| Objective:(1)Through the bioinformatics analysis of vascular dementia(Vascular Dementia,VaD)related data sets in the GEO database,the possible key genes with diagnostic significance for the disease were screened out,in order to provide a basis for the diagnosis of clinical cognitive impairment.(2)Through high-throughput sequencing,the expression profile of the hippocampal brain transcriptome of rats with multiple cerebral infarction-induced vascular dementia was constructed,and differential expression analysis was performed based on the sequencing results,and the relevant VaD driving factors—key mRNAs were screened in combination with key diagnostic genes to explore its correlation Genes and signaling pathways,and include clinical subjects to verify the screened diagnostic genes,key mRNAs and related signaling pathways to find possible targets for VaD intervention.(3)Evaluate the effect of Shenma Yizhi pills on rats with vascular dementia caused by multiple cerebral infarctions,and verify whether Shenma Yizhi pills can treat VaD through this target.Methods:(1)After searching and screening the cognitive impairment-related datasets in the GEO database,two suitable datasets were screened out,and the R package "limma" was used to analyze the data expression profiles in the datasets with a threshold value of |logFC|>0.5 and P value<0.05,carry out difference analysis on the two data sets respectively,and draw the volcano map and cluster map of the differential genes,take the intersection of the differential genes according to the up-regulated and down-regulated genes,and use the R package "ROCR" to draw.The ROC curve of the intersection genes in the dataset(the screening threshold with diagnostic significance is AUC score>0.7).Use the R package"clusterprofiler" to perform GSEA-GO and GSEA-KEGG functional enrichment for differential genes with diagnostic significance,obtain m6A-related genes according to the literature,use R’s cor.test according to the threshold |cor|>0.3,P value<0.05 calculate the pearson correlation between Hubgene and the expression of m6A-related genes in the VaD dataset,and draw a correlation heat map.(2)The VaD model caused by multiple cerebral infarctions was established by using the microsphere embolization method,and highthroughput transcriptome sequencing was performed on the model rats and the rats in the sham-operated group.After library construction and library quality inspection,it was put on the machine based on the Illumina HiSeq sequencing platform Sequencing,use HISAT2 for reference genome comparison analysis,use HTSeq to calculate gene expression,construct gene expression profile,use DESeq to filter differential genes according to |log2(FC)|>1,P<0.05 as threshold and draw clustering diagram and volcano picture.The GSEA official website software was used to perform GSEA enrichment analysis on differential genes between groups,and a protein interaction network was constructed based on the String database and Cytoscape software,and possible key mRNAs in the network were screened.Multiple differential genes screened by qPCR were verified in model rat hippocampal tissue.Use cor.test in R to calculate the pearson correlation between the genes with diagnostic significance obtained in the first part and the expression level of each gene after the qPCR result verification in the first part of the VaD dataset,and the genes with significant correlation are the key driving factors of VaD mRNA,and according to the enrichment analysis and literature search,determine its related signaling pathways,perform Western Blot(WB)verification at the protein level,perform Pearson correlation analysis on the key diagnostic genes and key genes and their related signaling pathways in the first two parts of the study,draw a heat map,and screen highly correlated genes as possible key diagnoses Gene.VaD subjects and healthy subjects were included,and the above-mentioned key genes,their pathway proteins,and key diagnostic genes were detected at the protein level by Elisa.(3)The VaD model caused by multiple cerebral infarctions was established by microsphere embolization,and the rats were divided into sham operation group(Sham),model group(Model),Shenma Yizhi low,medium and high dose groups,and donepezil hydrochloride group(DNPQ)and compound Cistanche deserticola group(FFCR)were administered orally for 3 months,and the Morris water maze test was administered to assess their spatial memory and learning ability after administration.HE staining was used to observe the neuron status in the CA1 area of the hippocampus,and transmission electron microscopy was used to examine the CA1 area.For the ultrastructural changes of neurons,Elisa and WB were used to verify the phosphorylation of Twistl,TNF-α,NF-κB p65,IL-1β,TGF-β1 and STAT,respectively,and qPCR was used to verify the mRNA.Results:(1)The VaD and MCI data sets screened in the GEO database were screened for differential genes,and the ROC curve was drawn for them,and finally three Hubgenes with diagnostic significance in the data set were obtained:C4BPA,HEMGN,STRN3.(2)Analysis of differentially expressed genes enrichment obtained by high-throughput sequencing,PPI network screening to obtain Top 10 genes and related enrichment pathways,construct rat hippocampal transcriptome expression profiles,and verify part of the sequencing results by qPCR detection,prove that its results are reliable.Combined with the correlation analysis between the diagnostically significant Hubgenes(C4BPA,HEMGN,STRN3)obtained in the first part and the genes verified by qPCR,Twist 1,a gene highly correlated with C4BPA,HEMGN,and STRN3,was screened out as the key gene in the second part.WB results showed that Twistl was significantly down-regulated in the hippocampus of rats in the Model group(P<0.01),confirming that its expression at the protein level was consistent with that at the gene level.Combined with the results of GSEA analysis of differentially expressed genes,the TNF signaling pathway and NF-κB signaling pathway were highly correlated with Twist1.Signaling pathways were positively correlated(P<0.05),so C4BPA and HEMGN were used as possible key diagnostic genes.(3)The Elisa test results of blood samples from VaD subjects and healthy subjects showed that:Twistl,TNF-α,and NF-κB p65 were all upregulated in the blood of VaD subjects,and the difference between Twist1 and TNF-α was statistically significant Significance(P<0.01),C4BPA was significantly down-regulated in the blood of VaD subjects(P<0.01),and the outcomes essentially corroborated those of the study’s first two phases.(4)The results of the water maze experiment on model rats showed:positioning navigation test:the average swimming speed of rats in each group had no significant difference(P>0.05).Compared with the Sham group,the latency period of rats in the Model group was significantly prolonged(P<0.05),compared with the Model group,the latency of the SMYZ-L and SMYZ-H groups were significantly reduced(P<0.05),although the rest of the groups were reduced,the difference,however,was not statistically significant.HE staining results showed that the structure of hippocampal CA1 area in Sham group was clear,the number of cells was normal,the neurons were neat without swelling,and the cells and neurons were not necrotic;the distribution of cells in hippocampal CA1 area of Model group was uneven,the number decreased,the intercellular space widened,and the swelling and necrosis of neuron degeneration were obvious;compared with the Model group,the neurons in each dose group of Shenma Yizhi prescription survived more and neatly,and the neuron degeneration,swelling and necrosis were not obvious.Compared with SMYZ-L,SMYZ-M group,FFCR group and DNPQ group,SMYZ-H group had relatively more neurons survived,arranged more neatly,and the state of neuron deformation,swelling and necrosis was not obvious.The transmission electron microscopy revealed that the neurons in the hippocampal CA1 area of rats in the Sham group had complete structures,clear envelopes,regular nuclear shapes,smooth and complete nuclear membranes,uniform distribution of chromatin in the nucleus,clear nucleoli,abundant organelles,and more mitochondria without swelling,visible lysosomes,rough endoplasmic reticulum,and no shedding of granules;the neurons in the hippocampal CA1 area of the rats in the Model group were significantly swollen,the nuclear membrane was incomplete,and only a small amount of mitochondria and other organelles were seen in the cytoplasm,with obvious edema,mitochondrial degeneration,endoplasmic reticulum expansion,granule fusion or shedding on the surface of the endoplasmic reticulum,and autophagy structure and lysosome;the neurons in the FFCR group,DNPQ group,and SMYZ-L group were better,the nuclear membrane was almost complete,and the chromatin distribution in the nucleus was more uniform.Although the mitochondria in the cytoplasm were partially swollen,the swelling degree was small,the endoplasmic reticulum was not expanded,the granules on the surface of the endoplasmic reticulum were less fused and shed,and a small amount of lysosomes could be seen;the SMYZ-M and SMYZ-H groups were more obvious than the Model group improvement,good neurons,complete nuclear membrane,uniform distribution of chromatin in the nucleus,almost no edema in the mitochondria and other organelle structures in the cytoplasm,no expansion of the endoplasmic reticulum,almost no fusion and shedding of granules on the surface of the endoplasmic reticulum,and no autogenesis,phagocytic structure,a small amount of lysosomes can be seen,tending to normal.The results of ELISA,WB,and qPCR showed that compared with rats in the Sham group,TNF-α,IL-1β,P-STAT3/STAT3 and TNF-α mRNA,NF-κB p65 mRNA,Twistl mRNA,IL-1β,STAT3 all were significantly upregulated in the hippocampal tissue of rats in the Model group(P<0.01),and NF-κB p65,Twistl,TGF-β1,and TGF-β1 mRNA were up-regulated(P<0.05).Compared with the Model group,TNF-α,NF-κB p65,Twistl,IL-1β,P-STAT3/STAT3 and TNF-α mRNA,NF-κB p65 mRNA,Twistl mRNA,IL-1β mRNA and STAT3 mRNA were down-regulated to a certain extent,and the high-dose group had the most obvious improvement in all indicators,with significant statistical differences(P<0.05),which was better than other dose groups and drug control groups.Conclusions:(1)The expression profile of the hippocampal transcriptome of VaD rats with multiple cerebral infarctions caused by microsphere embolization was constructed,and the key gene Twist1 and TNF-α/NF-κB/Twistl signaling pathway were preliminarily screened out.All samples were upregulated.Combined with the bioinformatics analysis of public databases,the possible key diagnostic gene C4BPA,which is highly related to Twistl and TNF-α/NF-κB/Twistl signaling pathway,was screened out,which was down-regulated in blood samples of VaD patients.(2)Shenma Yizhi prescription can improve the spatial learning and memory ability of multiple cerebral infarction-induced VaD model rats and the loss,necrosis,and swelling of neurons in the CA1 region of the hippocampus.Shenma Yizhi prescription can down-regulate TNF-α/NF-κB/Twist1 The signaling pathway and IL-1β can improve the inflammatory response in the process of VaD disease,which may be the therapeutic target of Shenma Yizhi prescription to improve the symptoms of VaD disease. |
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