| Objective1 To investigate the distribution of traditional Chinese medicine(TCM)syndromes in patients with hypertension complicated with atrial fibrillation(HTN-AF),and to analyze the clinical characteristics among different TCM syndromes of HTN-AF.2 To explore the whole transcriptome of peripheral blood mononuclear cells(PBMC)and immune-related proteome on membrane surface of HTN-AF patients by single-cell sequencing.To find the differential metabolites in HTN-AF patients by oxylipin targeted metabolomics.3 To probe the relationship between different TCM syndrome with differential expression of PBMC and differential lipid metabolites in HTN-AF patients,so as to provide the basis for TCM syndrome differentiation and classification.Method:1 Study on TCM syndrome distribution of HTN-AF patientsThe TCM syndrome scale of HTN-AF was developed,and the TCM information and clinical objective indicators of 305 patients were collected.Factor analysis and cluster analysis were used to obtain the TCM syndrome of HTN-AF,and the differences of clinical objective indexes in patients with different syndroms were analyzed.2 Analysis of single cell sequencing of PBMC and serum oxylipin metabolome in HTN-AF patients(1)Single cell sequencing analysis of PBMC in HTN-AF patients1)PBMC samples from 9 patients with HTN-AF,3 patients with hypertensive sinus rhythm and 3 healthy people were collected to prepare cell suspension.Single cell whole transcriptome sequencing(WTA)combined with surface proteomics(Abseq)detection technology was used to detect PBMC cell groupings,cell surface proteins and cell differentially expressed genes of the three groups of subjects.2)The boxplot.stats function was used for data preprocessing and normalization of each sample.The louvain algorithm was used for cluster analysis,and then the visual T-SNE and UMAP were constructed.Marker genes with high specific expression in each cluster and logFC>0.25 were screened by wilcoxon algorithm.The cell types were annotated by SingleR and marker genes.Using FC>1 and FDR<0.01 as screening conditions,the differential genes of HTN-AF,hypertensive sinus rate and healthy group were obtained,and the genes were analyzed by GO and KEGG respectively.3)The Seurat R package was used to search for interactions between ligand-receptor pairs and maj or cell types and establish cell communication networks.(2)Detection of serum oxidized lipid metabolome in HTN-AF patients1)Serum samples were collected from 45 patients with HTN-AF,15 patients with hypertensive sinus rate,and 15 healthy individuals.The UPLC-QQQ-MS was used to detect the content of 76 targeted metabolites of oxidized lipids in the three groups.2)R 4.0 and SPSS 22.0 were used to analyze the differences of metabolites among the three groups,and KEGG enrichment analysis was performed for the differential metabolites.3)Pearson correlation analysis was used to analyze the relationship between different metabolites and HTN-AF and atrial fibrillation.3 Analysis of differentially expressed and differentially oxidized lipid metabolites by single cell sequencing in different TCM syndrome of HTN-AF patients.(1)Based on the TCM syndrome of HTN-AF obtained in the above studies,PBMC samples of 9 patients with HTN-AF(3 cases for each syndrome type)were collected for Single-cell sequencing.Two types of differential genes were obtained by comparing one TCM syndrome type with the others and comparing HTN-AF with CON.The common genes of these two types of differential genes were found and analyzed.(2)Based on the TCM syndrome of HTN-AF obtained in the above studies,serum samples were collected from 45 patients with HTN-AF(15 for each syndrome type),and the contents of metabolomics of oxidized lipids in each syndrome type were detected by UPLC-QQQ-MS.The difference of metabolites among the three groups was analyzed by R 4.0 and SPSS 22.0.Pearson correlation analysis was performed for the differential metabolites of the three syndrome types.Results:1 Distribution of TCM syndromes in patients with HTN-AF(1)44 TCM syndromes with frequency>10%were collected,including 26 TCM symptoms,11 tongue signs and 7 pulse signs.Seventeen principal components were extracted and clustered into three syndrome types:deficiency of both vital energy and yin concurrently blood stasis syndrome(ZX1),Yin deficiency and effulgent fire with blood stasis syndrome(ZX2),yang-qi deficiency and turbid phlegm and blood stasis syndrome(ZX3).(2)The results of inter-group comparison showed that compared with ZX2,the HAS-BLED score and IgM level of patients with ZX1 were increased,while the ApoA1 level was decreased(P<0.05).Compared with ZX3,ApoAl level in ZX1 patients was decreased(P<0.05).Compared with ZX2,the levels of IgM in ZX3 patients were increased,and the levels of MCHC,WBC,EO,GLU,CK,CK-MB,TSA,Hcy and IgG were decreased(P<0.05).There were no significant differences in platelet,coagulation function,liver and kidney function,electrolyte parameters and cardiac ultrasound indexes among the three groups of TCM syndrome(P>0.05).2 PBMC single cell sequencing and serum oxylipin metabolome detection results of HTN-AF patients(1)Single cell sequencing analysis of PBMC in HTN-AF patients1)A total of 59,893 cells were detected,and 8 cell types(classical monocytes,B cells,CD4+T cells,CD8+T cells,DC,non-classical monocytes,NK cells,pDC)were obtained by combining according to cell annotation.2)Compared with the CON group,46,9,29,30,31,7,16,0 differential genes and 10,10,11,13,10,7,10,10,10,3 differential proteins were screened from 8 cell types,respectively.Compared with the HTN-SR group,4,3,5,16,97,36,8,0 differential genes and 7,15,11,15,14,5,10,0 differential proteins were screened from 8 cell types,respectively.3)Comparison between cell communication groups showed that,compared with CON group,the communication between B cells and other cells in HTN-AF group and HTN-SR group was weakened.Compared with CON group,the communication between CD4+T cells and CD4+T cells and CD8+T cells in HTN-AF group and HTN-SR group was weakened,and the communication between NK cells and CD4+T cells and CD8+T cells was weakened.Compared with CON group,pDC cells in HTN-AF group and NK cells in HTN-SR group had weakened communication with other cells.Pathway analysis in cellular communication showed that cells mainly communicated through CCL,IL6,BAFF pathways in the HTN-AF group,through TGF,CCL,IL2 pathways in CON group,and through CCL,IL16,TNF pathways in HTN-SR group.Through inter group comparison of cell communication pathways,it was found that compared to the CON group,the HTN-AF group showed an increase in IL16 pathway communication in DC cells and a decrease in other cells,while the ANNEXIN pathway and GRN pathway communication were enhanced in non classical monocytes.(2)Results of serum oxidative lipid metabolomics in HTN-AF patients1)A total of 42 metabolites of oxidized lipids were detected in each sample.The results of intergroup comparison showed that the levels of 11 β-prostaglandin F2a,11,12-diHET and 11,12-diHETE in HTN-AF patients were significantly higher than those in HTN-SR patients(P<0.05).Compared with CON group,the levels of differential metabolites 14-HDHA decreased in HTN-AF patients,while the levels of 11,12-EET、12,13-EpOME and 12-HETE increased(P<0.05).2)Compared with HTN-SR and CON groups,different metabolites of oxidized lipids in HTN-AF group were mainly enriched in the arachidonic acid metabolic pathway.3)Correlation analysis showed that in the comparison between the HTN-AF group and the CON group,8 differential metabolites were moderately positive correlated with the occurrence of HTN-AF,and 1 differential metabolite was moderately negative correlated.Compared with the HTN-SR group,7 differential metabolites were moderately positive correlated with the occurrence of atrial fibrillation,while 1 differential metabolite was not correlated.3 The results of differential expression of single cell sequencing and differential oxidized lipid metabolites analysis of different TCM syndrome in HTN-AF patients(1)Differential expression analysis of single cell sequencing in patients with different TCM syndromes of HTN-AFThe results of intergroup comparison showed that 126,194,136,225,137,188,507 and 5 differential genes were screened from 8 cell types in ZX1 and ZX2,respectively.Compared with ZX3,117,302,70,350,96,132,385 and 0 differential genes were screened for 8 cell types in ZX1,respectively.Compared with ZX3,15,45,24,114,18,16,25 and 3 differential genes were screened from 8 cell types in ZX2.There were 4,9,5 and 4 unique differential genes of ZX1 obtained in B cells,CD4+ T cells,CD8+T cells and NK cells,respectively.There were 2 and 15 unique differential genes of ZX3 obtained in CD4+T cells and classical monocytes,respectively.The unique differential gene of ZX2 was not obtained.(2)Study on the correlation between TCM syndrome type of HTN-AF and differential oxidized lipid metabolites1)A total of 42 metabolites of oxidized lipid were detected in each sample of patients with three syndrome types.Comparisons between the groups showed that levels of oxy lipid metabolites 14,15-EET、15-KETE、19(R)-HETE and Thromboxane B2 were significantly decreased in ZX1 patients compared with ZX2 patients.The contents of Arachidonic acid and Leukotriene B4 were significantly increased(P<0.05).Compared with ZX3 patients,the serum levels of differentially oxidized lipid metabolites 14,15-EET,15KETE,19(R)-HETE,5,6-diHETE,and Thromboxane B2 in ZX1 patients were significantly reduced,while the serum levels of Arachidonic acid were significantly increased(P<0.05).Compared with ZX3 patients,the differentially oxylipin metabolite 5,6-Dihete in ZX2 patients was significantly decreased(P<0.05).2)The correlation analysis results showed that ZX1 had a moderate negative correlation(0.3<|r|<0.6)with differential metabolites 14,15-EET,15-KETE,19(R)-HETE,and Thromboxane B2,while it had a moderate positive correlation(0.3<|r|0.6);ZX2 has a moderate negative correlation with differential metabolites Arachidonic acid and Leukotriene B4(0.3<|r|<0.6);ZX3 has a moderate negative correlation with the differential metabolite 5,6-diHETE(0.3<|r|<0.6).There is no correlation between each syndrome type and other differential metabolites(|r|<0.3).Conclusion:1 The common TCM syndrome of HTN-AF are deficiency of both vital energy and yin concurrently blood stasis syndrome,Yin deficiency and effulgent firewith blood stasis syndrome,yang-qi deficiency and turbid phlegm and blood stasis syndrome.There are differences in clinical objective indicators between different TCM syndrome in HTN-AF.2 Single cell sequencing differential expression and metabolome differential expression were found in HTN-AF patients.These differentially expressed substances can interact with each other and affect the occurrence and development of hypertension complicated with atrial fibrillation in multiple ways.3 There were differential expression of PBMC and differential oxylipin metabolites among different TCM syndromes of HTN-AF.The unique differential genes and differentially oxidized lipid metabolites of different syndrome types can be used as the basis for TCM syndrome differentiation of HTN-AF,which explains the material basis and scientific connotation of different syndrome types of HTN-AF,and promotes the development of TCM syndrome differentiation objectification. |
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