| Background: Due to the lack of understanding of the exact pathological mechanism,the research and development results of therapeutic drugs related to Alzheimer’s disease(AD)are not ideal,the prevention and treatment strategies cannot fully cope with the current situation,and the overall level of diagnosis and treatment cannot meet the clinical needs.How to deeply understand the pathological mechanism and find novel therapeutic targets is an urgent scientific problem to be solved.Objective: In this study,APP,PS1 and Tau Triple transgenic mice Alzheimer disease(3×Tg-AD)mice and Wild type(WT)mice were used as the research objects.Combined with transcriptional and nontargeted metabolomics analysis,gene regulation and endogenous metabolites in the hippocampus of 3×TgAD mice were analyzed to further verify the role of key Hub genes in the progression of AD disease.Besides,it also clarified the mechanism of Cystic fibrosis transmembrane conductance regulator(Cftr)in AD,which provided technical support and theoretical basis for analyzing the pathogenesis of AD and developing targeted prevention and treatment strategies.Methods:(1)Rat tail genomic DNA extraction kit was implemented for genotype identification of 3×Tg-AD mice while pathway behavior experiments were conducted to assess their spatial learning and memory ability.Thioflavin S staining was employed to detect amyloid plaque deposition in the brain of mice,and the Nissl staining was used to detect any neuronal lesions in the hippocampus of 3×Tg-AD mice.(2)The untargeted metabolomics of the hippocampal tissues of 3×Tg-AD mice was analyzed by ultra-high performance liquid chromatography-tandem time-of-flight mass spectrometry(UHPLC-TOF/MS).Principal component analysis(PCA)and quality control(QC)methods were employed to evaluate the reliability and stability of the experiment.By establishing an OPLS-DA(Oprthogonal partial least squares differentiation analysis)model,significant differential metabolites were screened,and cluster analysis,KEGG enrichment analysis,and correlation analysis were performed on the differential metabolites.(3)The hippocampal samples from 3×Tg-AD mice were analyzed using transcriptomics.The repeatability of the experimental data was evaluated using the PCA method.Genes were considered differentially expressed when the log2 Foldchange was less than-1 or greater than 1,with p value less than 0.05.The significantly differentially expressed genes were further subjected to cluster analysis,KEGGpathway and GO enrichment analyses,and correlation analysis.(4)Untargeted metabolomics and transcriptomics were combined to investigate the regulatorymechanism in 3×Tg-AD mice.Correlation analysis and KEGG pathway enrichment analysis were conductedfor the significantly different genes and metabolites.The top three signaling pathways that were mostinvolved in differential genes and differential metabolites were selected for further in-depth analysis,and theomics results were verified using q PCR.(5)Two AD models were established in vitro by inducing BV2 cells with LPS and Aβ25-25.To verifythe pathological mechanism of the targeted gene Cftr in AD,si Cftr,Cftr inhibitors,and Cftr enhancers wereutilized.The activation of the Nlrp3 inflammasome was measured using histological immunofluorescenceand Western blot analysis,while the expression of inflammatory factors(TNF-α,IL-1β and IL-6)wasdetermined by ELISA.NO content in cell supernatant was determined by Griess colorimetry.The contentsof SOD,GSH-Px and MDA in the supernatant of BV2 cells were determined by hydroxylamine method,colorimetric method and TBA method.Results:(1)The results of nucleic acid electrophoresis identification show that the genotype of the 3×Tg-AD mouse is correct,and it accurately carries the three pathogenic genes of APP,Tau and PS1.Pathological results show that compared with WT mice,3×Tg-AD mice show decreased short-term memory,long-term spatial memory,increased amyloid plaque load,and severe neuronal damage.(2)Through bioinformatics analysis,12 differential metabolites in positive ion mode and 49 differential metabolites in negative ion mode are screened out.The KEGG pathway enriches 48 differential metabolic pathways.These results reflect that the pathological mechanism of AD may be closely related to energy metabolism,biosynthesis,neurotransmitters,oxidative stress,inflammation and the formation of memory synapses.(3)Through RNA-seq data analysis,673 related differentially expressed genes are screened out.The KEGG pathway enriches 142 differential pathways,which are mainly related to physiological processes such as nerve injury,oxidative stress,inflammatory response,signal transduction,synaptic plasticity,and energy metabolism in AD.The functional enrichment of GO protein is mainly reflected in the difference of inflammatory response,oxidative stress and meiosis stage.(4)The comprehensive analysis of transcription and metabolism show that differential genes and differential metabolites participate in 43 metabolic pathways.It is also found that the Neuroactive ligandreceptor interaction pathway,Purine metabolism pathway and ABC transporters are most closely related to the pathological mechanism of AD.The Hub genes C5ar1,Gabrg1,Ptger1,Tac1,Lpar2,Pnp2,Cftr and Sstr3 in these three pathways were verified and analyzed by q PCR.The experimental results are consistent with the screening analysis.In order to further analyze the mechanism of the Hub gene in AD,it is necessary to study whether the changes of the Hub gene at the cellular level are consistent with the results in vivo.The experimental result shows that the expression of Cftr is decreased and Sstr3 is increased in Aβ25-35 and LPS induced vitro models,which is consistent with the change in vivo results.Since Sstr3 is well known,its role and mechanism on AD are relatively clear,while the role of Cftr in AD is rarely reported.Therefore,this study will focus on the role of Cftr in AD for in-depth research.(5)The expression of Cftr in animal and cell models is verified by immunofluorescence,immunohistochemistry and Western blot.The expression levels of Cftr gene and protein are significantly decreased,which is consistent with the results of RNA-seq.The knockdown or inhibition of Cftr activity aggravates the expression of inflammatory factors in BV2 cells induced by Aβ25-35 and LPS,activates the Nlrp3 inflammasome pathway,and triggers oxidative stress response,thereby promoting the pathological process of AD.In contrast,increasing the activity of Cftr can reduce these symptoms,which suggests that Cftr may reduce the expression of inflammatory factors and oxidative stress response by mediating the Nlrp3 inflammasome signaling pathway,thereby alleviating neuronal damage and improving cognitive function.Conclusion: This study reveals the intrinsic relationship between gene regulation and metabolome changes in the hippocampus of 3×Tg-AD mice and the pathophysiology of AD.It is found that Cftr is a novel potential therapeutic target for AD,which can inhibit neuroinflammation and oxidative stress by regulating the Nlrp3 inflammasome pathway to slow down the pathological process of AD.This newfound provides a new perspective for further understanding the pathological mechanism of AD and developing prevention and treatment strategies. |
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