signaling pathway and its associated protein carbonyl group has a close relationship with age-related aging and Neurodegenerative Parkinson’s diseases. The adaptive mechanism on regular aerobic exercise that anti-aging and delay neurodegenerative diseases is a sterss resilience mechanism. Sports induced energy-adaptive and steady oxygen stress changes, and AMPK signaling pathway and CaMK signaling pathway related protein carbonyl modification, miRNA and one of its target path CaMK signaling pathway was activated to regulate the expression of AMPK signaling pathway and P13K/Akt/mTOR signaling pathway related proteins with coordination. Thus the relationship of balanced and steady-state was regulated on neurogenesis/autophagy/apoptosis. And it improved or delaied the development of the neurodegenerative disease or Parkinson’s disease. |
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