The Neuroprotection Of The Novel Dual GLP-1 And GIP Receptor Agonists On MPTP-induced Parkinson’s Disease Mouse Model And Study On Its Mechanisims

Part one: The effects of the novel dual GLP-1 and GIP receptor agonist on MPTP-induced Parkinson’s disease mouse model: behavioral tests and tyrosine hydroxylase immunohistochemical evaluationObjects: To observe the effects of novel dual GLP-1 and GIP receptor agonist on MPTP-induced Parkinson’s disease mouse model by means of two behavioral tests and tyrosine hydroxylase immunohistochemical evaluation in the substantia nigra.Methods: Eight weeks old adult male C57BL/6 mice(15-20g)are chosen for this experiment.60 mice were divided into 6 groups,each group contained 10 mice,six groups as listed below :(A)control group: treated with saline;(B)MPTP group: intraperitoneal single MPTP injection for 1 day+ saline for 6 days starting the day after day 1;(C)liraglutide group: intraperitoneal MPTP injections for 1 day+liraglutide for 6 days after day 1;(D)DA-JC1 group: intraperitoneal MPTP injections for 1 day+DA-JC1 for 6 days after day 1;(E)DA-JC4 group: intraperitoneal MPTP injections for 1 day+ DA-JC4 for 6 days after day 1;(F)DA-CH5 group: intraperitoneal MPTP injections for 1 day+ DA-CH5 injection for 6 days after day 1.Blood was taken for glucose measurements from 5 mice per group,and the other 5 mice per group for the weight test.Behavioral evaluation start on day 2 after injection of each group:(1)The rotarod test : On days 2-7,5 mice per group were used in this test which started 1 h after drug injection.The experiment was repeated two times for each mouse with an interval of half an hour.(2)The traction test: In 2-7days,5 mice per group were used in this test after the drugs injection 1hour.On the 8th day after the MPTP and drugs treatment,mice were injected with lidocaine,then were perfused by saline,followed by 4% PFA.Brains were immediately removed and post-fixed in 4% PFA for overnight.Then they were paraffin embedded,and sections(5 um thick)were mounted onto slides for immune histochemical study of tyrosine hydroxylase.All of data from above should be recorded for statistical analysis.Results:1.When analysing body weights at day 1,7,a two-way repeated measures ANOVA found no difference between groups(P>0.05),When analysing plasma glucose levels on day 1,7,a one-way repeated measures ANOVA found no difference between groups on day 1(P>0.05)and on day 7(P>0.05).2.Rotarod test: A one-way repeated measures ANOVA found an overall difference over all groups(F=54.104 P<0.001),and a LSD-t post-hoc test found differences Between groups.The MPTP group and the Liragutide +MPTP group was impaired compared to controls(P<0.001).The MPTP+Liraglutide,DA-JC1+MPTP,DA-JC4+MPTP and DA-CH5+MPTP groups were performing at a better level compared to the MPTP group(P<0.001).The DA-JC4+MPTP group(P<0.01)and the DA5+MPTP group was performing better compared to the Liraglutide+MPTP group(P<0.001),The DA-JC4+MPTP group was performing better compared to the DAJC1+MPTP group(P<0.05),The DA-CH5+MPTP group was performing better compared to the DA-JC1+MPTP group(P<0.01).A two-way ANOVA found a difference between groups(P<0.001)and over time(P<0.05)when comparing performances for each day.There are 5 mice in per group,and the Rotarod test was repeated two times for each mouse after half an hour.Grip strength test: A one-way repeated measures ANOVA found an overall difference over all groups(F= 59.582 P<0.001),and a LSD-t post-hoc test found differences Between groups.The MPTP group,the Liragutide +MPTP group and the DA-JC4+MPTP group was impaired compared to controls(P<0.001),and The DA-CH5+MPTP group was improved compared to the DA-JC4+MPTP group(P<0.05).N=5 per group.A two-way ANOVA found a difference between groups(P<0.001)and over time(P<0.05)when comparing performances for each day.Part two: The effects of the novel dual GLP-1 and GIP receptor agonist on MPTP-induced Parkinson’s disease mouse model by inhibiting inflammation and study on its mechanismObjects: To observe the effects of novel dual GLP-1 and GIP receptor agonist on MPTP-induced Parkinson’s disease mouse model by immunohistochemical and Western blot analysis on several pro-inflammatory cytokines including IL-1β,TNF-α,NF-κB,IBA-1.Methods: Eight weeks old adult male C57BL/6 mice(15-20g)are chosen for this experiment.54 mice were divided into 6 groups,each group contained 9 mice,six groups as listed below as same treatment with Part one study :(A)control group;(B)MPTP group;(C)liraglutide group;(D)DA-JC1 group;(E)DA-JC4 group;(F)DA-CH5 group.On the 8th day after the MPTP and drugs treatment,mice were injected with lidocaine,then were perfused by saline,followed by 4% PFA.Brains were immediately removed and post-fixed in 4% PFA for overnight.(1)Then they were paraffin embedded,and sections(5 um thick)were mounted onto slides for immune histochemical study.The sections were pretreated with 3% H2O2 for 10 min at room temperature to remove endogenous peroxidase activity.Then,sections were incubated with primary antibodies rabbit anti-IBA-1(1:200,Abcam),the antibody diluted in PBS in 37°C for 2h.After these sections were incubated with their corresponding secondary antibodies for 60 min at 37°C.The immunoreactivity was visualized with DAB colour reaction in SNpc.Images were captured with a Zeiss Axioscope 1(G?ttingen,Germany)microscope and a digital Zeiss camera.All morphometric parameters were quantified using the image analysis computer program Image-Pro Plus Version 6.0(Media Cybernetics,USA).N=6 sections per brain were analysed,with n=6 per group.(2)For Western blotting,brains were snap frozen.Proteins were isolated from the ventral midbrain which have been homogenized in an ice cold RIPA buffer(the Boster Institute of Biotechnology,China)and phenyl-methylsulfonyl fluoride(PMSF).Protein concentration was measured by a BCA Protein Assay Kit,Then,equal amounts of proteins from each sample was subjected to SDS-polyacrylamide gel(PAGE)(12 %)for electrophoresis,The gel was run at 80 V for 30 min,and120 V for 1 h,and proteins were transferred to a polyvinylidene difluoride(PVDF)membrane by using a semidry transfer apparatus(Bio-Rad,Hercules,US).which was subsequently blocked for 2h with 5% milk in Tris-buffered saline(TBS)containing 0.05% Tween-20(TBST)at room temperature to block remaining protein binding sites,and then incubated with the rabbit anti-NF-κB(1:2,000;Cell Signaling Technology),the rabbit antibody against IL-1β(1:2,000;ABCAM),rabbit anti-TNF-α(1:1000;ABCAM),followed by washing six times in TBST for 5 min,the horseradish peroxidase(HRP)conjugated secondary antibody The immunoreactive bands were visualized with ECL Western blot kit(Boster Biotechnology,China)according to the manufacturer’s instructions.Densitometric analysis of protein bands in the Western blots and zymograms were done using Image Lab? Software version 3.0.N=4 per group.Results:1.Immunohistochemistry: A One-way ANOVA showed an overall difference between groups(F=72.437;P<0.001)followed by the The LSD-t post-hoc test.The MPTP group,Liraglutide +MPTP and DA-JC1+MPTP showed higher levels of IBA-1compared to the control group(P<0.001),but the other groups did not.The Liraglutide +MPTP,DA-JC1+MPTP,DAJC4+MPTP and DA-CH5+MPTP groups showed lower levels compared to the MPTP group(P<0.001),The DA-JC1+MPTP,DA-JC4 and DA-CH5 +MPTP groups showed lower levels compared to the liraglutide +MPTP group(P<0.001),The DA-JC4 and DA-CH5 +MPTP groups showed lower levels compared to the DA-JC1+MPTP group(P<0.001),The DA-CH5 +MPTP groups showed lower levels compared to the DA-JC4+MPTP group(P<0.05).2.Western blot: Western blot quantification of protein levels of IL-1β,TNF-α,NF-κB.A One-way ANOVA showed a difference between groups(F=11.664,F=140.208,F=23.816.F=10.458,F=21.932,P<0.001)followed by LSD-t post-hoc tests.Data are represented as mean±SD.Data are averages of 4 repetitions of blotting.IL-1β levels were higher in the MPTP group than in the control group(P<0.01).The Liraglutide+MPTP,DA-JC1+MPTP,DAJC4,DA-CH5 group showed lower levels compared to the MPTP group(P<0.05),and the DA5+MPTP groups showed lower levels compared to the DA-JC1+MPTP group and DA-JC4(P<0.05).Levels of NF-κB were lower in the Liraglutide+MPTP,DA-JC1+MPTP,DA-JC4+MPTP and DA-CH5+MPTP group showed lower levels compared to the MPTP group(P<0.001).The DAJC4+MPTP(P<0.05),DA-CH5+MPTP group(P<0.001)had lower levels compared to the Liraglutide+MPTP group,DA-CH5+MPTP group had lower levels compared to the DA4+MPTP group(P<0.05).TNF-α levels were higher in the MPTP group than in the control group(P<0.001).Liraglutide +MPTP,DA-JC1+MPTP,DA-JC4+MPTP and DA-CH5+MPTP group showed lower levels compared to the MPTP group(P<0.001).The DA-JC1+MPTP,DAJC4+MPTP group had lower levels compared to the Liraglutide+MPTP group(P<0.05),DA-CH5+MPTP group had lower levels compared to the DA4+MPTP group(P<0.01).Part three: The neuroprotective effects of the novel dual GLP-1 and GIP receptor agonist on MPTP-induced PD mouse model by increasing Synaptophysin and GDNFObjects: To observe the neuroprotective effects of novel dual GLP-1 and GIP receptor agonist on MPTP-induced Parkinson’s disease mouse model by immunohistochemical and Western blot analysis on Synaptophysin and GDNF,then to discuss its mechanism.Methods: Eight weeks old adult male C57BL/6 mice(15-20g)are chosen for this experiment.54 mice were divided into 6 groups,each group contained 9 mice,six groups as listed below as same treatment with Part one study :(A)control group;(B)MPTP group;(C)liraglutide group;(D)DA-JC1 group;(E)DA-JC4 group;(F)DA-CH5 group.On the 8th day after the MPTP and drugs treatment,mice were injected with lidocaine,then were perfused by saline,followed by 4% PFA.Brains were immediately removed and post-fixed in 4% PFA for overnight.(1)Immunohistochemical analysis on GDNF: Brain sections were incubated with primary antibody rabbit anti-mice GDNF(1:100);then were incubated with corresponding secondary antibody,finally the immunohistochemical analysis was done with DAB colour reaction.(2)Western blot analysis: Identical procedure as Part two study in this Western blot analysis except being incubated with the rabbit antibody against GDNF(1:1000;ABCAM),rabbit anti-SYN(1:10000;ABCAM).All data should be recorded for statistical analysis.Results:1.Immunohistochemistry: Levels of GDNF expression in the substantia nigra were different over all groups in a repeated measures one way ANOVA showed an overall difference between groups(F=71.369;P<0.001),followed by the LSD-t post-hoc tests.All the groups was significantly different from the control group(P<0.001).The liraglutide +MPTP(P<0.01)and DAJC1+MPTP(P<0.01),DA4+MPTP(P<0.001)and DA5+MPTP groups(P<0.001)showed higher levels compared to the MPTP group.The DA4+MPTP(P<0.01)and DA5+MPTP groups(P<0.5)showed higher levels compared to the liraglutide+MPTP group.The DA4+MPTP and DACH5+MPTP groups had higher levels compared to the DA-JC1+MPTP group(P<0.05).The DA4+MPTP had higher levels compared to the DACH5+MPTP group(P<0.05).2.Western blot: As a measure for the numbers and size of synapses,the level of the synaptophysin were evaluated.The MPTP(P<0.001),liraglutide +MPTP(P<0.01),DA-JC1+MPTP groups showed reduced levels synaptophysin compared to controls,DA-JC4+MPTP and DA-CH5+MPTP group showed higher levels compared to the MPTP group(P<0.01,P<0.001).The DA-CH5+MPTP group had higher levels compared to the Liraglutide+MPTP group(P<0.05),the DA-JC4+MPTP and DA-CH5+MPTP group was more protective compared to the DA-JC1+MPTP group(P<0.01).In GDNF level analysis.The MPTP,Liraglutide+MPTP,DA-JC1+MPTP,DAJC4+MPTP showed reduced GDNF levels compared to controls(P<0.001),The DA-CH5+MPTP also showed reduced GDNF levels compared to controls(P<0.01),Liraglutide+MPTP(P<0.01),DA-JC1+MPTP(P<0.05)and DACH5+MPTP group(P<0.01)showed higher levels compared to the MPTP group.The DA-JC4+MPTP had higher levels compared to the Liraglutide+MPTP group(P<0.01),the DA-JC4+MPTP(P<0.001)and DACH5+MPTP group(P<0.01)was more protective compared to the DA1+MPTP group.DA-CH4+MPTP group had higher levels compared to the DA4+MPTP group(P<0.05).Conclusions:1.It is confirmed that DA-JC4 and DA-CH5,as novel dual GLP-1 and GIP receptor agonist,are more neuroprotective to PD mouse model induced by MPTP than Liraglutide and DA-JC1 not only via improvement in behavioral evaluation from macrocosmic level,but via increase of Tyrosine Hydroxylase expression in the substantia nigra from microscopic level.These facts would provide evidences for further research on incretin and neurodegenerative disease,especially PD.2.It is proved that novel dual GLP-1 and GIP receptor agonist have two neuroprotection mechanism below: reducing chronic inflammatory responses via decreasing the levels of the pro-inflammatory cytokines including IL-1β,TNF-α,NF-κB in Western blot analysis,and going against the activation of microglia and astrocytes via down-regulation of IBA-1 expression of immunohistochemistry in the substantia nigra of PD model induced by MPTP.DA-JC4,DA-CH5 have better effects compared with Liraglutide and DA-JC1.3.It is demonstrated that synaptic activity is protected in PD model induced by MPTP after treatment through injecting novel dual GLP-1 and GIP receptor agonist via two aspects in our study: one is increasement of synaptophysin expression as a measure for the numbers and size of synapses,the other is enhancement of GDNF levels as an more effective way to protect synaptic activity compared with Liraglutide and DA-JC1.