Analysis Of The Expression Characteristics Of Constitutional Protein Of Yin Deficiency And Yang Deficiency Based On Mass Spectrometry Technology

Background and Purpose:In previous studies,studies of gene,transcription,epigenetics,protein,metabolism and other aspects showed that Yin deficiency and Yang deficiency constitution mainly exist metabolism,neuroendocrine,immune disorders on the micro-mechanism.Among them,the study of protein is weak,mainly on the specific function of the corresponding protein detection,there is no study of the overall protein characteristics.Constitution is stability,but also variability and adjustability,gene expression is stable,it is the foundation of constitution stability,and dynamic expression of protein can better reflect the variability and adjustability of constitution;The formation of constitution is the result of the interaction of nature and nurture.Genes are determined by nature,which reflects the inheritance of constitution,and proteins are influenced by the environment.The study of protein level combined with the study of gene can better reflect the characteristics and basic principles of constitution.Detect the protein spectrum expression of yin-deficiency and Yang-deficiency Constitution,to study the protein characteristics of Yin-deficiency and Yang-deficiency Constitution,so as to contribute to the study of microscopic identification of health state,provide the basis for distinguishing the population with different constitution,in addition,provide the molecular biology basis for the constitution conditioning,and provide the theoretical guidance for the prevention of its related diseases.Methods:According to the Classification and Determination Standards of Constitutions in Traditional Chinese Medicine,subjects identified as yin deficiency constitution(n = 12),yang deficiency constitution(n= 12)or balance constitution(n= 12)were selected.The age and gender baseline are comparable.Fasting peripheral venous blood was collected and serum was separated.Serum protein expression was detected by TMT technique.Pathway annotation based on the KEGG database and Reactome database,GO analysis based on Gene ontology database,is performed.The differential proteins are selected by previous studies,Western Blot is used to verify the results.Results:1.Based on |log2Fc|>0.263 and P<0.05,64 differentially expressed proteins in yang deficiency constitution are selected compared with balance constitution,including 35 up-regulation and 29 down-regulation.According to the classification of KEGG Pathway,metabolic pathway includes up-regulation of Terpenoid backbone biosynthesis,endocrine system pathway includes up-regulation of Renin-Angiotensinogen system,and digestive system includes up-regulation of Vitamin digestion and absorption and Fat digestion and absorption,immune system includes Leukocyte transendothelial migration.In addition,Reactome pathway enrichment and GO enrichment shows disorder of metabolism and protein metabolism,such as Terpenoid backbone biosynthesis,Metabolism of Angiotensinogen to Angiotensins,Regulation of Insulin-like Growth Factor(IGF)transport and uptake by Insulin-like Growth Factor Binding Proteins(IGFBPs);substance transport,such as VLDL biosynthesis;Hemostasis and cell cycle.2.Based on |log2Fc|>0.263 and P<0.05,100 differentially expressed proteins in yin deficiency constitution are selected compared with balance constitution,including 61 up-regulation and 39 down-regulation.According to the classification of KEGG pathway,the metabolic pathways includes up-regulation of Terpenoid backbone biosynthesis and down-regulation of Inositol phosphate metabolism and Primary bile acid biosynthesis;Endocrine system pathways includes up regulation of Renin-Angiotensin system and down regulation of Glucagon signaling pathway,Melanogenesis and Thyroid hormone signaling pathway;Immune system includes up regulation of Complement and coagulation cascades;Digestive system pathways includes up-regulation of Vitamin digestion and absorption and down-regulation of Pancreatic secretion,Salivary secretion and Gastric acid secretion.In addition,Reactome pathway enrichment and GO enrichment shows disorder of metabolism and protein metabolism,such as Metabolism of lipids and lipoproteins and Fatty Acids bound to GPR40(FFAR1)regulate insulin secretion;substance transport,such as VLDL biosynthesis and Lipoprotein metabolism;Hemostasis and cell cycle.3.In order to study the relationship between Yin-deficiency Constitution and Yang-deficiency Constitution,based on |log2Fc|>0.263 and P<0.05,further analysis reveals that there are 19 proteins statistically different between Yang-deficiency and Yin-deficiency Constitution,Yang-deficiency and Balance constitution,which is designated as the Yang Deficiency Constitution specific protein(Yasp);33 proteins statistically different between Yang-deficiency and Yin-deficiency Constitution,Yin-deficiency and Balance constitution,which is designated as the Yin Deficiency Constitution specific protein(Yisp);30 proteins statistically different between Yang-deficiency and Balance Constitution,Yin-deficiency and Balance constitution,but no difference between Yin-deficiency and Yang-deficiency Constitution,which is designated as the common protein;the three proteins were enriched.The analysis of KEGG pathway shows that pathways of common protein includes metabolic pathway,Terpenoid backbone biosynthesis;digestive system,Vitamin digestion and absorption and Fat digestion and absorption,and endocrine system,Renin-Angiotensin system.The TGF-signal pathway enriched by Yasp protein is related to diabetes,while Yisp protein enriched to Thyroid hormone signal pathway.In addition,Reactome pathway enrichment shows that common proteins were enriched into metabolic and protein metabolic pathways,such as Metabolism of protein,Metabolism of Angiotensinogen to Angiotensins,Regulation of Insulin-like Growth Factor(IGF)transport and uptake by Insulin-like Growth Factor Binding Proteins(IGFBPs);Yasp protein enriched in the pathway of Glycerophospholipid catabolism,Yisp protein enriched in Regulation of lipid metabolism by Peroxisome proliferator-activated receptor alpha(PPARalpha),Metabolism of lipids and lipoproteins,Recycling of bile acids and salts.Substance transport pathways,especiallyVLDL biosynthesis,is enriched by both common protein and specific protein.The GO enrichment shows similar results.4.Western Blot was used to identify the differential proteins involved in metabolism and substance transport,the results show that PTPRQ has statistical difference between Yin-deficiency Constitution and Yang-deficiency Constitution,Yin-deficiency Constitution and balance constitution,GDF6 has statistical difference between Yin-deficiency Constitution and Yang-deficiency Constitution,Yang-deficiency Constitution and balance constitution,DGAT2 has statistical difference between balance constitution and Yang-deficiency Constitution,balance constitution and Yin-deficiency Constitution,but there is no statistical difference between yin-deficiency and Yang-deficiency Constitution.The downstream of metabolism and endocrine system,G protein,was selected for WB detection,result shows there were statistical differences in the expression of G protein among the three constitutions.Conclusion:The characteristics of Yin deficiency and Yang deficiency constitution are disorder of metabolism and material transport,endocrine,digestive system and immunity,and may be related to abnormal cell cycle and coagulation function.The regulation mechanism of yin-deficiency and Yang-deficiency constitution has common part,which may be the common material basis of both of them;the regulation mechanism of yin-deficiency and yang-deficiency constitution has its own specificity,which may be the cause of the differences in clinical manifestations between the two.