Screening Serum Protein Markers Of People With Qi Deficiency Constitution And CSG Patients With Pi-Qi

BackgroundChronic superficial gastritis is one of the common digestive system diseases.The incidence rate of the general population is as high as 80%.It can be caused by irregular diet,Helicobacter pylori infection,alcohol consumption,drug damage and emotional stimulation,etc.Chronic superficial gastritis recurrent attacks,and if you do not take immediate treatment,it is easy to develop into chronic atrophic gastritis and gastric cancer,and make the patients extremely painful.At present,the diagnosis of chronic superficial gastritis in our country relies on gastroscopy and pathological examination.The process is cumbersome and expensive,and it will bring great pain and risk to the patients.It is imminent to find a convenient,effective and inexpensive method for the early diagnosis of chronic superficial gastritis.Clinical cases show that the number of clinical patients with chronic superficial gastritis of Pi-Qi deficiency syndrome is huge,and has become a fixed pattern of disease and syndrome;literature data show that Qi deficiency constitution is the physiological basis of Pi deficiency syndrome,and Qi deficiency constitution is easy to develop into chronic superficial gastritis of Pi-Qi deficiency syndrome under specific causes.Guided by the idea of"preventive treatment of disease" in Chinese medicine,the purpose of this study is to start from the early stage of chronic superficial gastritis,namely Qi deficiency constitution,and explore the specific protein markers and their intrinsic associations between Qi deficiency constitution and chronic superficial gastritis with Pi-Qi deficiency syndrome.ObjectiveThis study used Label-free quantitative proteomics technique to find proteins between healthy people with normal constitution,healthy people with Qi deficiency constitution and chronic superficial gastritis patient with Pi-Qi deficiency syndrome.Study uses GO analysis and KEGG analysis methods for bioinformatics analysis of these differential proteins,to find the biological processes and signaling pathway involving these proteins.Find the biomarker and inner connection between Qi deficiency constitution and CSG with Pi-Qi deficiency syndrome to provide a new idea about clinical diagnosis of CSG among Qi deficiency people.MethodsAccording to Classification and Determination of Constitution in TCM(2009),Guiding Principle of Clinical Research on New Drugs of Traditional Chinese Medicine(2002),Criteria for Classification and Treatment of Chronic Gastritis by Endoscopic Classification and Treatment and Consensus Opinion of Chinese Chronic Gastritis in Shanghai(2012)to recruit 3 cases of healthy people with normal constitution,3 cases of healthy people with Qi deficiency constitution,and 3 cases of Chronic Superficial Gastritis patients with Pi-Qi deficiency syndrome,and collect their serum.Use Bradford to exam the quality of total serum protein.Use Label-free proteomic technique to do qualitative and quantitative detection on the serum protein.Use MeV software to do cluster analysis of the identified serum proteins.Use online software Venn diagrams to categorize the identified serum proteins.Use Gene Functional Annotation(GO)analysis to find the main biological functions of those differentially expressed proteins.Use Kyoto Encyclopedia of Genes and Genomes Database(KEGG)pathway analysis to find the main metabolic pathways of the differentially expressed proteins.ResultsRecruiting subjects and quality identification of serum samplesAll 9 subjects signed informed consent and volunteered to work with all questionnaires,physical examination items and collecting serum samples.9 total protein samples purified from serum were well preserved without degradation.The total amount of purified protein could satisfy at least one time of Label-Free unlabeled quantitative proteomic analysis.All serum protein samples were qualified.Proteomics study on serum proteinsAfter Label-free detection,623 kind of proteins were identified in 9 samples,among which 322 kind of proteins are found in healthy people with normal constitution(group A),504 proteins are found in healthy people with Qi deficiency constitution(group B),and 498 kind of proteins are found in Chronic Superficial Gastritis patients with Pi-Qi deficiency syndrome(group D).There are 216 differentially expressed proteins between group A and group B(fold change>1.2 or fold change<0.833),24 significant differential proteins(p<0.05),in which 16 are up regulated,and 8 are down regulated.There are 214 differentially expressed proteins between group A and group D(fold change>1.2 or fold change<0.833),28 significant differential proteins(p<0.05),in which 22 are up regulated,and 6 are down regulated.Bioinformatics analysis of serum proteinClustering result shows that those identified differentially expressed proteins have both rationality and accuracy,and it can be used to split group A,group B and group C.After GO enrichment analysis,the result shows that the differentially expressed proteins between group A and group B are mostly associated with:negative regulation of cellular metabolic process,negative regulation of nitrogen compound metabolic process,negative regulation of immune system process,negative regulation of protein metabolic process and lipid transport.The differentially expressed proteins between group A and group D are mostly associated with:regulation of proteolysis,regulation of protein activation cascade,negative regulation of cellular metabolic process,negative regulation of catalytic activity and negativeregulation of immune system process.After KEGG enrichment analysis,the result shows that the differentially expressed proteins between group A and group B are mostly associated with the following pathways:complement and coagulation cascades,thyroid hormone synthesis and pertussis.The differentially expressed proteins between group A and group D are mostly associated with the following pathways:complement and coagulation cascades,prion disease and systemic lupus erythematosus.Compare group A vs.group B with group A vs.group D,there are 10 significant differential proteins,among which C4BPB rises in the order of A to B to D.C4BPB is mainly involved in:blood coagulation,complement activation,classical pathway and innate immune response.Conclusion(1)In the develop process of Pi-Qi-deficiency syndrome,the serum expression of protein C4BPB has a continuous upward trend,meanwhile it has no significant differential expression in the Damp-Heat constitution and Damp-Heat syndrome.And the protein C4BPB participates in the negative regulation of the classical pathway of complement activation in the body’s innate immune system.The higher C4BPB expressed,the weaker the innate immunity is.This is related to the fact that Qi-deficiency and Pi-Qi-deficiency syndrome people are easy to get ill and have slow recovery after the disease.It is believed that protein C4BPB may be the serum bio-marker of Qi-deficiency constitution and CSG Pi-Qi-deficiency syndrome population.(2)The cluster analysis of serum proteins of people with different physical types can get the same type of physique together,and separate the people of different physical types.Our results provide large data support for the Constitution Theory’s core theory "constitution can be divided".(3)The mutual serum proteins between Qi-deficiency constitution and CSG Pi-Qi-deficiency syndrome are confirmed its consistency and progressiveness in the negative regulation of cell metabolism pathway,negative regulation of immune system pathway,and complement and coagulation cascade pathway,which provided large data support for the Constitution Theory’s core theory "diseases are related to constitution".