| Objective:Brain derived neurotrophic factor(BDNF)is one of the important members of the neurotrophic factor family.Its function of promoting the growth and survival of neurons is crucial to the formation of learning and memory.It changes in many neurodegenerative diseases and mental diseases.The abnormality of PI3K/Akt-mTOR signaling pathway is related to the pathological changes of many human neurodegenerative diseases,especially Alzheimer’s disease(AD).The purpose of this study is to explore the relationship between the plasma expression level of BDNF protein and cognitive function through clinical research,and further study whether the traditional Chinese medicine compound GAPT can regulate the expression of BDNF through PI3K/Akt-mTOR pathway to play a role in AD through animal experiments.Methods:1.The clinical study included 41 subjects in the outpatient department of encephalopathy,Dongzhimen Hospital,Beijing University of traditional Chinese medicine,including 16 males and 25 females.They were divided into normal control group(NC,n=16),amnestic mild cognitive impairment group(aMCI,n=7)and ad group(n=18).General data were collected and neuropsychological evaluation was performed.The expression level of plasma BDNF protein was detected by enzyme linked immunosorbent assay(ELISA).The correlation between the expression level of plasma BDNF protein and cognitive function in each group was studied by Pearson linear correlation analysis.The efficacy of plasma BDNF in identifying subjects with different cognitive functions was analyzed by receiver operating characteristic(ROC).2.Three month old APPswe/PS1dE9 double transgenic mice were randomly divided into 5 groups,including model group,donepezil group,high,medium and low dose groups of jinsishui,with 19 mice in each group.Nineteen c57/bl6j mice of the same age and background served as the normal control group.Donepezil group was given donepezil hydrochloride(0.92mg/kg/d)by gavage;The high-dose group of GAPT group was given GAPT(20g/kg/d)by gavage;GAPT(10g/kg/d)was administered by gavage to the middle dose group of GAPT;The low-dose group of GAPT was given GAPT(5g/kg/d)by gavage;The normal control group and the model group were given equal volume 0.5%CMC by gavage once a day for 8 months.Y maze and Morris Water Maze experiments were used to study the effect of traditional Chinese medicine compound GAPT on cognitive behavior of AD transgenic mice.Transmission electron microscope was used to observe the effect of traditional Chinese medicine compound GAPT on the structure of neurons,synapses and astrocytes in hippocampal CA1 region of AD transgenic mice.Western blot was used to detect the expression of PI3K,Akt,p-Akt,mTOR,p-mTOR,BDNF and p-tau proteins in mouse hippocampus.The mRNA expression of PI3K,Akt and mTOR in mouse hippocampus was detected by RT-qPCR.Results:1.Relationship between plasma BDNF and cognitive function in human samples(1)Comparison of plasma BDNF expression level between groups:the plasma BDNF protein level in AD group was significantly lower than that in NC group and aMCI group,and gradually decreased among NC,aMCI and ad groups,and the difference between any two groups was statistically significant(P<0.05);(2)Correlation between BDNF level in plasma and cognitive function of subjects in each group:after adjusting for age factors,there was a significant positive correlation between BDNF in plasma protein level and comprehensive cognition(MMSE score)in the cognitive impairment group(aMCI+AD)(β=0.491,P<0.05),which was significantly negatively correlated with dementia severity(CDR)and ADL score(β=-0.595,P<0.01;β=-0.502,P<0.01);there was a significant positive correlation between BDNF levels in plasma of all subjects(NC+aMCI+AD)and MMSE(β=0.538,P<0.01)、MoCA(β=0.539,P<0.01)、DSR(β=0.557,P<0.01),which was significantly negatively correlated with CDR(β=-0.605,P<0.01)and ADL(β=-0.473,P<0.01).(3)Diagnostic performance of BDNF in plasma:BDNF in plasma has medium accuracy in differentiating cognitive impairment(AD+aMCI)from NC(AUC=0.823),with sensitivity of 0.950 and specificity of 0.563;The accuracy of BDNF in plasma in differentiating AD from NC was the best(AUC=0.861),the sensitivity was 0.944,and the specificity was 0.625.The accuracy of BDNF in plasma in differentiating aMCI from AD was low(AUC=0.762).However,the accuracy of BDNF in plasma in differentiating aMCI from NC was not statistically significant(P=0.109).2.Effect of traditional Chinese medicine compound GAPT on the behavior of APPswe/PS1dE9 miceThe traditional Chinese medicine compound GAPT improved the short-term working memory of mice to a certain extent,that is,in the Y maze spontaneous alternation experiment,the spontaneous alternation rate of mice in the low-dose GAPT group was significantly higher than that in the model group(P<0.05,P=0.029),and that of the middle-dose and high-dose GAPT groups were higher than that in the model group,but the difference was not statistically significant.The learning and memory abilities of mice were significantly improved by the traditional Chinese medicine compound GAPT:the average escape latency of donepezil group(P<0.01)and GAPT low-dose group(P<0.05)were significantly shorter than that of the model group on the first day of the test period of Morris water maze navigation experiment(the fourth day of the experiment).On the second day of the test period(the fifth day of the experiment),the average escape latency of all treatment groups were significantly shorter than that of the model group(P<0.01 or P<0.05).On the first day of the test period(the fourth day of the experiment),the average swimming distance of the low-dose GAPT was significantly lower than that of the model group(P<0.05).On the second day of the test period(the fifth day of the experiment),the average swimming distance of donepezil group,GAPT high-dose group and GAPT low-dose group were significantly shorter than that of the model group(P<0.01 or P<0.05).There was no significant improvement in the spatial exploration ability of mice by the traditional Chinese medicine compound GAPT:in the spatial exploration experiment,there was no significant statistical difference in the number of crossing the platform between each group(P>0.05),but the number of crossing the platform between the normal control group and all treatment groups were higher than that of the model group.There was no significant statistical difference in the residence time of target quadrant between groups(P>0.05).3.Effect of traditional Chinese medicine compound GAPT on the structure of hippocampal neurons and astrocytes in the CA1 region of APPswe/PS1dE9 miceThe results of transmission electron microscope showed that the neurons in the CA1 area of hippocampus in the model group were pyknosis,obvious degeneration and necrosis,cell shrinkage,irregular morphology,nuclear pyknosis and nuclear membrane rupture;Morphological changes of organelles in cytoplasm,more lipofuscin,obvious swelling of mitochondria,breakage,dissolution and deletion of mitochondrial cristae in varying degrees,swelling and degranulation of endoplasmic reticulum;The number of synapses is small,the ultrastructure of synapses is fuzzy,the synaptic vesicles are significantly reduced,the postsynaptic density is thin,the mitochondria in synapses are swollen,and the mitochondrial cristae are broken;Vacuoles appeared in the cytoplasm of hippocampal astrocytes,and the lamellar structure of endoplasmic reticulum disappeared and degranulated.Compared with the model group,the ultrastructures of hippocampal neurons,synapses and astrocytes in the GAPT and donepezil groups were improved to varying degrees:in the traditional Chinese medicine group,the ultrastructure of the GAPT medium-dose group showed the best improvement in terms of neuron structure,synapse number and astrocyte structure,followed by the GAPT high-dose group and the GAPT low-dose group.4.Effect of traditional Chinese medicine compound GAPT on the expression of p-tau protein,BDNF protein and PI3K/Akt-mTOR pathway related proteins in the hippocampus of APPswe/PS1dE9 miceWestern blot detection of hippocampal p-tau protein showed that compared with the normal control group,the expression of p-tau protein in the model group was significantly increased(P<0.01);Compared with the model group,the expression of p-tau protein in donepezil group,GAPT medium-dose group and GAPT high-dose group decreased significantly(P<0.01,P<0.05,P<0.05).Western blot detection of BDNF protein in hippocarnpus showed that the expression of BDNF protein in the model group was significantly lower than that in the normal control group(P<0.01);Compared with the model group,the expression of BDNF protein in donepezil group,GAPT middle-dose group and GAPT high-dose group was significantly increased(P<0.01,P<0.01,P<0.05).Western blot detection of hippocampal PI3K protein showed that the expression of PI3K protein in the model group was significantly lower than that in the normal control group(P<0.01);Compared with the model group,the PI3K protein expression was significantly increased in the high-dose GAPT group(P<0.05);Western blot detection of Akt and p-Akt protein in hippocampus showed that the expression of Akt protein in the model group was significantly higher than that in the normal control group(P<0.05);The expression of p-Akt protein in the model group was significantly lower than that in the normal control group(P<0.05);The expression of p-Akt protein in GAPT medium-dose group was significantly higher than that in model group(P<0.05).The ratio of p-Akt/Akt in the model group was significantly lower than that in the normal control group(P<0.05);The ratio of p-Akt/Akt in the GAPT high-dose group was significantly higher than that in the model group(P<0.05).Western blot detection of mTOR and p-mTOR protein in hippocampus showed that the expression of mTOR protein in the model group was significantly higher than that in the normal control group(P<0.01),and the expression of mTOR protein in donepezil group,GAPT medium-dose group and GAPT high dose group was significantly lower than that in the model group(P<0.01,P<0.05,P<0.05);The expression of p-mTOR protein in the model group and donepezil group was significantly lower than that in the normal control group(P<0.01,P<0.01);The p-mTOR/m FOR ratio of the model group,donepezil group and high-dose group of GAPT was significantly lower than that of the normal control group(P<0.05,P<0.05,P<0.05),and the p-mTOR/mTOR ratio of donepezil group,low-dose group of GAPT,medium-dose group of GAPT and high-dose group of GAPT was significantly higher than that of the model group(P<0.05,P<0.05,P<0.05,P<0.05).5.Effect of traditional Chinese medicine compound GAPT on mRNA expression of PI3K/Akt-mTOR pathway in hippocampus of APPswe/PS1dE9 mice:The expression of PI3K mRNA in the model group was lower than that in the normal control group,with no statistical difference(P>0.05);Compared with the model group,the expression of PI3K mRNA was significantly increased in the GAPT low-dose group(P<0.05).The expression of Akt mRNA in the model group was higher than that in the normal control group,with no statistical difference(P>0.05);Compared with the model group,the expression of Akt mRNA in the middle-dose group and high-dose group of GAPT were significantly lower(P<0.05,P<0.05).The expression of mTOR mRNA in the model group was higher than that in the normal control group,with no statistical difference(P>0.05);Compared with the model group,the expression of mTOR mRNA in the low,medium and high dose groups of GAPT were significantly lower(P<0.05,P<0.05,P<0.05).Conclusion:1.BDNF levels in plasma in patients with aMCI and AD were significantly lower than those in NC.The decrease of cognitive function is related to the decrease of BDNF level in plasma.The level of plasma BDNF has certain predictive value for AD,aMCI and cognitive impairment,and the accuracy of plasma BDNF in diagnosing AD is high.2.The traditional Chinese medicine compound GAPT can significantly improve the learning and memory ability of APP/PS1 double transgenic AD model mice;It can obviously repair the ultrastructural damage of hippocampal neurons and astrocytes in mice;Its neuroprotective mechanism may be playing a role by regulating PI3K/Ak-mTOR pathway and BDNF expression. |
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