Analysis Of Peripheral Blood TCR Immune Repertoire And Quantitative Proteomics Research In People With Different Traditional Chinese Medicine Constitutions

Objective:Assessment of traditional Chinese medicine constitution(TCMC)types is fundamental and essential to the TCMC research.How do we carry out the assessment in a standardized and objective way so as to facilitate the application and generalization of TCMC theory?The problem remains to be solved.To make the assessment more objective,one way is to identify potential laboratory test indicators through routine physical examination.Previous studies have found the differences in immunity,metabolism and other physical functions among people of different TCMC types.This study is aimed at identifying different gene expression patterns and specific markers of gene rearrangement in adaptive immunity of people of different TCMC types,by the means of applying the immune repertoire sequencing,a major technological breakthrough,to the TCMC research.Plasma proteomic variation may occur in one or many TCMC types.It is important to identify serological biomarker profile from healthy people in the TCMC research.Methods:A total of 157 healthy subjects of nine TCMC types were included in this study based on inclusion criteria,and received a routine physical examination,with fasting peripheral venous blood and first morning urine sample collected.1.After collecting information on gender,age,height,weight,routine blood and urine parameters,biochemical parameters and other clinical data,we conducted statistical analysis of routine blood and urine parameters and serum biochemical parameters,then performed multiple stepwise logistic regression analysis of variables with statistical difference,and calculated the value of risk factors.We identified the score of independent risk factor based on odd ratio(OR)value and established prediction models.The receiver operating characteristic(ROC)Curve was created and area under the curve(AUC)was calculated to assess the constitution prediction performance of each index.2.Thirty-four female subjects of nine TCMC types in corresponding age groups were selected and their peripheral blood was collected for high-throughout sequencing of the T cell receptor(TCR).We analyzed the differences of the TCR diversity and characteristics of immunity maps of different TCMC types,including diversity indexes,CDR3 length distribution,V-region and J-region gene sub-family usage,and the diversity of V-J combinations,so as to identify high-frequency CDR3 amino acid sequences.Then ROC curve was constructed according to the random forest model established depending on V-J combinations data.3.Plasma protein expression was determined by using digital image analysis(DIA)for the analysis of protein expression level,GO annotation and KEGG enrichment analysis.The aim is to deduce the association of differentially expressed genes,their protein expression variation,functions and pathways with the nine TCMC types,and explore the biological mechanism of the nine TCMC types at the macromolecular level and identify biomarkers.Results:1.Compared with balanced constitution(BC)group,physical examination results of healthy subjects in other groups are in the normal ranges.Specifically,in the yang-deficiency constitution(YADC)group,body mass index(BMI),neutrophil count(NC),alpha-fetoprotein(AFP)level,blood uric acid(UA)level and albumin percentage(ALB%)are comparatively lower,while high-density lipoprotein(HDL)and Apolipoprotein A1(APOA1)level are relatively higher.In the yin-deficiency constitution(YIDC)group,calcium(CA)level and platelet-lymphocyte ratio(PLR)are comparatively lower,while NC,potassium ion(K)level and blood phosphorus(P)level are relatively higher.In the qi-deficiency constitution(QDC)group,lactate dehydrogenase(LDH)and ?-Hydroxybutyrate dehydrogenase(?-HBDH)levels are comparatively lower.In the qi stagnation constitution(QSC)group,total cholesterol(TC)level is relatively higher,while Apolipoprotein B(APOB)level is comparatively lower.In the phlegm-dampness constitution(PDC)group,weight,BMI and TC level are relatively higher.In the dampness-heat constitution(DHC)group,K and ?-HBDH levels are relatively lower.In the inherited special constitution(ISC)group,ALB%and LDH level are relatively higher,while blood platelet(PLT),procalcitonin(PCT),PLR,total protein(TP)and CA levels are comparatively lower.Those differences are statistically significant,P<0.05.ROC curve is generated on the basis of examined variables selected from the logistic regression model and AUC is calculated.AUCs are 0.698,0.881,0.840,0.716,0.856,0.769,0.760 and 0.894 respectively for the BC,YADC,YIDC,QDC,QSC,PDC,DHC,and ISC groups.2.A total of28600 ± 16300 specific TCRB CDR3 clonotypes are identified through sequencing.Analysis of the immune repertoire diversity shows that the D50,diversity index(DI)and Shannon index of the BC group were 13.40 ± 5.32,23.52 ± 53.97 and 11.95 ± 0.68 respectively;the three indexes of the PDC group were 2.60 ± 2.52,13.63 ± 6.76 and 9.45 ± 1.32 respectively,which were significantly decreased compared with the BC group,P<0.05.No significant differences were detected in other groups.Characteristics of CDR3 length distribution:A CDR3 generally contains 5-21 amino acids to compose 17 types of peptides of different lengths,and is normally distributed.Amongst them,7 types of peptides comprising 9-15 amino acids account for 95%of all CDR3s.The TCR? chain CDR3 length of in healthy people is normally distributed.CDR3 length distribution of different TCMC types varies.The PDC group showed a higher proportion of shorter CDR3.The average proportions of CDR3 length of 9aa were(6.58±1.82)%,(4.95±0.58)%and(5.22±0.94)%respectively in the PDC group,the BC group,and the other groups.The average proportions of CDR3 length of 10aa were(13.35±2.85)%,(10.75± 0.83)%and(11.11± 1.48)%respectively in the PDC group,the BC group,and the other groups.However,the PDC group showed a lower proportion of longer CDR3.The average proportions of CDR3 length of 13aa were(19.05±2.13)%,(21.05±0.25)%and(20.69±1.28)%respectively in the PDC group,the BC group,and the other groups.The average proportions of CDR3 length of 14aa were(9.45±1.52)%,(11.17 ± 0.65)%and(10.91±1.10)%respectively in the PDC group,the BC group,and the other groups.No significant differences were observed among other groups.There were differences in the frequency of V-region gene use between the BC group and other seven unbalanced constitution groups,most notably the PDC group.The frequency of V7-8 gene use increased in the YADC group,but no significant difference was observed in the YIDC group.However,in the QDC group,the frequency of V6-3,V6-5 and V6-6 gene use increased,that of V12-5 gene use increased,and that of V4-1 gene use showed a decrease tendency.In the QSC group,the frequency of V11-2 gene use increased,and that of V3-1 and V4-1 gene use showed an increasing tendency.In the PDC group,the frequency of V9,V29-1 and V30 gene use increased,but that of V7-9,V10-3,V12-5,V20-1 and V28 gene use decreased and that of V2 and V6-4 gene use showed a decrease tendency.In the DHC group,the frequency of V5-1 and V18 gene use increased,but that of V12-5 and V28 gene use decreased,and that of V7-2 gene use showed a decrease tendency.In the ISC group,the frequency of V27 and V28 gene use decreased.There were differences in the frequency of J-region gene use between the BC group and other seven unbalanced constitution groups.Compared with the BC group,the frequency of J1-4,J1-5 and J1-6 gene use in the QDC group increased,and that of J2-1 gene use in the PDC group decreased,P<0.05.Frequency of J gene subtype use of different TCMC types displayed different tendencies.A decrease tendency in J1-1 and an increasing tendency in the J2-3 were found in the QSC group.On the contrary,in the QDC group,the frequency of TRBJ2-5 and hTRBJ2-6 gene use decreased,while that of hTRBJ2-7 gene use increased.This study found no shared CDR3 sequences among unbalanced constitution groups.Compared with the BC group,random forest classifier was adopted to identify the seven unbalanced constitution types.Results showed that AUCs of the YADC,YIDC,QDC,QSC,PDC,DHC and ISC groups were 1,1,0.778,0.772,0.833,and 0.944 respectively.3.Proteomic analysis showed that compared with the BC group,the YADC group had 16 differentially expressed proteins(DEPs)(6 up-regulated and 10 down-regulated),including Keratin,type ? cytoskeletal 2 epidermal,Transgelin-2 and Talin-1,which were only differentially expressed in this group.In the YIDC group,there were 201 DEPs(70 up-regulated and 131 down-regulated),and differential expressions of ten proteins including IGKV4-1,HSPA8,NCAM1,ANPEP,ORM2,OGN,FLT4,SH3BGRL3 and PROCR were only detected in this group.The QDC group had 195 DEPs(70 up-regulated and 125 down-regulated),including IGLV2-8,MMP2 and LGALS3BP,which were only differentially expressed in this group.In the QSC group,there were 11 DEPs(3 up-regulated and 9 down-regulated),including ATP2A1,HIST1H2BK,APCS and MYH7,which were only differentially expressed in this group.In the PDC group,there were 171 DEPs(65 up-regulated and 106 down-regulated),including TXN,PZP and COLEC11,which were only differentially expressed in this group.In the DHC group,there were 185 DEPs(65 up-regulated and 120 down-regulated),including S100A9,KRT1 0,CRTAC1 and FCGBP,which were only differentially expressed in this group.In the BSC group,there were 185 DEPs(66 up-regulated and 119 down-regulated),including B2M and IL1RAP,which were only differentially expressed in this group.In the ISC group,there were 168 DEPs(65 up-regulated and 103 down-regulated),including LCAT and PON1,which were only differentially expressed in this group.KEGG analysis showed that significant differences were detected in some immune and metabolism signaling pathways in all eight unbalanced constitution types,including complement and coagulation cascades,NF-?B signaling pathway,autoimmune thyroid disease signaling pathway,glycosamine vitamin binding protein,phospholipase D signaling pathway and cGMP-PKG signaling pathway.Significant enrichment was observed in the thyroid hormone,estrogen,dilated cardiomyopathy,hypertrophic cardiomyopathy,arrhythmia and atherosclerosis signaling pathways in the YADC group,which were the key mechanisms that distinguish this group from others.Conclusion:Indicators obtained from routine physical examination have the potential to facilitate objective assessment of TCMC types.People of different TCMC types have different immune functions.In the PDC group,the immune repertoire diversity decreased,and CDR3 length distribution tilted.Immune repertoire sequencing can be used to assess TCMC types,but no specific CDR3 is identified in different TCMC types.The difference in immune repertoire of different TCMC types may be reflected in the V-J combination abundance.Protein expression variation is observed in the unbalanced constitution types.All eight unbalanced constitution types may show protein expression variation of C9,IGHG3,AQR,SOD1,TF,CD44,APOL1,APOC2 and TUBB1 in complement and coagulation cascades,B-cell receptor signaling pathway,natural killer cell-mediated cytotoxicity,NF-?B signaling pathway and other immunity-related signaling pathway in common.It indicates that people of unbalanced constitution types have reduced immune function represented by complement and coagulation cascades.Transgelin-2,HSPA8,ATP2A1,TXN and S100A9 may be the potential biomarkers for the YADC,YIDC,QSC,PDC and DHC respectively.A discrimination model for identifying a group of biomarkers is needed for objective assessment of TCMC types based on multi-dimensional analysis.