| Objective: To observe the effects of GLP-1/GIP/Gcg receptor(Triagonist)on the cognitive behaviors and brain pathological hallmarks in 3xTg Alzheimer's disease mice and explore its possible signal pathway mediating the neuroprotection of the Triagonist.Methods: 9 months old APPSwe/PS1M146V/tau P301 L 3xTg-AD female mice and wild-type C57BL/6J mice were divided into four groups: C57+NS,C57+Triagonist,3xTg-AD+NS and 3xTg-AD+Triagonist.In triagonist group,the drug triagonist dissolved with normal saline was injected intraperitoneally at a dose of 10 nmol/kg per day.In control group,the same volume of 0.9% Na Cl was given.Behavioral tests were performed 28 days after continuous administration.(1)Open filed test Mice were individually placed in the centre of open filed,and allowed to move spontaneously for 5 min.Recorded the trace and total distance during 5 min,and counted the percentage of time spend in the centre area.(2)Y maze test Mice were placed in the centre of Y maze and allowed to move freely through the maze for 8 min.The total arm entries and the order of arm entry were recorded,and the rate of correct spontaneous alternation of mice was calculated.(3)Elevated plus-maze task Mice were individually placed in the centre of the maze facing closed arm,and allowed to explore freely for 10 min.The total arm entries,open arm entries and closed arm entries were recorded,and the ratio of open arm entries to closed arm entries was counted.(4)Classical Morris water maze and reversal water maze test Mice were placed in the maze for hidden platform test,probe trials and visible platform test.The escape latency in 1 min,the time in the target quadrant and the time to find visible platform were recorded.After classical Morris water maze test,the platform was moved to reversal quadrant,and mice were placed in the water for similar hidden platform test and probe trails as mentioned above.(5)Radical arm-maze task Before the maze task,the mice were kept on restricted diet for 24 hour.Then,the mice were placed in the centre of the maze to explore freely for 10 min,including adaption and test phase.Recorded the non-baited arm entries(reference memory errors)and batied-arm re-entries(working memory errors)in the test phase.(6)Tail suspension test Mice were suspended by the tail from a white-painted box,the camera should be as close as possible in order to obtain the highest possible resolution of the animals.Recorded the immobility time in 6 min.(7)Forced swimming test Mice were individually placed into cylindrical recipients containing 20 cm of water.The mice were left to swim for 6 min before being removed.Recorded the immobility time in the last 4 min.(8)Immunohistochemistry After the behavioral experiments,the mice were anesthetized with chloral hydrate by intraperitoneal injection and then fixed with paraformaldehyde.Put the 30? m of frozen section in PBS,blocked with BSA,followed by adding the primary antibody,second antibody and DAPI.The A? plaques,phosphorylated tau protein,synaptophysin and PSD95 were observed by confocal laser scanning microscopy.(9)Western blot After the behavior tests,mice were anesthetized with chloral hydrate,cut the head,took the brain,isolated the hippocampal tissue and extracted the protein,and then carried out the SDS-PAGE electrophoresis.The protein was transferred to the PVDF membrane after electrophoresis.Added the primary antibody,second antibody and ECL after 5% BSA treatment.The relative gray values of target protein(SYP,PSD95,PKAc,PKAR2,p-CREB,t-CREB)were analysied by Alpha SA software.(10)ELISA The extraction of the protein were added to the c AMP monoclonal antibody coated microplates to determine the c AMP level in accordance with the method of ELISA kit.Results:(1)Open filed test There was no statistical difference in total distance and time spend in the centre area among four groups(P>0.05).(2)Y maze test The percentage of correct spontaneous alternation in 3xTg-AD+NS group was lower than C57+NS(P<0.001),and in triagonist treated 3xTg-AD mice,the percentage significantly increased(P<0.01).The total arm entries has no significant difference among four groups(P>0.05).(3)Classical Morris water maze test In the 1-5 days of hidden platform test,the mean escape latency of mice in four groups was decreased with the increase of training day,From the second day,the escape latency in 3xTg-AD+NS group increased significantly(P<0.01,P<0.001)compared to that in the C57+NS group,From the third day,the escape latency of mice in 3xTg-AD+Triagonist group was significantly less than the 3xTg-AD+NS mice(P<0.01).In the probe trails,the percentage of time in target quadrant in 3xTg-AD+NS group was significantly lower than the group of C57+NS(P<0.001),while the percentage was significantly increased in 3xTg-AD+Triagonist group compared with 3xTg-AD+NS group(P<0.001).There was no significantly difference in the escape latency among the four groups in the visual platform test(P>0.05).(4)Reversal morris water maze test In the reversal morris water maze,the escape latency of mice in the 3xTg-AD + NS group was significantly higher than that in the C57+NS group from the first day(the seventh day in total water maze test)(P<0.01,P<0.001),and the Triagonist decreased the escape latency in 3xTg-AD mice(P<0.05,P<0.001).In the probe trails,compared with the C57 + NS group,3xTg-AD + NS group has a significant decrease in the percentage of time in target quadrant(P<0.001).Compared with the 3xTg-AD+NS group,3xTg-AD+Triagonist group had a significant increase(P<0.001).(5)Radical arm-maze task In the test phase of radical arm-maze,the average working memory errors in 3xTg-AD+NS group significantly increased during 10 days(P<0.001)compared with C57+NS group,which was significantly reduced after treatment with Triagonist in 3xTg-AD+Triagonist group(P<0.001).In addition,compared with the C57+NS group,the 3xTg-AD+NS group showed a significant increase in reference memory errors(P<0.01),and the reference memory error was also decreased by Triagonist in the 3xTg-AD+Triagonist group(P<0.01).(6)Tail suspension test The percentage of immobility time in the 3xTg-AD+NS group was higher than that in the C57+NS group(P<0.01),but Triagonist had no significant effect on the immobility time in the 3xTg-AD mice(P>0.05).(7)Forced swimming test The percentage of immobility time in the 3xTg-AD+NS group were significantly increased compared with the C57+NS group(P<0.001),and the Triagonist also did not affect the immobility time of 3xTg-AD mice(P>0.05).(8)Immunohistochemistry The positive staining of A? plaques and phosphorylated tau in the hippocampus of 3xTg-AD+NS mice had a significant increase compared with that in the C57+NS(P<0.001),which was prevented by the treatment with Triagonist in 3xTg-AD + Triagonist group(P<0.001).(9)Western blot Compared with the C57+NS group,the levels of SYP,PSD95,PKAc PKAR2 and p-CREB in 3xTg-AD+NS mice hippocampus were significantly decreased(P<0.05,P<0.001),while the expression levels of these signal molecules were restored to some extent after Triagonist treatment(P<0.05,P<0.01,P<0.001).(10)ELISA The expression level of c AMP in hippocampus of 3xTg-AD+NS mice was significantly lower than that in C57+NS mice(P<0.01),and the Triagonist treatment partly restored the expression of c AMP in 3xTg-AD mice(P<0.01).Conclusion:(1)The spatial working memory,reference memory,re-learning ability and cognitive flexibility were damaged in 9 month age of 3xTg-AD female mice,and chronic administration of Triagonist effectively improved the cognitive behaviors.(2)The 9 month age of 3xTg-AD female mice did not show any disorder in locomotor and exploratory performance,also without obvious anxiogenic-like behavior,but presented a depressive-like behavior,which could not be improved by the treatment with Triagonist.(3)Chronic administration of Triagonist alleviated the pathological damages including A? plaques and phosphorylated tau in the hippocampus of 3xTg-AD female mice,and increased the expression of synaptic transmission related proteins such as SYP and PSD95.(4)The neuroprotection of Triagonist to 3xTg-AD female mice may be mediated by up-regulation of hippocampal c AMP/PKA/p-CREB signal pathway. |
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