ITRAQ-based Proteomic Analysis Of Plasma Proteins In Patients With CHB And Noninvasive Diagnostic Models Of Liver Fibrosis And Objective Study Of TCM Syndromes

BackgroundChronic hepatitis B is the most common infectious disease in China,and liver fibrosis is the early stage of transition from chronic hepatitis B to cirrhosis.Early diagnosis and timely treatment of liver fibrosis can control or even reverse the progression of cirrhosis.Liver biopsy is a"gold standard"for the diagnosis of liver fibrosis,but as an invasive test,it is clinically limited.In this study,based on the pathology of liver puncture,we could study the correlation,sensitivity and diagnostic value of non-invasive diagnostic testing commonly indicators used in different fibrosis grades,and analyze the correlations between various indicators and different TCM syndrome types.The role and diagnostic value of commonly used clinical indicators in fibrosis staging and TCM syndromes were studied.A non-invasive diagnostic model with multiple clinical indicators as variables was constructed and investigated the diagnostic efficiency for chronic hepatitis B liver fibers.Several studies had demonstrated that there were some abnormal glycosylation phenomena in the serum of patients with different stages of liver disease,and were closely related to the development of the disease.Therefore,exploring and discovering the biomarkers of diagnosis of hepatic fibrosis has become one of the new research hotspots?QuantitativeproteomicsusingiTRAQcombinedwithliquid chromatography and tandem mass spectrometry analysis can provide us with specific markers for potential hepatic fibrosis and the pathogenesis of hepatic fibrosis.Objective(1)Based on liver biopsy and the pathological staging of hepatitis,to explore the correlation betweenroutine and biochemical indicators,new serological markerWFA~+-M2BP,spleen thickness,hepatic elasticity index,and severity of liver fibrosis in patients with chronic hepatitis B,and to analysis the diagnostic effect of single indexes on different stages of liver pathology,and the diagnostic value of a non-diagnosis model of liver fibrosis on liver fibrosis and early cirrhosis.(2)According to the classification of TCM syndromes,to investigate the routine and biochemical indicators,WFA~+-M2BP,liver fibrosis,spleen thickness,hepatic elasticity index,liver reserve function and liver fibrosis different syndromes in each group.(3)Based on iTRAQ combined with liquid chromatography and tandem mass spectrometry,comparing serological proteomics in subjects with different stages of liver fibrosis.Methods(1)To collect liver tissue of liver biopsy in Hospital from 2016 to 2018,a small part of which was collected from splenectomy and liver transplantation,a total of 36 patients with chronic hepatitis B confirmed by pathological diagnosis were collected at the same time.There were 10healthy people in the center of outpatient examination.Patients with chronic hepatitis B were divided into four groups according to pathological diagnosis of hepatic fibrosis:non-hepatic fibrosis(S0-S1)group(n=12);mild hepatic fibrosis(S2)(n=9);prominent hepatic fibrosis(S3)(n=8);early cirrhosis group(?S4)(n=7).To investigate the correlations between blood routine,liver function,liver fibrosis,coagulation,WFA~+-M2BP,aspartate aminotransferase and platelet ratio(APRI),hepatic elasticity index,spleen thickness,and other pathological stages of hepatic fibrosis in these groups.To diagnose the sensitivity,specificity,and AUROC at the different stages of liver fibrosis.Comebined with the data,we screen out three non-invasive liver fibrosis diagnosis model:S index model,ASPRI index model,FSM model to assess the three non-invasive diagnostic model for the diagnosis of liver fibrosis.(2)Thirty-six patients with liver fibrosis were divided into three groups:hepatobiliary damp-heat type(n=13),liver-stagnancy and spleen-deficiency type(n=11),and blood stasis-responsive type(n=12).The routine indexes,WFA~+-M2BP,hepatic elasticity index,liver function,and coagulation function were studied.The correlations of four items of liver fiber,hepatic color Doppler ultrasound-portal vein and spleen thickness with classification of TCM syndromes were compared.(3)Quantitative proteomics techniques based on iTRAQ combined with liquid chromatography and tandem mass spectrometry were used to detect the differential expressions of plasma proteins between healthy patients and subjects?Results1.We found that PLT?WBC?neutrophil count?PT%?FIB were negatively positively correlated with liver fibrosis stages;but TBIL?DBIL?PT?APTT?HA?P?NP were significantly correlated with the staging of liver fibrosis;WFA~+-M2BP?spleen thickness?elastic index?APRI and liver fibrosis staging were significantly positively correlated.The differences were statistically significant.2.We also found that the AUROCs of APRI,elasticity index,spleen thickness,hyaluronic acid(HA),and type III collagen amyloid peptide(PIIINP)in the diagnosis of early cirrhosis of chronic hepatitis B were0.968?0.922?0.954?0.854?0.971.The above indicators had high diagnostic value in the severe hepatic fibrosis and early hepatic cirrhosis.3.The predictions of WFA~+-M2BP on the AUROCs of patients in severe hepatic fibrosis and in early hepatic cirrhosis was 0.765?0.973,the above indicator had high diagnostic value in early hepatic cirrhosis.4.The predictions of S index model?ASPRI and FSM on the AUROCs of patients in severe hepatic fibrosis(stage S3-4)were 0.854?0.844?0.898,and in early hepatic cirrhosis(S4)were 0.867?0.975?0.966.The above indicators had high diagnostic value in the severe hepatic fibrosis and early hepatic cirrhosis.5.The levels of platelet?hyaluronic acid?PIIIP?WFA~+-M2BP?APRI?spleen thickness?elastic index and liver fibrosis stagingwere significantly higher in the blood stasis-responsive typethan in hepatobiliary damp-heat type,liver-stagnancy and spleen-deficiency type.The levels of spleen thickness?elastic index and liver fibrosis staging were significantly higher in the blood stasis-responsive typethan other type,and were positively correlated with the syndromes of TCM.6.(1)The Protein Group was identified as 762 by iTRAQ-LC-MS based proteomics study.Using P value<0.1,Fold Change<0.83 or Fold Change>1.2,and log Change>2 as the reference range,we found that there were a lot of differential protein among the different stage of liver fibrosis.(2)Screening differential proteins closely related to the different staging of liver fibrosis and analysis of the differential protein expression level,we found the protein of ATXN2?TUFM and ZNF407 were correlated with different stages of liver fibrosis.(1)The levels of ATXN2in different stages of fibrosis(S0-1,S2,S3,and S4)were 1.109,0.682,0.455,and 0.179;(2)The levels of TUFM in different stages of fibrosis0.641?0.449?0.099?0.118;(3)The levels of ZNF407 in different stages of fibrosis(S0-1,S2,S3,and S4)were 0.641?0.449?0.099?0.118.Conclusion1.There were closely correlation between white blood cell count,neutrophil count,PLT,HA,PIIIP,WFA~+-M2BP,APRI index,spleen thickness,hepatic elasticity index with liver fibrosis staging severity.These indicators were of great value in diagnosing early cirrhosis of chronic hepatitis B,with high sensitivity and specificity.2.the serological marker of WFA~+-M2BP had the had high diagnostic value in early hepatic cirrhosis;The S index model,ASPRI model,and FSM model were valuable in the diagnosis of chronic hepatitis B S0-2/S3-4 stage and S0-3/S4 stage.3.The levels of platelet,hyaluronic acid,PIIIP?WFA~+-M2BP?spleen thickness?elastic index and liver fibrosis staging were significantly higher in the blood stasis-responsive typethan other type,and were positively correlated with the syndromes of TCM.4.The iTRAQ-based proteomics study can identify differential proteins ATXN2?TUFM and ZNF407 with clinical stages and different syndrome types.This differential proteinscould be used as new targets for the early diagnosis of hepatic fibroblast markers and to investigate the molecular mechanisms of liver fibrosis.