| Objective:To study the distribution of TCM Syndromes of paroxysmal atrial fibrillation patients with coronary heart disease（CHD-PAF）,and to compare the clinical routine test results between CHD-PAF and coronary heart disease patients with sinus rhythm（CHD-SR）,as well as among its different TCM syndromes.To detect the serum bile acids（BAs）profile of CHD-PAF and its different TCM syndromes.To detect the structure and function of gut microbiota（GM）of CHD-PAF and its different TCM syndromes.To analyze the correlation between GM-BAs axis and CHD-PAF,as well as its different TCM syndromes.To detect PGC-1α/PPARγ,CPT-1β and NF-κB protein or gene expression levels in peripheral blood of CHD-PAF.Finally,to provide objective biological evidence for TCM syndrome differentiation of CHD-PAF,and to provide a new idea for the exploration of novel biomarkers for CHD-PAF and the pathogenesis of CHD-PAF.Methods:1.Collection of clinical data from CHD-PAFFrom October 2019 to November 2021,365 CHD-PAF and 122 CHD-SR who met the respective diagnostic and inclusion criteria were collected from the Department of Cardiology and General Department of Guang’anmen Hospital,Chinese Academy of Chinese Medical Sciences.Case report forms and a database built by Epidata3.1 were used to collect and save clinical data of the subjects.SPSS 26.0 was used to process the data and compare the clinical routine test results between CHD-PAF and CHD-SR.2.Study on the distribution of TCM syndromes in CHD-PAFThe TCM symptom rating scale for CHD-PAF was developed.Based on the scale,information about TCM symptoms,tongue and pulse conditions was collected from the 365 CHD-PAF.Factor analysis and cluster analysis methods were used to explore the major TCM symptoms of CHD-PAF patients,and to compare the clinical routine test results among CHD-PAF with different TCM syndromes.3.Detection of serum bile acid profile in CHD-PAF and its different TCM syndromes 60 CHD-PAF（15 patients with each TCM syndrome）,30 CHD-SR and 20 healthy controls（HC）were selected randomly.The concentration and content of 43 kinds of serum BAs in the three groups were detected by ultra-high performance liquid chromatography-mass spectrometry.R 4.0 and SPSS 26.0 were used to process the data and compare BAs profile between CHD and HC,CHD-PAF and CHD-SR,and among CHD-PAF with different syndromes.4.Detection of gut microbiota in CHD-PAF and its different TCM syndromes60 CHD-PAF（15 patients with each TCM syndrome）,30 CHD-SR and 20 HC were selected randomly.The structure and biological function of GM in the three groups were detected by next-generation sequencing platform.R 4.0 and SPSS 26.0 were used to process the data and compare the diversity and abundance of GM between CHD and HC,CHD-PAF and CHD-SR,and among CHD-PAF with different syndromes.5.Correlation analysis between GM-BA axis and CHD-PAFPearson correlation analysis,Spearman correlation analysis,η Coefficient and logistic regression analysis were used to analyze the correlation between BAs and clinical indicators,between BAs and CHD-PAF as well as CHD-PAF with different TCM syndromes,and between GM and BAs.6.Detection of PGC-1α/PPARγ,CPT-1β and NF-κB expression levels in peripheral blood of CHD-PAFThe serum level of PGC-1α/PPARγ,CPT-1β and NF-κB protein in peripheral venous blood of CHD-PAF were measured by ELISA.The mRNA expression levels of CPT1β and NF-κB in peripheral blood mononuclear cell（PBMC）of CHD-PAF were measured by q-PCR.Results:1.Comparison of clinical routine test results between CHD-PAF and CHD-SR365 CHD-PAF were included,including 184 males and 181 females.The youngest was 39 years old and the oldest was 85 years old,with higher age groups resulting in a larger distribution.There were statistically significant differences in erythrocyte parameters,leukocyte parameters,platelet parameters,serum lipid level,total bile acid,urea,Cr,serum electrolyte level,serum enzymes,Hcy,NT-proBNP,ECG parameters and cardiac ultrasound parameters between CHD-PAF and SR（P<0.01 or P<0.05）.Correlation analysis showed that most of the above differential indicators were correlated with the occurrence of CHD-PAF to some degree.LAD,CHE,PDW,NTproBNP,heart rate,and RAD were the strongest and positively correlated（0.25<r<0.5）.2.Distribution of TCM Syndromes of CHD-PAF and comparison of clinical routine test results among its different TCM syndromesFour major TCM syndromes of CHD-PAF were concluded by factor analysis and cluster analysis.They were heart-spleen yang deficiency and stagnant phlegm syndrome（ZX1）,Qi stagnation and blood stasis in heart and blood vessels syndrome（ZX2）,Deficiency of Qi and Yin leading to failure of nourishment of the heart spirit syndrome（ZX3）,hyperactivity of liver Yang disturbing the heart spirit syndrome（ZX4）.Significance tests of the clinical routine test results among 4 groups showed that CHDPAF with ZX1 syndrome and ZX2 syndrome had more abnormal results.3.Serum bile acid profile in CHD-PAF and its different TCM syndromesCompared with HC,the ratio of secondary BAs（SBA）to primary BAs（PBA）and the serum level of unconjugated BAs（UCBA）,6α-hydroxylated bile acid(6α-HBA,HDCA,βMCA,NorCA,et al increased significantly in CHD,but the ratio of conjugated BAs（CBA）to UCBA and the serum level of taurine BAs（T-BA）,TDCA,GCA,TβMCA et al decreased significantly in CHD（P<0.01 or P<0.05）.Compared with CHD-SR,the serum level of total BAs（TBA）,SBA,UCBA,CBA,HBA,TCDCA,TCA,apoCA et al increased significantly in CHD-PAF（P<0.01 or P<0.05）.Compared with CHD-PAF with ZX1 syndrome,the serum level of TBA,SBA,PBA,GUDCA,GHDCA,et al in CHD-PAF with ZX2 syndrome decreased significantly（P<0.01 or P<0.05）,and TBA,SBA,UCBA,glycine BAs（G-BA）and 7β-HBA in CHD-PAF with ZX3 syndrome decreased significantly（P<0.01 or P<0.05）,and 7βHBA in CHD-PAF with ZX4 syndrome decreased significantly（P<0.01）.Compared with CHD-PAF with ZX2 syndrome,the serum level of TBA,PBA,SBA,GUDCA,TCDCA,THDCA et al in CHD-PAF with ZX4 syndrome increased significantly（P<0.01 or P<0.05）.CHD-PAF with ZX3 syndrome also had significantly lower TBA compared to CHD-PAF with ZX4 syndrome（P<0.05）,while no significant differences were found in serum BAs levels compared to CHD-PAF with ZX2 syndrome.4.Results of gut microbiota in CHD-PAF and its different TCM syndromesCompared with HC,the abundance of Atopobium parvulum,Streptococcus parasanguinis,Akkermansia muciniphila et al in CHD-PAF increased significantly,but Bacteroides coprocola,Prevotella copri（P.copri）,Eubacterium rectale et al in CHDPAF decreased significantly.Compared with CHD-SR,the abundance of P.copri,Lactobacillus fermentum（L.ferment um）,Streptococcus salivarius（S.salivarius）et al in CHD-PAF increased significantly,but Bacteroides salyersiae,Paraprevotella clara,Dorea formicigenerans（D.formicigenerans）decreased significantly.Compared with CHD-PAF with ZX1 syndrome,Alistipes putredinis（A.putredinis）,Bacteroides faecis（B.faecis）,Streptococcus gordonii et al in CHD-PAF with ZX2 syndrome increased significantly,but Alistipes indistinctus（A.indistinctus）,Ruminococcus torques（R.torques）,Acidaminococcus intestine（A.intestine）decreased significantly.Compared with CHD-PAF with ZX1 syndrome,A.putredinis,Klebsiella pneumoniae,Actinomyces odontolyticus（A.odontolyticus）et al in CHD-PAF with ZX3 syndrome increased significantly,but its Bacteroides uniformis（B.uniformis）,Alistipes unclassified and A.intestine decreased significantly.Compared with CHD-PAF with ZX1 syndrome,A.odontolyticus,S.salivarius and Clostridium bartlettii（C.bartlettii）in CHD-PAF with ZX4 increased significantly,but B.uniformis,Bacteroides fragilis,D.formicigenerans et al decreased significantly.Compared with CHD-PAF with ZX2 syndrome,P.copri,Clostridium leptum,Roseburia hominis et al in CHD-PAF with ZX3 syndrome increased significantly.But Veillonella parvula（V.parvula）,B.faecis,L.fermentum decreased significantly.Compared with CHD-PAF with ZX2 syndrome,C.bartlettii and Ruminococcaceae bacterium D16（R.bac.D16）in CHD-PAF with ZX4 syndrome increased significantly,but Collinsella aerofaciens（C.aerofaciens）,B.faecis,Parabacteroides distasonis et al Compared with CHD-PAF with ZX3 syndrome,P.copri in CHD-PAF with ZX4 syndrome decreased significantly.Flavonifractor plautii,Lachnospiraceae bacterium 2158FAA and V.parvula in CHD-PAF with ZX4 syndrome increased significantly,but P.copri,Coprococcus catus,D.formicigenerans decreased significantly.The Shannon indexes of CHD from phylum to genus were significantly different from those of HC（P<0.01 or P<0.05）.The Shannon index of CHD-PAF was not significantly different from that of CHD-SR.The Shannon index was significantly higher in CHD-PAF with ZX2 syndrome than in CHD-PAF with ZX2 syndrome（P<0.05）,and there was no more significant difference in the Shannon index among the 4 groups.Enrichment analysis showed that the differential genes were mainly enriched in metabolism related GO terms and KEGG pathways,including amino acid metabolism,carbohydrate metabolism,fatty acid metabolism and so on.5.Correlation between GM-BA axis and CHD-PAFCHD-PAF had a moderate positive correlation with serum TBA,SBA,6α-HBA,3DHCA,NorDCA,and 7,12-diketoLCA（0.3<r<0.5）.CHD-PAF with ZX1 syndrome had a relatively strong positive correlation with TBA,UCBA and SBA（0.6<r<0.7）,and a moderate positive correlation with SBA,CBA,G-BA,HBA,DCA,GCDCA and GDCA（0.3<r<0.6）.CHD-PAF with ZX2 syndrome had a relatively strong negative correlation with TBA（r=-0.623）,and a moderate negative correlation with serum PBA,SBA,CBA,GUDCA,DCA and TCDCA（-0.6<r<-0.3）.CHD-PAF with ZX4 syndrome had a moderate positive correlation with serum CBA,isoLCA and NorDCA（0.3<r<0.6）.CHD-PAF with ZX3 syndrome had no significant correlation with any serum BA.Binary logistic regression analysis showed that 7,12-diketoLCA,3-DHCA and PT%may be potential risk factors for CHD-PAF.Compared with CHD-SR,there was a moderate positive correlation between V.parvula enriched in CHD-PAF and 3-DHCA（r=0.373）,and a moderate positive correlation between L.fermentum enriched in CHD-PAF and TCDCA（r=0.366）.When ZX1 compared with ZX2,there was a moderate positive correlation between A.intestine,A.indistinctus and R.torques enriched in ZX1 and TBA,CBA,HBA,GDCA,GUDCA,ωMCA,TCDCA,TUDCA,THDCA,GCA,UDCA or βMCA（0.4<r<0.6）,and a moderate negative correlation between A.putredinis and Lachnospiraceae bacterium 8157FAA enriched in ZX2 and SBA,UCBA or 7β-HBA（-0.5<r<-0.4）.When ZX1 compared with ZX4,there was a moderate or relative strong positive correlation between Megasphaera unclassified and D.formicigenerans enriched in ZX1 and TBA,SBA,CBA,6α-HBA,ωMCA,ACA or GCA（0.5<r<0.7）When ZX2 compared with ZX4,there was a moderate negative correlation between C.aerofaciens enriched in ZX2 and 7β-HBA and βMCA（former r=-0.373,later r=-0.389）,and a moderate positive correlation between C.bartlettii and R.bac.D16 enriched in ZX4 and 7β-HBA,UDCA,GDCA,GUDCA,NorDCA,βUDCA,TCDCA,βMCA or alloLCA（0.3<r<0.6）.6.PGC-1α/PPARγ,CPT-1β and NF-κB expression in peripheral blood of CHD-PAFPeripheral blood PGC-1α/PPARγ and CPT-1β protein levels were lower in CHDPAF compared with CHD-SR（P<0.05）,but NF-κB levels were not significantly different.In addition,q-PCR results also showed that CPT-1β mRNA expression in PBMC were also decreased in CHD-PAF compared to CHD-SR patients（P<0.01）,but NF-κB mRNA expression in PBMC were also not significantly different.Conclusions:1.The nature of CHD-PAF is the deficiency root and excess tip.There are four major syndromes of CHD-PAF:heart-spleen yang deficiency and stagnant phlegm syndrome,Qi stagnation and blood stasis in heart and blood vessels syndrome,Deficiency of Qi and Yin leading to failure of nourishment of the heart spirit syndrome,hyperactivity of liver Yang disturbing the heart spirit syndrome.2.The changes of serum bile acid profile in CHD-PAF were the most significant in the syndrome of ZX1 and ZX4,and the levels of various serum bile acids increased significantly.3.CHD-PAF has GM dysbiosis,and the number and abundance of species varies between its different TCM syndromes.The marker species for CHD-PAF with ZX1 syndrome may be A.intestine,and for CHD-PAF with ZX2 syndrome may be B.faecis,and the marker genus for CHD-PAF with ZX4 syndrome may be Collinsella.The increase of serum bile acid profile is related to the dysbiosis.4.GM-BA axis is correlated to the occurrence of CHD-PAF.It can be used as potential biomarkers for syndrome differentiation of CHD-PAF.Elevated serum bile acid profile may contribute to AF by inhibiting myocardial energy metabolism and promoting myocardial inflammatory responses via the PGC-1α/PPARγ pathway. |
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