| Objective: 16 S rDNA and untargeted metabolism tests were performed on the stool and blood of the normal group,stroke group,and post-stroke depression group,for preliminarily exploring the relationship among changes in intestinal microbes,blood metabolites and post-stroke depression.At the same time,the correlation analysis was performed among differential gut microbes,differential metabolites and transcriptomics,we further explored the biological pathogenesis of gut microbes involved in post-stroke depression.Our findings may provide new ideas and laboratory evidence for the pathogenesis of post-stroke depression,also provide new targets for the prevention and treatment of post-stroke depression.Methods:(1)Research subjects were collected that meet the requirements for inclusion in Affiliated Hospital of Youjiang Medical Unwenity for Nationalities.A total of 36 patients with post-stroke depression(26 stools,36 serum)were considered as the post-stroke depression group(group C),and 34 patients with stroke(34 stools,32 serum)as the stroke group(group B),A total of 30 healthy peopel cases(30 feces and 30 serum)were regarded as the normal group(group A).Stool and fasting venous blood were collected from each group of subjects.Stool is used for 16 S rDNA sequencing.Serum was extracted from fasting venous blood is used for untargeted metabolome sequencing.(2)The transcriptome data of stroke patients and depression patients were downloaded separately from the National Center for Biotechnology Information(NCBI)website.Differential expression analysis was used to identify differentially expressed genes in stroke patients and depression patients compared with healthy people.And through paired sample analysis,the overrecovery molecules and the recovery but incomplete molecules in the process of stroke recovery-mediated depression have been identified.(3)The clusterprofiler package was used for gene ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis.GSEA software was used to perform gene set enrichment analysis(GSEA).(4)The collected blood is analyzed for untargeted metabolomic,based on liquid chromatography-multi-stage mass spectrometry(liquid chromatograph/mass spectrometer-mass spectrometer,LC/MS-MS).The differential metabolites of stroke patients and post-stroke depression patients were identified through differential analysis,and further bioinformatics analysis was performed.(5)The gut microbial ecological landscape of stroke and post-stroke depression patients was explored,based on α and β diversity analysis,LEf Se(LDA Effect Size)analysis,Linear discriminant analysis(LDA)and STAMP difference analysis.(6)Non-targeted metabolome data,16 S rDNA sequencing data,and transcriptome data were combined to construct a comprehensive regulatory network,for exploring the possible mechanism of gut microbes participating in post-stroke depression.Results:(1)Comparison of general conditions of subjects: age,body mass index(BMI),and gender: there was no statistically significant difference(P> 0.05)in the pairwise comparison between the three groups(normal group,stroke group,and post-stroke depression group).NIHSS score,the normal group was compared with the stroke group or the post-stroke depression group,the difference was statistically significant(P<0.05).Comparing the stroke group and the post-stroke depression group,the difference was not statistically significant(P>0.05).HAMD score,there was no statistically significant difference between the normal group and the stroke group(P>0.05).Compared post-dtroke depression group with normal group/stroke patients group,the difference was statistically significant(P<0.05).(2)After differential expression analysis,we found that compared with the control group,stroke patients included a total of 1492 differentially expressed genes,while depression patients included 4376 expressed genes.The comorbid molecules that were up-regulated in stroke and depression still maintained a low level of high expression during the stroke recovery process.The comorbidity molecules,which were down-regulated in stroke and depression,still maintained a low level of low expression during the stroke recovery process.Molecules whose expression was down-regulated in stroke and up-regulated in depression were over-recovered during stroke recovery,and the abundance of molecules that were significantly under-expressed recovers to exceed the expression level of normal human cerebral cortex.Molecules whose expression was up-regulated in stroke and down-regulated in depression are over-recovered during stroke recovery,and their abundance was restored to lower than the expression level of normal human cerebral cortex.(3)The enrichment analysis results showed that biological processes,in which in the comorbid molecules of stroke and depression,the molecular features that mediate depression by excessive changes during stroke,and the depression-specific genes were involved,mainly were related to metabolism,immunity,microbes and nerves.(4)Through untargeted metabonomics detection and bioinformatics analysis,there were3443 dysregulated metabolites in stroke patients compared with the control group,under the condition of P<0.05.Disordered metabolites were mainly involved in two metabolic pathways: ascorbate and alginate metabolism and the biosynthesis of valine,leucine and isoleucine.(5)There were 5408 differential metabolites in post-stroke depression patients compared with the control group,under the condition of P<0.05.There are 1749 differential metabolites in post-stroke depression patients compared with stroke patients,under the condition of P<0.05.Among them,53 metabolites were dysregulated in stroke patients and post-stroke depression patients.Acamprosate was significantly reduced during the development of depression.The dysregulated metabolites were mainly involved in valine,leucine and isoleucine biosynthesis.(6)The α diversity results showed that the species richness and diversity of the control group were the highest,followed by the stroke group,and the lowest in the post-stroke depression group.The β diversity results showed that the species diversity of the stroke group was the highest,followed by the control group,and the lowest in the post-stroke depression group.The results of STAMP difference analysis showed that Salviae UCG-003 was the most significant difference species in the control group and the stroke group.Pseudomonas was the most significant difference specie in the control group and the post-stroke depression group.Pseudomonas was the most significant difference species in the stroke group and the post-stroke depression group.(7)Correlation analysis results show that 59 dysregulated metabolites and 518 dysregulated OTUs were involved,and there were a total of 3038 significant related pairs,under the condition of P<0.05.It shows that there is a wide range of interactions between dysfunctional metabolites and dysfunctional microorganisms in PSD patients.Correlation analysis results show that 59 dysregulated metabolites and 518 dysregulated OTUs were involved,and there were a total of 3038 significant related pairs,under the condition of P<0.05.Constructing a global regulatory network for dysregulated microorganisms,dysregulated metabolites and dysregulated genes.The results showed that there are extensive interactions among dysregulated microorganisms,dysregulated metabolites(inosine,guanosine,α-tocopherol,adenine,adenosine and thymine),dysregulated genes(GNAO1 、PRKCB 、 HRAS 、 GRIA2 、 KRAS 和 MAPK1 genes),and dysregulated phenotypic functions(Salomonas infection,shigellosis,pathogenic Escherichia coli infection,Alzheimer’s disease,c AMP signaling pathway,Huntington’s disease,neuroactive ligand receptor interaction,Ras signaling pathway,MAPK signaling pathway,MAPK signaling pathway,long-term depression and apoptosis).Conclusion:In this study,we found that gut microbiota are mainly dysregulated by regulating the metabolites(inosine,guanosine,alpha-tocopherol,adenine,adenosine and hymine)of stroke patients.It then mediates the dysregulation of GNAO1,PRKCB,HRAS,GRIA2,KRAS,MAPK1 genes.It eventually induce the occurrence of post-stroke depression. |
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