| Parkinson’s disease（PD）is the second most common neurodegenerative disease after Alzheimer’s disease（AD）,and the disease often occurs in middle-aged and elderly people.As an increasingly common neurodegenerative disease,Parkinson’s disease has typical clinical features including motor disorders,motor retardation and resting tremor,etc.Meanwhile,it also causes a series of non-motor features,mainly including mood disorders,sleep disorders,autonomic nerve dysfunction,cognitive disorders,dementia and neuropsychiatric symptoms.For half a century,drugs for Parkinson’s,notably levodopa,have been available,but current treatments fail to improve the underlying progression of the disease,resulting in more severe motor and cognitive impairment in many patients.Therefore,exploring the underlying pathogenesis of Parkinson’s disease and developing ideal therapeutic drugs are essential conditions for the treatment of Parkinson’s disease.The main clinical feature of Parkinson’s disease is the accumulation of intracellular alpha-synuclein in the form of Louie bodies and Louie neurites.It has been suggested that apoptosis is the main mechanism of neuron loss in Parkinson’s disease.Autopsies of Parkinson’s disease patients showed that DNA fragments and apoptotic chromatin of dopaminergic neurons were changed,and activation of Caspase-3 was increased in the substantia nigra dense region.Similarly,anti-apoptotic proteins such as Bcl-2 have been shown to inhibit the death of dopaminergic neurons in vitro.All these evidences directly or indirectly indicate that apoptosis is the main mechanism of neuron loss in PD.Autophagic vacuoles were also found in the brain tissues of patients with Parkinson’s disease.At present,the relationship between autophagy and apoptosis in Parkinson’s disease still needs further study.Rotenone,as an environmental toxin,is often used in the preparation of Parkinson’s disease models.In rotenone induced Parkinson’s disease models,a large number of experiments have observed the occurrence of apoptosis,and the aggregation of autophagy vesicles has also been found in this model.Calcium ion,as an important second messenger,has been shown to be closely associated with cell apoptosis and autophagy,and increased intracellular calcium ion concentration has also been observed in rotenone induced Parkinson’s disease models.Ca2+/Calmodulin-dependent protein kinase kinaseβ,CaMKKβ/AMP-activated protein kinase,AMPK/Rapamycin target protein,mTOR signaling pathway are closely related to neuron function,calcium overload caused by high calcium ion concentration in neurons can lead to increased sensitivity to excitotoxicity and apoptosis,leading to brain injury.Meanwhile,activation of CaMKKβ/AMPK/mTOR signaling pathway has also been found to induce autophagosome formation in late onset Parkinson’s disease caused by LRRK2 gene mutation.ST2-104 polypeptide is a small-molecule derived peptide based on Collapsin respon mediator protein 2（CRMP2）.Based on the design of the CBD3 region of CRMP2 protein and the structure of the nine arginine transmembrane peptides,CRMP2 has specificity for N-methyl-D-Aspartate receptors（NMDARs）and enhanced transmembrane ability.Previous studies have shown that ST2-104polypeptide has a protective effect on both Alzheimer’s disease and neuronal damage caused by cerebral ischemia.Previous studies have shown that ST2-104 polypeptide has a protective effect on both Alzheimer’s disease and neuronal damage caused by cerebral ischemia.On this basis,this study will further explore the effect of ST2-104polypeptide on neuron injury in Parkinson’s disease and its possible mechanism.Method:At animal level:To determine the effect of ST2-104 polypeptide on Parkinson’s disease neurons in vivo and its mechanism,rotenone was used to treat mice to establish Parkinson’s disease model.Animals were divided into Con group,ROT group,ROT+ST2-104 group.The expression of Bax,Bcl-2,Caspase-3,Beclin-1,LC3-II and CaMKKβ,p-AMPK,p-mTOR were detected by WB.The rigidity strength of mice was evaluated by grid test,and the pole climbing test was used to detect the motor function of mice.At the cellular level:In order to preliminatively determine the protective effect and mechanism of ST2-104 polypeptide on rotenone-treated cell damage,this study first used rotenone-treated SH-SY5Y cells to establish an in vitro model of Parkinson’s disease,and then applied ST2-104 polypeptide to intervene on this basis.Cells were divided into Con group,ROT group,ROT+ST2-104 group and ST2-104group,respectively.Cell activity was detected by MTT assay,Hoechst 33258fluorescence staining assay was used to evaluate cell apoptosis,autophagy was evaluated by MDC fluorescence staining assay,and intracellular calcium concentration was evaluated by Flow Cytometry.The expression of Bax,Bcl-2,caspase-3,LC3-II,beclin-1 and CaMKKβ,p-AMPK,p-mTOR were detected by Western Blot.To determine the relationship between autophagy and apoptosis in rotenone-treated cell models,the autophagy agonist rapamycin was first used to intervene,and cells were divided into Con group,ROT group ROT+ST2-104 group ROT+ST2-104+RAPA group and RAPA group respectively.Apoptosis and autophagy levels were evaluated by Hoechst 33258 fluorescence staining and MDC fluorescence staining,respectively.Bax,Bcl-2,Caspase-3,Beclin1,and LC3-II expression were detected by Western Blot.Then the autophagy inhibitor 3-MA was used for intervention,and cells were divided into Con group,ROT group,ROT+ST2-104group,ROT+ST2-104+3-MA group and 3-MA group respectively.The experimental content was the same as that of rapamycin group.To determine whether ST2-104polypeptide exerts cell protective effects through the CaMKKβ/AMPK/mTOR signaling pathway,STO-609（CaMKKβinhibitor）was used for intervention,cells were grouped as follows:blank group rotenone group,rotenone+ST2-104 group,rotenone+ST2-104+STO-609 group,STO-609 group,Hoechst 33258 fluorescence staining and MDC fluorescence staining were used to determine cell apoptosis and autophagy levels respectively,and Western Blot was used to detect the expression of Bax,Bcl-2,Caspase-3,LC3-II,beclin-1,CaMKKβ,P-AMPK and p-mTOR.Result:At the animal level:compared with the Con group,the climbing time of ROT group was significantly increased in the pole climbing experiment,indicating that rotenone could cause motor dysfunction in mice.In the grid experiment,the duration of paralysis in rotenone group was significantly increased,indicating that rotenone could cause increased intensity of paralysis in mice.In contrast to ROT group,the climbing time and duration of paralysis were significantly reduced in ROT+ST2-104group,indicating that ST2-104 polypeptide could inhibit rotenone-induced motor dysfunction in mice and improve the enhanced degree of paralysis caused by rotenone.Compared with the Con group,Western Blot experiment in ROT group showed that the expression of apoptosis-related proteins Bax and C-caspase-3 were significantly increased,and the expression of Bcl-2 was significantly decreased.The expression of autophagy related proteins Beclin-1 and LC3-II was significantly enhanced,suggesting that rotenone could enhance cell apoptosis and autophagy.Compared with rotenone,the expression of apoptosis-related proteins Bax and C-caspase3 were significantly decreased and the expression of Bcl-2 was significantly increased in ROT+ST2-104 group in WB experiment.The expression of autophagy-related proteins Beclin-1 and LC3-II was significantly decreased,indicating that ST2-104polypeptide could inhibit rotenone-induced apoptotic autophagy at animal experimental level.Compared with the control group,the expression of CaMKKβ,p-AMPK in ROT group was significantly increased（P<0.05）,the expression of p-mTOR protein was significantly decreased（P<0.05）,suggesting that rotenone can activate CaMKKβ/AMPK/mTOR signaling pathway,and compared with the ROT group,the expression of signaling pathway related proteins in the ROT+ST2-104group was completely opposite to that in the ROT group,and the results showed that ST2-104 polypeptide could inhibit the activation of rotenone on this signaling pathway.At the cellular level,MTT results showed that compared with the Con group,the cell viability of ROT group was significantly decreased after treatment with 5μM rotenone for 24h（P<0.01）.Fluorescence staining results of Hoechst 33258 showed that the fragment nucleus and fluorescence intensity were significantly increased,and Western Blot results showed that the expression of apoptosis-related proteins Bax and C-caspase-3 were significantly increased（P<0.01）.The expression of Bcl-2 protein decreased significantly（P<0.01）,indicating that rotenone-induced PD cell model was successfully established.The results of MDC fluorescence staining showed that cell fluorescence intensity was significantly increased,and the expression of autophagy related protein Beclin-1,LC3-II protein was significantly increased by Western Blot（P<0.01）,indicating that rotenone can significantly enhance the level of autophagy.FCM results showed that intracellular calcium concentration was significantly increased,and CaMKKβ,p-AMPK protein expression was significantly increased（P<0.01）,the expression of p-mTOR protein was significantly decreased（P<0.05）,suggesting that rotenone can significantly increase intracellular calcium concentration and simultaneously activate CaMKKβ/AMPK/mTOR signaling pathway.Compared with ROT group,ROT+ST2-104 group（co-incubated with rotenone for 24h after 1h pretreatment）significantly increased cell viability（P<0.01）.Fluorescence staining of Hoechst 33258 showed that the fluorescence intensity was decreased and the cell morphology was significantly improved.Meanwhile,Western Blot results showed that the expression of apoptosis-related proteins Bax and C-caspase-3 was significantly decreased（P<0.01）,and the expression of Bcl-2 protein was significantly increased,suggesting that ST2-104 polypeptide could significantly inhibit rotenone-induced apoptosis;MDC fluorescence staining results showed that cell fluorescence intensity was weakened,and autophagy related proteins Beclin-1and LC3-II were significantly decreased in Western Blot results,indicating that ST2-104 polypeptide can significantly inhibit the enhancement of autophagy induced by rotenone.FCM detected a significant decrease in intracellular calcium ion concentration,and Western Blot assay showed a significant decrease in CaMKKβp-AMPK protein expression（P<0.05,P<0.01）,p-mTOR protein expression increased significantly（P<0.05）.These results showed that ST2-104 polypeptide significantly inhibited the increase of intracellular calcium ion concentration induced by rotenone,and also significantly inhibited the activation of CaMKKβ/AMPK/mTOR signaling pathway by rotenone.After the intervention with autophagy activator RAPA,compared with the ROT+ST2-104 group,Hoechst 33258 fluorescence staining showed that the nucleus of the ROT+ST2-104+RAPA group was obviously pellucid,broken and fluorescently enhanced.Meanwhile,Western Blot experiment showed that the expression of Bax and C-caspase-3 protein was significantly enhanced（P<0.05,P<0.01）,Bcl-2 protein expression was significantly decreased（P<0.01）,suggesting that enhanced autophagy could inhibit the protective effect of ST2-104 polypeptide on rotenone-induced apoptosis.After intervention with autophagy inhibitor 3-MA,fluorescence staining of Hoechst 33258 in ROT+ST2-104+3-MA group was compared with that in ROT+ST2-104+3-MA group.Western Blot experiment showed that Bax,C-caspase-3 protein expression decreased significantly（P<0.05,P<0.01）,Bcl-2 protein expression increased significantly（P<0.01）,indicating that inhibition of autophagy can enhance the protective effect of ST2-104 on rotenone-induced apoptosis.To further determine whether ST2-104 protects rotenone-induced cell damage through CaMKKβ,STO-609 cells were pretreated with CaMKKβinhibitor.Hoechst in ROT+ST2-104+STO-609 group was compared with that in ROT+ST2-104group.The fluorescence staining experiment of 33258 showed that the fluorescence intensity of fine nucleus decreased obviously.Western Blot assay also showed that the expression of apoptosis-related proteins Bax and C-caspase-3 was significantly enhanced（P<0.05,P<0.01）,Bcl-2 protein expression was significantly decreased（P<0.01）,demonstrating that STO-609 can significantly increase the protective effect of ST2-104 polypeptide on rotenone-induced cell damage.The fluorescence intensity of the cells and the dotted structure dyed green particles were decreased by MDC fluorescence staining,and the expression of Beclin-1,LC3-II protein was significantly decreased by Western Blot（P<0.05,P<0.01）,indicating that STO-609 can enhance the inhibitory effect of ST2-104 polypeptide on autophagy.Western Blot results showed that the expression of CaMKKβ,p-AMPK was significantly decreased（P<0.05）,the expression of p-mTOR protein was significantly increased（P<0.05）,indicating that STO-609 can significantly enhance the inhibitory effect of ST2-104polypeptide on CaMKKβ/AMPK/mTOR signaling pathway.Conclusion:ST2-104 polypeptide has a protective effect on rotenone-induced Parkinson’s disease injury,possibly by inhibiting rotenone-induced intracellular calcium overload,which in turninhibits autophagy and further inhibits apoptosis by inhibiting CaMKKβ/AMPK/mTOR signaling pathway. |
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