| ObjectiveIn this study,abnormal expression of selenium-related signaling pathways were screened out in brain tissues by Gene Set Enrichment Analysis(GSEA)based on GEO data set GSE84422 human brain tissue gene expression profile and Se-related gene database in Comparative Toxicology Genomics Database(CTD)of Alzheimer’s disease compared to normal people.The results showed that selenium related pathway BDNF/Trk B signaling pathway may be related to AD.Furthermore,dietary selenium was used to intervene APP/PS1/tau 3×Tg-AD model mice aged 25-26 weeks.Behavioral cognition,electron microscopy,reverse transcription PCR(RT-PCR),Western blot and other methods were used.From the aspects of RNA,protein level and behavioral cognition,whether selenium treatment can effectively improve the injury of learning and memory ability caused by AD,whether it can relieve the anxiety of mice,and whether it can reverse the damage of synapses in target tissues.Furthermore,the expression levels of BDNF,CREB,TRKB,P-CREB and other molecules in mouse brain tissue were detected to explore whether the improvement of cognitive function in AD patients with selenium is related to the regulation of BDNF/Trk B signaling pathway.Methods1.Screening of AD-Se differential pathways: Using AD brain tissue microarrays and Se-associated gene bank to screen AD-Se associated genes,GSEA was used to screening differential expression genes and pathways of AD-Se.2.Groups of experimental animals: Female APP/PS1/tau 3×Tg-AD mice and wild-type(WT)mice with the same heritage background were divided into four groups with or without selenium supplementation: 3×Tg-AD with selenium supplement diet(0.5ppm Se/Kg3×Tg+H),3×Tg-AD with standard diet(0.15 ppm Se/Kg 3×Tg+C),wild-type with selenium supplement diet(0.5ppm Se/Kg WT+H)and wild-type with standard diet(0.15 ppm Se/Kg WT+C).Each groups had 7 mice.The changes of the relevant indicators would be observed after six months of dietary intervention.3.Behavioral tests: Novel object recognition(NOR),Y maze,shuttle box,Morris water maze(MWM),Nesting,Open field test(OFT),and Tail suspension test(TST)were conducted by the fifth month of dietary intervention.4.Electron Microscopy Observation: Ultrastructure of neurons in hippocampal tissue was observed by Transmission electron microscope.5.Detection of selenium and GSH-Px activity: The content of selenium and GSH-Px activity in whole blood and brain tissue was detected by ICP-MS and detection kit respectively.6.RT-PCR and Western-blot:The expression levels of Aβ and tau proteins and gene and protein levels of key molecules BDNF,Trk B and p CREB/CREB involved in BDNF/Trk B pathway were detected.Results1.Screening of AD-Se differential pathways: GSEA analysis showed that two gene sets were up-regulated in AD expression(FDR<25%,P<0.05).BDNF/Trk B signaling pathway was one of them,the enrichment score was 0.59.2.Behavioral tests: The results of NOR test showed that the time for new objects exploration activity in response to novelty increased in the 3×Tg+H compared to the 3×Tg+C group(P=0.019).The results of Y maze experiment showed that the changes of novel abnormal arm time proportion had statistically significant difference among four groups with the increase of training days(P<0.001);The results of the seventh novel abnormal arm time proportion showed that the 3×Tg+C had more time proportion than the WT+C and there was statistically significant difference in time proportion between 3×Tg+H and 3×Tg+C(P<0.001).In the shuttle box experiment,the ability to avoid noxious stimuli of 3×Tg-AD mice was weaker than the wild mice(P=0.019);there was statistically significant difference in ability to avoid stimulation between 3×Tg+H and 3×Tg+C(P=0.002).The results of Morris water maze showed that the time of the escape latent time was reduced in all groups during the acquired training.The fifth day analysis results showed that the escape latency period of the 3×Tg+H group was significantly shorter compared with the 3×Tg+C group mice(P<0.05).During exploration training,the platform passage increased significantly in the 3×Tg+H compared to the 3×Tg+C(P<0.01).In the nesting experiment,the 24-hour nesting scoring results showed that statistical significance of WT+C and WT+H groups was found(P=0.025),but no statistical significance was found among the 3×Tg+C and 3×Tg+H groups(P>0.05).In OFT experiment,the analysis of the activity time in the central area showed that the difference between 3×Tg+C group and 3×Tg+H group was statistical significant(P<0.05).The results of the TST showed that none of the four groups had depressive tendencies(P>0.05).3.Electron Microscopy Observation: The 3×Tg+H group neuronal nuclei were more regular,relatively less heterochromatin compared to the 3×Tg+C,but more than wild-type mice,less ribosome attachment was visible in the endoplasmic reticulum,a large number of glial cells repaired nerve cells.4.Detection of selenium and GSH-Px activity: The results of whole blood selenium level showed that the whole blood selenium level in WT group and 3×Tg+H group were higher than that in 3×Tg+C,but the difference was not significant(P>0.05).Brain selenium level in WT group and 3×Tg+H group were significantly higher than that of 3×Tg+C(P<0.05);Pairwise comparisons found that it has a significant difference in selenium levels in brain tissues between 3×Tg+H group and 3×Tg+C group(P=0.009).The whole blood GSH-Px activity showed no significant difference in the four groups(P>0.05),but the difference of brain GSH-Px viability was significant(P<0.05),Pairwise comparison found,GSH-Px activity was higher in WT+C and 3×Tg+H than in 3×Tg+C,with a statistically significant difference(P=0.001 and 0.027).5.RT-PCR: No significant differences for p-Tau and BDNF m RNA were among the four groups was observed(P>0.05),but there were significant differences for Aβ,TRKB and CREB m RNA(P<0.05).Further pairwise comparison: Aβ and CREB m RNA in brain in WT+H group were not significantly different compared to WT+C group,while TRKB m RNA expression was significantly increased(P=0.047).Compared with the 3×Tg+C,the m RNA level of Aβ in the brain in 3×Tg+H group was lower and TRKB and CREB m RNA were significantly higher(P<0.05).6.Western-blot:There was no significant difference in the expression of BDNF protein in the brain among the four groups(P=0.776),but there was statistically significant differences for the expression of Aβ,p-Tau,TRKB,CREB and p-CREB(P<0.05).Further comparison found that compared with WT+C,the expression of Aβ in brain decreased in WT+H group(P<0.001),while for p-Tau,TRKB,CREB and p-CREB there were no significant differences(P>0.05);the expression level of p-Tau in brain was increased in3×Tg+C group(P<0.05),TRKB and CREB were decreased(P<0.05),and there were no significant differences for Aβ and p-CREB(P>0.05).The expression trend of related proteins in 3×Tg+C and 3×Tg+H group were consistent with the results of RT-PCR.Conclusion1.Selenium supplementation might improve cognitive dysfunction in mice model of Alzheimer’s disease.2.The effect of selenium supplementation on cognitive function of AD model mice may be related to the BDNF/Trk B signaling pathway.It has been found that the expression levels of key molecules TRKB,CREB and p-CREB proteins in BDNF/Trk B signaling pathway were increased after selenium supplementation in mice model of AD,which might affect the occurrence and development of cognitive function. |
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