The Role Of PHB2 In The Mechanism Of MPTP-induced Mitochondria In Mice With Parkinson's Disease Under The Intervention Of Pre-exercise Training

Objective: In this study,an MPTP-induced model of Parkinson's disease in mice was established.During the onset of Parkinson's disease and the intervention of pre-exercise training to Parkinson's disease,the relationship between the changes of antiproliferative protein 2 content,mitochondrial autophagy and the content of fusion-splitting related proteins was observed.,To further reveal the pathogenesis of Parkinson's disease,and observe whether pre-exercise treadmill training can further alleviate the symptoms of Parkinson's disease.Methods:Methods: 120 male C57 BL/6 mice(young mice,8 weeks old)weighing20-25 g were randomly divided into two groups: control group(C,n = 60),pre exercise training group(PM,n = 60),cage feeding and free diet.The feeding environment is 20-25 ?,humidity 55%,natural light.In the PM group,the animals were first trained on the adaptive running platform for two days to make them familiar with the running environment and the training process.After that,they were trained on the medium-intensity running platform every morning,with the intensity of 12 meters/minute and the duration of 30 minutes for six weeks.After training,group C was randomly divided into control saline group(n,n = 30)and control MPTP group(P,n = 30).PM group was randomly divided into pre exercise training saline group(E,n = 30)and pre exercise training MPTP group(EP,n = 30).The dose of MPTP in group P and EP was 30 mg/kg once a day for 5 days.At the end of all groups,four suspension tests and climbing bar tests were conducted and scored one day before MPTP injection and the second,fifth and eighth days after MPTP.After that,the midbrain and striatum of mice were taken,and the following related indexes were detected and analyzed: 1.The scores of suspension and climbing bar test before and after MPTP injection.2.The number of th neurons in the substantia nigra of mice was detected by immunofluorescence.3.The protein expression of phb2,Mfn2,Drp1,FIS1,and LC3,beclin-1,parkin and PINK1 were measured by Western blot.Using Photoshop CS6 software for protein gray value analysis,using graphpad prism 8.0software for chart making,using SPSS 22.0 software for statistical analysis,using mean ± standard deviation for statistical results,nonparametric test for non normal data analysis,chi square test for differences between groups in the same period,k independent samples for differences between two groups.One way ANOVA is used to compare the differences between groups,and two way ANOVA is used to explain whether the combination of two factors will produce interaction.Results:1.Successfully established a mouse PD model with 6-week pre-exercise intervention.(1)6-week pre-exercise training can slow down the MPTP-induced decline in rod climbing scores There were no differences between group N and group E for four days under different days in the same group.In group P,compared with the first day,the scores on the second and fifth days decreased significantly(p <0.01),and the scores on the eighth day decreased and there were differences(p <0.05).In the EP group,compared with the first day,the scores on the second day decreased significantly(p <0.01),and the scores on the fifth day decreased and there were differences(p <0.05);on the same day and in different groups,the first day and the first day were different.No difference in eight days.On the second day,compared with the N group,the scores of the P group and the EP group decreased significantly(p <0.01).On the fifth day,compared with the N group,the scores of the P group and the EP group decreased significantly(p <0.01).(2)6 weeks of pre-exercise training can slow down the drop of suspension scores induced by MPTP in Parkinson mice There were no differences between group N and group E for four days under different days in the same group.In group P,compared with the first day,the scores on the second and fifth days decreased significantly(p <0.01).In the EP group,compared with the first day,the scores on the second day decreased significantly(p<0.01),and the scores on the fifth day decreased and there were differences(p <0.05);on the same day and in different groups,the first day and the first day were different.No difference in eight days.On the second day,the scores of the P group and the EP group decreased significantly(p <0.01).On the fifth day,compared with the N group,the score in the P group decreased significantly(p <0.01).(3)6-week pre-exercise training can slow down the number of substantia nigra TH neurons in MPTP-induced Parkinson mice Compared with the N group,there was no difference in the number of TH neurons in the E group.The number of TH neurons in the P group decreased significantly(p <0.05).The number of TH neurons in the EP group decreased significantly(p <0.05).Compared with the P group,The number of TH neurons in the EP group increased significantly(p <0.05).2.Expression changes of midbrain striatum-associated proteins in MPTP-induced Parkinson mice under pre-exercise training(1)Increased expression of PHB2 protein in midbrain striatum of MPTP-induced Parkinson mice and MPTP-induced Parkinson mice under the intervention of 6 weeks pre-exercise Compared with group N,PHB2 in group E was significantly increased(p <0.05),PHB2 in group P was significantly increased(p <0.05),and PHB2 in EP group was significantly increased(p <0.01).Compared with group P,PHB2 in EP group was significantly increased.Up-regulation(p <0.05);compared with E group,PHB2 in EP group was significantly up-regulated(p <0.05).(2)Levels of mitophagy proteins in the midbrain striatum of MPTP-induced Parkinson mice and MPTP-induced Parkinson mice under 6-week pre-exercise training Beclin1: Compared with the N group,Beclin1 in the P group increased significantly(p <0.05),and Beclin1 in the EP group increased significantly(p <0.01);compared with the P group,Beclin1 increased significantly in the EP group(p <0.01);compared with the E group,In comparison,Beclin1 in the EP group increased significantly(p<0.01).LC3?/? ratio: Compared with N group,LC3?/? in group E increasedsignificantly(p <0.05),LC3?/? in group P increased significantly(p <0.05),and LC3?/? in EP group increased significantly(p <0.01);Compared with group P,LC3?/? in group E was significantly increased(p <0.05),and LC3?/? in EP group was significantly increased(p <0.05);compared with group E,LC3?/? was significantly increased in group EP(p <0.05).(3)MPTP-induced Parkinson's striatum fusion and cleavage protein increase in6-week pre-exercise training intervention.MPTP-induced Parkinson's striatum fusion protein level decreases and mitosis Increased protein levels Mfn2: Compared with the N group,Mfn2 in the P group was significantly decreased(p <0.05);compared with the P group,the Mfn2 was significantly increased in the E group(p <0.01),and the Mfn2 in the EP group was significantly increased(p <0.05);Compared with group E,Mfn2 in group P was significantly decreased(p <0.01).Drp1: Compared with the N group,Drp1 in the E group was significantly increased(p<0.05),Drp1 was significantly increased in the P group and the EP group(p <0.01);compared with the P group,Drp1 was significantly increased in the EP group(p <0.05);Compared with the E group,Drp1 in the EP group increased significantly(p<0.01).Fis1: Compared with the N group,Fis1 in the P group increased significantly(p<0.05),and Fis1 in the EP group increased significantly(p <0.01);compared with the P group,Fis1 in the E group increased significantly(p <0.05),EP Fis1 increased significantly in the group(p <0.01);compared with the E group,Fis1 increased significantly in the EP group(p <0.01).3 PHB2 is significantly related to mitochondrial autophagy,mitochondrial fusion and the expression of mitotic proteins Through Pearson correlation analysis,it can be found that the correlation between phb2 and Beclin1,Drp1,FIS1,Mfn2,lc3 ii/I is less than 0.01,which indicates that phb2 is positively correlated with mitochondrial autophagy,mitochondrial fusion and mitosis to some extent.Conclusion:Six weeks of pre exercise training improved the PD model of mice.Phb2 can protect the nerves and indirectly regulate PD by regulating the expression of mitochondrial autophagy related protein and fusion division related protein.