| Objective:In this study,APPswe/PS1dE9(APP/PS1)transgenic mice were used as the research objects to explore the effect of Breviscapine(Bre)on the behavior and pathology of APP/PS1 transgenic mice,and to explore whether this is regulated by multiple complex signaling pathways mediated by the interaction of multiple genes,so as to provide molecular targets for Bre in the treatment of Alzheimer's disease(AD),and to optimize the effect of Bre on AD by genetic modification in this study.Methods:Thirty male APP/PS1 transgenic mice were randomly divided into two groups:APP/PS1 group(Tg,0.01 ml/g/d,0.9%sodium chloride solution intraperitoneal injection),Bre treatment group(Tg+Bre,0.01 ml/g/d,50 mg/kg/d Bre intraperitoneal injection),and fifteen wild-type mice of the same sex and the same age that were used as a normal control group(WT,0.01 ml/g/d,0.9%sodium chloride solution intraperitoneal injection).Mice in the three groups were given the drug intraperitoneally from the age of 6 months,once a day for 3 months.Morris water maze test,Y-maze test and open field test were used to detect the cognitive function of mice.The mice were anesthetized and sampled.Congo red staining was used to detect the A? plaques in hippocampus and cortex of mice,and WB was used to detect the expression levels of BACE1,RAGE and IDE.Niss1 staining was used to detect the survival of hippocampal neurons in mice,and WB was used to detect the expression levels of PSD95 and SYP.Changes of differentially expressed genes were detected by microarray.GO database and KEGG database were used to analyze the correlation between differentially expressed genes.To verify the microarray results,the key genes related to the p38 MAPK signal pathway were validated at mRNA and protein levels using RT-qPCR and WB,respectively,and the efficacy and mechanism of Bre on AD were comprehensively evaluated.Results:Morris water maze test revealed that in the place navigation test,the inner-group escape latency day effect was significant in all three groups when compaed with WT ones,respectively(F=13.518;P<0.05),there was also a marked difference in escape latency between any two groups among the three(F=12.891;P<0.05).At days 3-5,the escape latency of Tg group was substantially longer than that of WT group(P<0.05).Dramatically,the escape latency in mice of Tg+Bre group was significantly shorter than that of Tg one(P<0.05).In the spatial probe test,compared with WT group,the frequency of Tg group mice passing through the target platform and the percentage of time in the target quadrant were significantly reduced(P<0.05).However,compared with the Tg group,the frequency and percentage of time in the target quadrant of the Tg+Bre group were significantly increased(P<0.05)In Y-maze test,compared with the WT group,the percentage of times and percentage of time that the Tg group entered the new arm were significantly reduced,and the total number of arm approaches was also significantly less than that of the WT group(P<0.05).However,compared with the Tg group,the percentage of times and percentage of time that the Tg+Bre group entered the new arm were significantly increased,and the total times of arm entry were also significantly higher(P<0.05).In open field test,compared with WT group,Tg group significantly reduced the peripheral time and total detection distance,while significantly increased the time in the center(P<0.05).Compared with Tg group,the peripheral time and total detection distance of Tg+Bre group significantly increased,while the time in the center significantly decreased(P<0.05).Congo red staining showed that the number and area percentage of A? plaques in the hippocampus and cortex of the Tg group were significantly higher than those in the WT group(P<0.05).However,compared with the Tg group,the number and area percentage of A? plaques in the hippocampus and cortical areas of the Tg+Bre group were significantly decreased(P<0.05).WB analysis showed that compared with WT group,the expression levels of BACE1 and RAGE protein in Tg group increased significantly,and IDE protein expression levels decreased significantly(P<0.05).Compared with Tg group,The expression levels of BACE1 and RAGE proteins in the Tg+Bre group were significantly reduced,and the expression level of IDE protein was significantly increased(P<0.05).Nissl staining showed that compared with WT group,the number of surviving neurons in the hippocampal DG and CA3 regions of Tg group was significantly reduced(P<0.05).However,compared with Tg group,the number of surviving neurons in DG and CA3 regions of the hippocampus in Tg+Bre group increased significantly(P<0.05).WB analysis results showed that compared with WT group,PSD95 and SYP protein expression levels of Tg group were significantly reduced(P<0.05).Compared with Tg group,PSD95 and SYP protein expression levels of Tg+Bre group were significantly increased(P<0.05).Microarray results showed that compared with the mice in the Tg group,there were 17,327 up-regulated genes and 14,692 down-regulated genes in the brain of the Tg+Bre group.The results of GO analysis showed that biological processes(BP)involved 940 types,including neural system processes,synaptic assembly and G protein coupled receptor signal transduction pathways,etc.Cell components(CC)involved 99 types,including NMDA selective glutamate receptor complex,axon initiation portion and projection neurons,etc.Molecular function(MF)involves 155 types,including nerve growth factor binding,platelet-derived growth factor receptor binding and MAP kinase activity,etc.KEGG analysis results show that the enriched signaling pathways involve 22 types,including MAPK signaling pathway,PI3K-AKT signaling pathway and neuroactive ligand-receptor interaction signaling pathways,etc.The above results indicated that the therapeutic effect of Bre on APP/PS1 transgenic mice may be mediated by the interaction of multiple genes involved in the regulation of multiple signaling pathways.On the basis of the microarray results,we selected three genes,NT4,p38 MAPK and p53 that have been widely accecpted in playing an important role in the treatment of AD to verify the mRNA and protein levels.RT-qPCR showed that compared with the Tg group,the expression level of NT4 mRNA in the Tg+Bre group was significantly increased,and the expression levels of p38 MAPK and p53 mRNA were significantly decreased(P<0.05).WB showed that compared with Tg group,the expression level of NT4 protein in the Tg+Bre group was significantly increased,the total protein expression level of p38 MAPK was not different,and the expression levels of p-p38 MAPK and p53 protein were both significantly decreased(P<0.05).The results are consistent with the microarray results.Conclusion:In this study,Bre improved the ability of APP/PS1 transgenic mice to learn,remember and explore new things in the outside world.At the same time,it alleviated the deposition of A? plaque and the damage of hippocampal neurons.The action mechanism of Bre may be related to multiple signaling pathways mediated by the interaction of multiple genes,including MAPK signaling pathway,PI3K-AKT signaling pathway and neuroactive ligand-receptor interaction signaling pathways,etc.The results of this study explored the therapeutic effect of Bre on AD from the perspective of molecular mechanism,which will provide molecular targets for clinical development of Bre in the treatment of AD,which has important scientific significance and clinical application value. |
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