The Effects Of Allicin On Neuropathological Changes In Alzheimer’s Disease Transgenic Mice

Objective:The main purpose of this study was to clear the effect of allicin on behavior,the pathogenesis and relevant mechanisms in Alzheimer’s disease mouse model.The implementation of this experiment is expected to allicin as a new target for prevention and treatment of AD,provide experimental and theoretical basis for effectively delay or inhibit Alzheimer’s disease progression.Materials and methods:In this study,the AD model mice（APP/PS1 transgenic mice）were used as a mouse model with double transfection of human APP695swe gene and human premature 1 mutant gene（presenilin,PS-1）.APP/PS1 transgenic mice were identified by PCR technology,then treated with allicin,and Morris water maze and mouse nest test were used to detect the learning and memory ability of APP/PS1transgenic mice.Immunohistochemical technique was used to detect the distribution of senile plaque in the brain tissue of APP/PS1 transgenic mice.The content of Aβ_1-40-40 and Aβ_1-42-42 in the brain of APP/PS1 transgenic mice was analyzed by ELISA technique.Western blot was used to detect APP lyase expression in the mice brain.The effects of allicin on the neuronal apoptosis in the brain of APP/PS1 transgenic mice were detected by Real-Time PCR or Western blot.At the same time,the effects of allicin on oxidative stress in the brain of APP transgenic mice were detected.In addition,we selected tau transgenic mice with high TauP301S mutation gene,and observed the effects of allicin on tau phosphorylation in tau transgenic mice brain by Western blot and immunofluorescence.Results:1.Allicin reduces the formation of senile plaques in APP/PS1 transgenic mice brain.A beta immunohistochemical staining showed that a large number of A beta positive neurons and positive plaques were found in the cortex and hippocampus of APP/PS1 transgenic mice.The plaque reactant is brown,precipitated outside the cell,unequal size,the shape is round or irregular shape,and the boundary is clearer.After administration of allicin to APP/PS1 transgenic mice,the number of plaques in the cortex and hippocampus decreased significantly,and the coloring of plaques was shallower and the volume was reduced.The number and relative area of senile plaques from the same A beta sections in immunohistochemical staining of each mouse were counted,and the Image-Pro Plus 6 software was used for statistical analysis,the results showed that the number of senile plaques in the cortex and hippocampus of the allicin treated group was significantly lower than that of the normal control group,compared with the control group,the decrease was 40%（P<0.05）and 29%（P<0.05）,the experimental results were statistically significant.2.The effect of allicin on the content of A beta in APP/PS1transgenic mice brain.ELISA results showed that the content of A beta 1-42 in APP/PS1transgenic mice brain was significantly higher than that in the wild type mice.（4.21±0.45）pg/ml of the control group increased to（18.30±1.60）pg/ml in AD group.The difference was statistically significant compared with the control group（P<0.05）.After treatment of APP/PS1 transgenic mice with allicin,the content of A beta 1-42 in the treated group mice brain decreased to（10.22±1.08）pg/ml,compared with the AD group,the difference was statistically significant（P<0.05）.The results of A beta 1-40 ELISA detection showed that the content of A beta 1-40 in APP/PS1 transgenic mice brain was significantly higher than that in the wild type mice.（3.33±0.66）pg/ml of the control group increased to（12.98±1.32）pg/ml in group AD,the results was significant difference.After treatment with allicin in APP/PS1 transgenic mice,the content of A beta1-40 in the treated group mice brain decreased,which was（8.56±0.98）pg/ml,and the difference was statistically significant compared with AD group.3.The effect of allicin on APP metabolism in APP/PS1 transgenic mice brain.The expression of APP secretase（including BACE1,NCT,PS1,PS2）in the three groups mice brain was detected by Western blot.The results showed that the APP lyase BACE1,NCT,PS1 and PS2expressiones in AD group mice brain was significantly higher than that in the control group（.P<0.05）,the secretase expressiones in allicin treatment group was significantly lower than that in the AD group（.P<0.05）.4.The effects of allicin on the behavior of APP/PS1 transgenic mice.In the water maze test,statistical results show that the average incubation period of allicin treated group mice in the third days and the fourth days was（41.05±2.80）s and（35.22±3.21）s.The average search route was（1.58±0.14）m and（1.25±0.12）m.It was significantly lower than the model group,but it was higher than the control group.This showed that the spatial memory ability of the allicin treatment group mice was improved,but it had not yet reached the level of the wild type mice（P<0.05）.The space orientation ability test was carried out at the next fifth days.In the space exploration experiment,mice and wild type mice were treated with allicin is mainly concentrated in the trajectory of the corresponding platform position,and the model group of APP/PS1 mice along the wall and away from the corresponding position of moving platform.The number of allicin treated mice was significantly higher than that of the model group in 1 min,it indicated that the spatial memory ability of allicin treated mice was improved.In the nest construction experiment,after seven days of observation,it was found that the allicin treated group had better ability to bite and slice the paper at the sixth day than in the AD group.5.The effect of allicin on tau protein phosphorylation in tau transgenic mice brain with Alzheimer’s disease.Tau protein phosphorylation levels in Ser202,Ser396,Ser400/Thr403/Ser404 and Thr181 sites in the three groups mice were detected by Western blot and immunofluorescence.It was found that at Ser202,Ser396,Ser400/Thr403/Ser404 and Thr181 sites,tau protein phosphorylation level in AD group mice brain was significantly higher than that in the control group.Tau protein phosphorylation level at Ser202,Ser396 and Ser400/Thr403/Ser404 sites in allicin treated mice brain was significantly lower than that in the AD group（P<0.05）,but there was no significant difference between at the Thr181 site compared with the AD group.6.The effects of allicin on apoptosis related proteins in neurons of APP/PS1 mice brain.Real Time PCR and Western blot results showed that compared with the control group,Bax expression in the brain of AD group increased and Bcl-2 expression decreased significantly（P<0.05）.Compared with the AD group,Bax expression in the allicin intervention group was significantly decreased,and Bcl-2 expression was increased（P<0.05）.The Western blot method was used to detect JNK and its downstream molecules c-Jun and Caspase-3 expression.The results showed that compared with the control group,the expression level of p-JNK1/2 in AD group increased significantly,while the expression level of p-JNK1/2 in allicin treated group was significantly lower than that in AD group（P<0.05）.c-Jun is an important target in the downstream of JNK,and its activation is related to many cell activities,including Caspase dependent apoptosis.The results showed that the expression level of p-c-Jun and Caspase-3 increased in the AD group,and the expression of c-Jun and Caspase-3 in allicin treated group was significantly lower than that in the AD group（P<0.05）.In addition,Western blot method was used to detect the expression of the cholinergic neuron marker CHAT in the three groups of mice brain.The results showed that CHAT expression in the allicin treated group was significantly higher than that in the AD group（P<0.05）,it indicated that allicin had a protective effect on cholinergic neurons.7.The effects of allicin on oxidative stress in the APP transgenic mice brain.The results showed that the SOD activity in APP/PS1 transgenic mice brain decreased significantly,and the MDA content increased significantly,the difference was statistically significant（P<0.05）.After the treatment of APP/PS1 transgenic mice with allicin,the SOD activity in the mice brain was significantly higher than that in the AD group（P<0.05）.The MDA content was significantly lower than that in the AD group（P<0.05）.Conclusion:1.Allicin could reduce the number and volume.of A beta senile plaques in the cerebral cortex and hippocampus of APP/PS1 transgenic mice.2.Allicin could reduce A beta deposition by reducingβ-andγ-secretase expression in the brain of APP/PS1transgenic mice.3.Allicin could significantly improve the spatial learning and memory ability of APP/PS1 transgenic mice.4.Allicin treatment could reduce the expression of protein kinase related to the tau protein phosphorylation in the brain of tau transgenic mice.5.Allicin could significantly reduce the phosphorylation level of tau protein in the brain of tau transgenic mice at Ser202,Ser396 and Ser400/Thr403/Ser404 sites.6.Allicin could reduce the expression of Bax,JNK1/2,c-Jun and Caspase-3 in the APP/PS1transgenic micebrain,increase the expression of Bcl-2,and ultimately play a protective role in the cholinergic neurons in mice brain.7.Allicin reduced the content of MDA and increased the activity of SOD,improved the oxidative stress reaction in the brain of APP/PS1 transgenic mice.