Study Of Neuroprotection And Mechanism Of Active Peptide On MPTP/MPP~+-induced Parkinson’s Disease Model

Objective:Parkinson’s disease(PD)is a common neurodegenerative disease that occurs more frequently in the elderly people.The prominent pathological changes in PD are degenerative death of dopaminergic neurons in the substantia nigra of the midbrain and the presence of eosinophilic inclusions in the cytoplasm of residual neurons in the substantia nigra,namely Lewy body.The clinical manifestations of the patient are mainly a series of dyskinesias such as resting tremor,bradykinesia,and muscular rigidity.With the aging of today’s society,the incidence of PD has increased year by year,which causes great burden to patients and society.However,at present,the commonly used therapeutic drugs have limited effect,so it is of great significance to research and develop drugs for preventing and treating PD.This study aimed to explore the neuroprotective effect and specific mechanism of the active peptide on PD through the MPP~+-induced SH-SY5Y cell injury model and MPTP-induced Parkinson’s disease mouse model,providing a theoretical basis for the treatment of PD.Methods:In this study,SH-SY5Y cells injury was induced by MPP~+to establish a cell model of PD,and C57BL male mice were intraperitoneally injected with MPTP to establish an animal model of PD.The neuroprotective effect of active peptide on PD and related mechanisms were elucidated.The cell experiments:SH-SY5Y cells were pretreated with peptides of different concentrations for 24 h,and then MPP~+(4 mmol/L)was added for 24h to damage the cells.MTT assay was used to detect the cell survival rate;Hoechst 33258 staining was used to observe the changes of cell nuclear morphology;Flow cytometry and DCFH-DA staining were used to detect the generation of reactive oxygen species in cells.Annexin-V/PI double staining was used to detect the apoptotic rate of cells;JC-1 staining was used to detect the change of mitochondrial membrane potential;The levels of MDA,SOD,GR,and GSH-Px were detected by the corresponding kits.The animal experiments:C57BL male mice were randomly divided into control group,MPTP(30 mg/kg)model group and peptide group(25,50 mg/kg).Behavioral tests were performed through traction test,pole test and open field test.The levels of inflammatory factors TNF-α,IL-1βand IL-6 in serum and midbrain were detected by ELISA.The levels of MDA,SOD,GR and GSH-Px in mouse midbrain tissues were detected by the corresponding kits.The expression of tyrosine hydroxylase(TH)in the substantia nigra of the midbrain was detected by immunohistochemistry and Western blot.Ionized calcium bindingadaptor molecule-1(Iba-1),a marker of microglia activation,was detected by Western blot.Western blot was used to detect the expression of apoptosis-related proteins Bax,Bcl-2,Caspase-3,PARP,and Cyto-c;Expression of NF-κB mediated inflammation-related proteins iNOS,COX-2,TNF-α,and IL-1β,and Expression of Nrf2 antioxidant pathway related proteins HO-1 and NQO1.Results:The cell experiments:MTT assay results showed that the survival rate decrease of SH-SY5Y cells induced by MPP~+could be significantly inhibited after pretreatment with PP-1 for 24 h at concentrations of 50 and 100μmol/L.The results of Hoechst 33258 staining showed that the nucleus was densely stained and fragmented after MPP~+treatment,while the peptide pretreatment could significantly improve the apoptotic state of the nucleus.Flow cytometry and DCFH-DA staining showed that peptide could reduce MPP~+induced ROS overproduction.Annexin-V/PI double staining results showed that the apoptosis rate increased after MPP~+injury,while the peptide pretreatment group could reduce the apoptosis rate.The results of mitochondrial membrane potential test showed that peptide could inhibit the decrease of mitochondrial membrane potential caused by MPP~+.After treatment with MPP~+,MDA content increased,SOD,GR and GSH-Px activity decreased.Peptide pretreatment significantly improved these indicators,thereby reducing oxidative stress damage caused by MPP~+.The animal experiments:The results of animal behavioral test showed that peptide PP-1 could improve the dyskinesia of PD mice,which was manifested in the improvement of grip ability of mice in traction test,the reduction of the time required to complete the climbing rod in pole test,and the increase of total distance and the number of times entering the central area in open field experiment.ELISA results showed that peptide could inhibit the increase of inflammatory factors TNF-α,IL-1βand IL-6 in PD mice.The results of oxidative stress-related indicators detection showed that the peptide reduced the increase of MDA levels in PD mice induced by MPTP and increased the activities of SOD,GR and GSH-Px.The results of immunohistochemistry showed that compared with the model group,the number of TH-positive cells increased in the peptide treatment group,indicating that the peptide could inhibit MPTP-induced damage of dopaminergic neurons in PD mice.Western blot results showed that the expression of Iba-1 increased in the model group,while peptide could inhibit the expression of Iba-1 protein,suggesting that peptide could inhibit the activation of microglia.Western blot analysis of related proteins showed that the peptide treatment group could inhibit the expression of apoptotic proteins,activate the Nrf2 pathway,and inhibit the NF-κB inflammatory pathway.Conclusion:The cell experiments in vitro indicated that the peptide could alleviate MPP~+-induced injury of SH-SY5Y cells,ameliorate oxidative stress,inhibit apoptosis and stabilize mitochondrial membrane potential.Meanwhile,the results of animal experiments in vivo demonstrated the peptide was able to improve the behavioral disorder and protect dopaminergic neurons in mice with PD induced by MPTP.Its mechanism may be related to inhibition on TH positive cells decrease in substantia nigra of PD mice;antagonizing apoptosis with inhibition of the expression of pro-apoptotic protein Bax and promotion of the expression of anti-apoptotic protein Bcl-2;oxidative stress reduction in midbrain,Nrf2 pathway activation,the expression promotion of downstream antioxidant proteins;reducing the expression of inflammatory factors in midbrain tissue and serum,inhibiting the activation of NF-κB,and then hindering the expression of downstream pro-inflammatory factors and pro-inflammatory proteases.In summary,the peptide was provided with a neuroprotective effect on MPTP/MPP~+-induced PD model,and its mechanism was related to anti-apoptosis,reducing oxidative stress and inhibiting inflammation.