| Background:China is the hardest hit area of hepatitis B.About 120 million people are carriers of Hepatitis B surface Antigen(HBsAg),and up to 20 million patients with chronic hepatitis B,a considerable part of which will develop into cirrhosis and liver cancer.Current guidelines for the prevention and treatment of chronic hepatitis B at home and abroad have put forward the importance of antiviral therapy,and standardized antiviral treatment should be carried out in patients with immune clearance(IC)or HBeAg-negative chronic hepatitis B(ENH).Through antiviral therapy,it can inhibit viral replication,reduce inflammation,necrosis and fibrosis of hepatocytes,and delay and reduce the occurrence of cirrhosis,liver cancer and complications.However,the existing interferons and nucleic(acid)analogues are still difficult to achieve the ideal end point of HBsAg negative treatment.At present,hepatitis B cure is still a problem,so it is very important to establish a new antiviral/immunomodulatory therapy to improve the functional cure rate of hepatitis B.Current anti-hepatitis B drugs can not completely eliminate the covalently closed cyclic DNA,or cccDNA,which is the main culprit of HBV persistent infection in hepatocytes.Although it is possible that the new combined antiviral immunomodulatory therapy can improve the serological conversion rate of e antigen and S antigen in hepatitis B antiviral therapy,it is still difficult to achieve very satisfactory results.It is hoped to find out the predictive indicators of curative effect before and after treatment,in order to provide individualized treatment according to the specific conditions of patients,which are the difficulties and hot issues of the field of antiviral therapy and also good ways to improve curative effect and save medical resources.Metabolomics detects changes in metabolites under disease conditions on a holistic level.Therefore,metabonomics technology plays a significant role in the early diagnosis,pathogenesis,and efficacy prediction of liver diseases.In this study,a serum metabolic method based on ultra-high performance liquid chromatography mass spectrometry(UPLC/MS)and gas chromatography mass spectrometry(GC/MS)was established to search for biomarkers in serum that can predict the efficacy of antiviral therapy,so as to guide the antiviral treatment of chronic viral hepatitis B.Part I:A new method of antiviral/immunomodulatory therapy to improve the functional cure rate of chronic hepatitis BMethods:Patients with initial HBeAg-positive chronic hepatitis B were randomly and prospectively divided into two groups to receive new antiviral/immunomodulatory therapy:(1)interferon a(IFN-a)+ADV(Adefovir dipivoxil),(2)IFN-a+ADV+GM-CSF(granulocyte macrophage knockdown stimulating factor).52 treatment Groups with IFN-a+ADV treatment and 66 treatment Groups with IFN-a+ADV+GM-CSF treatment were treated for 48 weeks.New antiviral/immunomodulatory treatment regimen:GM-CSF treatment that injects 75 ug subcutaneously once a day on three consecutive days Wednesdays,Thursdays and Fridays in the week 0,4,12,24,36 and 48.Drug use regimen:IFN-a subcutaneous injection of qod(quaque omni die),ADV 10mg qd(Oral on an empty stomach,once a day).The time points of follow-up were at 0,4,8,12,24,36,48 weeks of treatment.The recovery of liver function and HBV negative conversion rate(HBV DNA<20IU/ml),e antigen serological conversion rate and s antigen negative conversion rate(HBsAg<0.05IU/ml)at 24 and 48 weeks after treatment were analyzed.Results:The general data of the two groups were not statistically different at baseline.At the 24th week of treatment,the ALT recurrence rate was 75.76%in the IFN+ADV+GM-CSF group,which was higher than the 57.69%in the IFN+ADV group(p<0.05).HBV DNA negative conversion rate(HBV DNA<20IU/ml)in IFN+ADV+GM-CSF group was 77.27%,higher than the 69.23%in IFN+ADV group,there was no significant between two groups(p=0.324).The seroconversion rate of e antigen(e antigen turns negative,e antibody appears)in the IFN+ADV+GM-CSF group was 33.33%,significantly higher than the 19.23%in the IFN+ADV group(p=0.041).The negative conversion rate of S antigen in IFN+ADV+GM-CSF group(s antigen negative or S antigen seroconversion rate)was 1.51%,higher than the 0%in the IFN+ADV group(p=0.373).At the 48th week of treatment,the ALT recurrence rate was 91.93%in the IFN+ADV+GM-CSF group,which was higher than the 82.69%of IFN+ADV group(p=0.134).HBV DAN negative conversion rate in the IFN+ADV+GM-CSF group was 89.39%,which was higher than the 84.61%of IFN+AD group(p=0.439).The e antigen seroconversion rate of the IFN+ADV+GM-CSF group was 53.03%,which was higher than the 30.77%of IFN+ADV group(p=0.015).The s antigen negative conversion rate of IFN+ADV+GM-CSF group was 6.06%,which was higher than the 1.92%of IFN+ADV group(p=0.268).The proportion of s antigen titer in IFN+ADV+GM-CSF group decreased by more than 90%compared with baseline was 54.55%,which is higher than the 34.62%of IFN+ADV group(p=0.031).The proportion of s antigen titer in IFN+ADV+GM-CSF group decreased by less than 50%compared with baseline was 13.64%,lower than the 38.46%of IFN+ADV group(p=0.002).Part II:Application of serum metabolomics methods based on UPLC/MS and GC/MS in the prediction of Antiviral effect of chronic hepatitis B.Methods:A new method for improving the functional cure rate of chronic hepatitis B for antiviral/immunomodulatory therapy was selected to study the enrolled patients as the subject of this study.According to different curative effects,it was divided into e antigen serological conversion group and non-e antigen serological conversion group.Among them,T1 treatment group(IFN+ADV),16 patients with e-antigen serological conversion and 36 patients with non-e-antigen serological conversion;In the T2 treatment group(IFN+ADV+gm-csf),there were 35 patients with e-antigen serological conversion and 31 patients with non-e-antigen serological conversion.At the 0th,24th,and 48th week of the study,the serum samples of the investigators were placed in an EP tube at-80℃ for storage.All serum samples were thawed from-80℃to 4℃ the night before treatment.The same sample was divided into two parts,all samples were tested randomly,one part was tested by liquid chromatography mass spectrometry,and the other part was tested by liquid chromatography mass spectrometry.After normalization,LC/MS data were imported into Simca-P 13.0 for PC A and OPLS-DA analysis.Screening markers were based on P<0.05 and fold change>1.5 rules.The database retrieval is based on the local HMDB database.The GC/MS mass spectrometry data was analyzed by NIST17 database and then normalized.SIMCA13.0 was used for further OPLS-DA analysis.SPSS software(16.0)and Kruskal-Wallis test were used for multi-factor nonparametric difference analysis,selection of differential metabolites(p<0.05).Spearman correlation analysis was applied to the metabolites with significant difference and HBsAg,HBsAb,HBeAg,HBeAb,HBcAb and HBV DNA.Results:A serum metabolomics diagnostic model based on UPLC/MS and GC/MS was established to predict e antigen seroconversion.Metabolomics analysis was performed on sera at weeks 0,24,and 48 of chronic hepatitis B patients receiving antiviral therapy using UPLC/MS techniques.PCA was performed at metastases at weeks 0,24 and 48 for different treatment groups according to efficacy(whether e antigen seroconversion occurred).It was found that there were significant differences in the metabolic fingerprints of patients with and without e-antigen serological conversion in different treatment groups.Further analysis of the samples using OPLS-DA revealed that the efficacy(whether e antigen seroconversion occurred)was better differentiated at week 0,week 24,and week 48 for different treatment groups.This indicates that the OPLS-DA model can be used to predict the efficacy of e antigen seroconversion.Metabolites with a large contribution(p<0.05)were identified,and found that patients with e antigen conversion had a low concentration of 5Z-dodecenoic acid at week 0,and high concentration of TG(16:1/16:1/16:1)and SM(d18:1/24:0)at week 24 and 48.Analysis of serum metabolites in patients at week 0,week 24 and week 48 for OPLS-DA in patients with e antigen serology or without e antigen seroconversion in different treatment groups,and found that patients receiving e antigen seroconversion were well differentiated at 0,24,and 48 weeks of serum metabolites regardless of whether they received T1 or T2.Serum metabolites at weeks 0,24,and 48 were also well differentiated in patients without e-antigen seroconversion.This indicates that the OPLS-DA model can be used to predict the efficacy of e antigen seroconversion.Metabolites with large contribution(p<0.05)were identified and found that the concentration of PC(18:1/18;1),、PC(20:1/14:1),DG(14:1/22:4/16:0),SM(18:0/14:0)and SM(17:1/24:1)in the serum of patients with e antigen seroconversion was progressively decreased;the concentration of 2-Arachidonylglycerol,8-Hydroxydesmethylclomipramine,3,4-Methylenesebacic acid,N-Acetylglutamine,TG(16:1/16:1/16:1),SM(18:1/24:0),SM(17:1/24:0),2-Arachidonylglycerol and 8-Hydroxydesmethylclomipramine was progressively increased.Metabonomic analysis of sera at weeks 0,24 and 48 of chronic hepatitis B patients receiving antiviral therapy was analyzed using GC/MS.The original data of the metabolome were analyzed and compared with the database NIST17.A total of 70 metabolites with reliable results were identified.OPLS-DA was used to analyze the serum metabolites of patients with or without e-antigen seroconversion at week 0,24 and 48 in different treatment groups and found that serum metabolites of patients with e antigen seroconversion were well differentiated at 0,24,and 48 weeks regardless of T1 or T2 treatment.Serum metabolites were also well differentiated at 0,24,and 48 weeks in patients without e-antigen seroconversion.This indicated that the GC/MS-based OPLS-DA model can be used to predict the efficacy of e antigen seroconversion.Kruskal-Wallis test for multivariate nonparametric difference analysis,selection of differential metabolites(p<0.05),and spearman correlation analysis was applied to the metabolites with significant difference and HBsAg,HBsAb,HBeAg,HBeAb,HBcAb and HBVDNA.The concentration of fumaric acid((E)-2-Butenedioic acid)and Ethanolamine in the serum of patients with e antigen seroconversion was found to decrease progressively,and D-Talose and D-Allose to increase progressively;the concentrations of fumaric acid((E)-2-Butenedioic acid),3-a-mannobiose and glycine(Glycine)were positively correlated with the concentration of HBeAg;the concentration of D-Allose was negatively correlated with the concentration of HBeAg;the concentrations of fumaric acid((E)-2-Butenedioic acid),3-a-mannobiose and glycine(Glycine)were positively correlated with the concentration of HBsAg.Conclusion:IFN-a+ADV+GM-CSF antiviral/immunomodulatory antiviral treatment can improve the e-antigen serological conversion and s antigen conversion rate.By conducting metabolomics studies on the serum of chronic hepatitis B patients receiving new antiviral/immunomodulatory treatment regimens,combined with PCA and OPLS-DA multivariate statistical methods,it was found that metabolomics has a good predictive effect on antiviral efficacy.It can be used to predict the efficacy before or during treatment,thereby further improving the efficacy of antiviral. |
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