| BackgroundAcute-on-chronic hepatitis B liver failure(ACHBLF)is a clinical syndrome,characterized by the rapid deterioration of liver function in patients with chronic liver disease associated with hepatitis B virus(HBV)infection.ACHBLF has a high incidence in patients with chronic hepatitis B in China,its main manifestations are coagulopathy,jaundice,hepatic encephalopathy,ascites,etc.The disease progresses rapidly,eventually leading to multiple organ dysfunction and high mortality.The pathogenesis of ACHBLF is very complex.Excessive inflammatory response is an important pathophysiological mechanism of disease progression in patients with liver failure.Therefore,the role of inflammatory related cytokines in the pathogenesis of liver failure has received increasing attention.In recent years,studies have found that inflammatory markers such as macrophage inflammatory protein 3α,interleukin,soluble protein CD 163 and mannitol receptor are predictive factors for the development of liver failure disease,but their sensitivity and specificity are low,resulting in delaying the judgment of the prognosis of liver failure.Therefore,there is an urgent need to find new indicators to judge the outcome and therapeutic effect of patients with liver failure.Thymosin β4(Tβ4)has attracted much attention as a new biological marker of hepatocyte inflammation damage.Tβ4 is a major actin sequestration protein,which participates in many biological processes,including angiogenesis,tissue remodeling,wound repair and cancer progression.Another important biological function of Tβ4 is anti-inflammatory effect.Its inflammatory regulatory effect mainly includes inhibiting the migration of macrophages and the production of inflammatory cytokines,and can also alleviate the inflammatory response by reducing the infiltration of inflammatory cells.Tβ4 is closely related to a variety of liver diseases.Recent studies have shown that Tβ4 ameliorates acute hepatocyte injury and liver fibrosis by reducing oxidative stress and inflammatory responses.It has been reported that the serum level of Tβ4 was correlated with the severity of patients with liver failure.Tβ4 might be involved in mediating the release of cellular inflammatory factors and might become one of the disease evaluation and early warning indicators of liver failure.DNA methylation is a process in which add a methyl group to a specific base with S-adenosylmethionine as the methyl donor under the action of DNA methyltransferases(DNMTs).Aberrant methylation of gene promoters leads to abnormal gene expression.DNA methylation is associated with many diseases,such as tumors,nervous system diseases and autoimmune diseases.Emerging evidence suggests that DNA methylation plays a crucial role in immune regulation.Our previous study has confirmed that abnormal methylation of genes such as glutathione S-transferase M3,suppressor of cytokine signaling 1 and estrogen receptor 1 plays an important role in the occurrence and development of HBV-related liver failure.The expression of Tβ4 gene was normal in healthy people,but decreased in patients with liver failure,which can be used as an evaluation index of liver failure.It has been reported aberrant promoter methylation of Tβ4 gene can lead to its abnormal expression in hepatocellular carcinoma.However,it remains unclear whether abnormal expression of Tβ4 gene in serum of patients with liver failure is due to altered DNA methylation status.Therefore,the methylation status of Tβ4 gene in serum of patients with ACHBLF and the related regulatory mechanisms need to be further investigated.Glucocorticoids(GCs),as effective anti-inflammatory agents,can inhibit the phagocytosis of antigens by macrophages,inhibit the production of inflammatory cytokines,rapidly inhibit excessive immune response,and play an important role in blocking the progression of disease.The application of GCs in the early stage of liver failure can reduce the inflammatory response of the body and reduce the damage to tissues and organs,thereby delaying the progress of hepatitis.Therefore,GCs has a theoretical basis for the treatment of liver failure.However,so far,there still has no unified conclusion on the efficacy of GCs in the treatment of ACHBLF.How to screen out the advantaged population with GCs sensitivity and optimize therapeutic indications is the focus of controversy.It has been confirmed that GCs treatment can affect the expression and methylation status of several genes,which has an important hint role in the evaluation of the efficacy of GCs.Based on this,we believe that it is reasonable to screen the advantaged population of GCs therapy based on the molecular characteristics of methylation markers.However,the methylation status of Tβ4 promoter in ACHBLF and the effect of GCs have not been reported.Therefore,this study was divided into three parts,mainly to investigate the expression of Tβ4 in patients with ACHBLF,evaluate the early predictive value of Tβ4 methylation status on the prognosis of patients with ACHBLF,observe the efficacy of glucocorticoids on ACHBLF and its effect on the methylation status of Tβ4,and explore its possible mechanism.The main content is shown in the general design roadmap of this project.PART Ⅰ Expression of Thymosin β4 Gene and its Promoter Methylation in Patients with Acute-on-Chronic Hepatitis B Liver FailureBackgroundHepatitis B virus(HBV)infection is a major global health problem.Globally,it is estimated that more than 20 billion people have serological evidence of previous or current hepatitis B virus infection,of which 2.57 million are chronically infected.The clinical syndrome of acute liver decompensation and liver failure on the basis of chronic hepatitis B(CHB)in the short term is called acute-on-chronic hepatitis B liver failure(ACHBLF).The condition of ACHBLF was severe,with many complications,difficult treatment and high fatality rate.Early warning,diagnosis,prognosis assessment and timely diagnosis and treatment are very important to reduce the mortality of patients with ACHBLF.Therefore,it is urgent to find effective indicators for early assessing the severity of patients with liver failure.Thymosin β4(Tβ4)is a highly conserved small acidic peptide,which is involved in the occurrence of a variety of diseases.In recent years,it has been found that Tβ4 is closely related to a variety of liver diseases.In patients with viral hepatitis,the more severe the inflammatory response of hepatocytes,the lower level of serum Tβ4,which can be used as an evaluation index of the condition of hepatitis patients.It has been confirmed that in ACHBLF,once inflammatory state of hepatocytes was activated,a large number of inflammatory cytokines are released,and the expression level of Tβ4 is reduced.Previous studies from our team have shown that the abnormal expression of several genes in patients with chronic liver failure was due to aberrant methylation of their promoter regions.Aberrant methylation of Tβ4 gene has been found in patients with chondrosarcoma or hepatocellular carcinoma to regulate its expression and participate in the development of the disease.However,the methylation status of Tβ4 in the serum of patients with ACHBLF and the related regulatory mechanism are still unclear,and no report of this at home or abroad.Objectives1.To determine the expression and methylation status of Tβ4 in peripheral blood mononuclear cells(PBMCs)from patients with ACHBLF.2.To analyze the correlation between Tβ4 methylation and inflammatory cytokines(interleukin-6(IL-6),IL-10,and tumor necrosis factor-α(TNF-α))and disease assessment indicators of patients with ACHBLF.3.To investigate the role and clinical significance of Tβ4 promoter methylation in the progression of liver failure.MethodsIn this study,we enrolled a total of 120 patients with ACHBLF,45 patients with CHB and 32 healthy controls(HCs)from the Department of Hepatology,Qilu Hospital of Shandong University from January 2019 to December 2020.Real-time quantitative polymerase chain reaction(RT-qPCR)was used to detect the relative expression of Tβ4 mRNA in peripheral blood mononuclear cells(PBMCs),methylation-specific polymerase chain reaction(MSP)was used to determine the methylation status of Tβ4,and enzyme-linked immunosorbent assay(ELISA)was used to detect the concentration of Tβ4 and inflammatory cytokines in plasma.Firstly,the expression levels of Tβ4 and inflammatory cytokines in ACHBLF,CHB and HCs groups were compared.Secondly,the correlation between inflammatory cytokines(IL-6,IL-10,TNF-α)and clinical evaluation indexes(total bilirubin(TBIL),prothrombin activity(PTA),alanine aminotransferase(ALT),model for end-stage liver disease(MELD)scores)of hepatitis was analyzed.Finally,the correlation of Tβ4 mRNA level and its methylation status with the levels of diseases assessment indicators and inflammatory cytokines were tested,and the levels of clinical evaluation indexes under different methylation status of Tβ4 were compared.Statistical analysis was performed using SPSS23.0 software(SPSS,Chicago,IL,USA)and GraphPad Prism 9.0 software(San Diego,CA,USA).Results1.The concentrations of inflammatory cytokines(IL-6,IL-10 and TNF-α)and the expression of Tβ4 gene in different groups1.1 The plasma concentrations of IL-6,IL-10 and TNF-α in ACHBLF group and CHB group were higher than in HCs group,and those in ACHBLF group were higher than CHB group,the differences were significant(P<0.05).1.2 The Tβ4 methylation frequency of patients with ACHBLF was significantly higher than that in CHB group and HCs group(P<0.001).In addition,that in CHB group was also higher than HCs group(P<0.001).The expression of Tβ4 mRNA and its plasma concentration was detected in ACHBLF group,CHB group and HCs group.The mRNA level and plasma concentration of Tβ4 in HCs group were the highest,which were significantly higher than those in CHB and ACHBLF group,and those in CHB group were also higher than ACHBLF group(P<0.001).2.Relationships of inflammatory cytokines(IL-6,IL-10 and TNF-α)with disease assessment indicatorsSpearman correlation analysis tested the correlation between the level of cytokines(IL-6,IL-10 and TNF-α)and clinical evaluation indexes of patients with ACHBLF,indicating that serum TBIL level,serum ALT level and MELD score were positively correlated with the plasma concentrations of cytokines IL-6,IL-10 and TNF-α(P<0.05),and serum PTA level was negatively correlated with those(P<0.05).3.Relationships of Tβ4 mRNA with inflammatory cytokines and disease assessment indicators3.1 The expression of Tβ4 mRNA in PBMCs from patients with ACHBLF was negatively correlated with the concentrations of cytokines IL-6,IL-10 and TNF-α(P<0.05).3.2 Tβ4 mRNA level was negatively correlated with serum TBIL level(r=-0.256,P=0.005),serum ALT level(r=-0.244,P=0.007)and MELD score(r=-0.207,P=0.023),but positively correlated with PTA(r=0.227,P=0.013).4.Relationships of Tβ4 methylation with inflammatory cytokines and clinical indicators4.1 The plasma concentrations of IL-6,IL-10 and TNF-α in the methylated group were all significantly higher than those in unmethylated group(P<0.001),and Tβ4 methylation status was positively correlated with the concentrations of cytokines(IL-6,IL-10,and TNF-α)(IL-6:r=0.293,P=0.001;IL-10:r=0.330,P<0.001;TNF-α:r=0.318,P<0.001).4.2 There were no significant relationships with regard to age,sex,ALT,albumin(ALB),creatinine(Cr),cholinesterase(CHE),pre-albumin(pre-ALB),hepatitis B e antigen(HBeAg),hepatitis B virus DNA(HBV-DNA),hepatic encephalopathy(HE)and ascites between the patients from methylated and unmethylated groups of ACHBLF(P>0.05),but the serum TBIL level,serum PTA level,MELD score and the level of Tβ4 mRNA were significantly different between the two groups(P<0.05).4.3 Correlation analysis showed that Tβ4 methylation status was negatively correlated with its mRNA level(r=-0.593,P<0.001),and were highly correlated with serum TBIL level,serum PTA level and MELD score(r=0.464,-0.376,0.248,P<0.01).4.4 TBIL and PTA are independent risk factors for Tβ4 promoter methylation.ConclusionsThe expression level of Tβ4 was negatively correlated with the severity of hepatocyte inflammation.Tβ4 methylation may be involved in the inflammatory reaction process of liver failure by regulating the release of cellular inflammatory cytokines(IL-6,IL-10 and TNF-α),which can be used as an effective indicator to evaluate the progression of disease in patients with ACHBLF.Part Ⅱ Early Predictive Value of Thymosin β4 Promoter Methylation on the Prognosis of Patients with Acute-on-Chronic Hepatitis B Liver FailureBackgroundThere are four phases of acute-on-chronic hepatitis B liver failure(ACHBLF):ascending,plateau,descending,and recovery.However,before the ascending stage,a period of severe liver failure,namely acute-on-chronic hepatitis B pre-liver failure(pre-ACHBLF),is experienced.Once it progresses to liver failure,the mortality rate of patients is extremely high.This period is also called "golden window" for effective clinical intervention.During this golden period,there is less degeneration or necrosis of liver cells,and if the excessive immune response can be inhibited,the progression to ACHBLF may be prevented.Early warning and intervention of the patients with pre-ACHBLF are essential to reduce the morbidity and mortality of ACHBLF.Therefore,early sensitive and accurate predictive biomarkers for the prognosis of patients with ACHBLF are urgently needed.Our previous studies have shown that thymosin β4(Tβ4)is involved in the process of hepatocyte inflammatory response,and its expression level is closely related to the severity of inflammation.Previous studies have shown that Tβ4 combined with model for end stage liver disease(MELD)score and Tβ4 combined with alpha-fetoprotein(AFP)have certain predictive value for the prognosis of patients with ACHBLF.In conclusion,detection of Tβ4 gene is instructive for the prognosis of patients with ACHBLF.However,previous studies only used enzyme-linked immunosorbent assay to measure the level of serum Tβ4,and there is still a lack of relative quantitative evaluation of the relationship between the expression of Tβ4 and the prognosis of patients with ACHBLF,and its early predictive value for the prognosis of ACHBLF patients is still unclear.Our study has confirmed that Tβ4 methylation could be used as an noninvasive indicator of the severity of patients with liver failure.Detection of Tβ4 methylation status in pre-ACHBLF stage and evaluation the early predictive value for the prognosis of ACHBLF patients,may serve as an important reference for the early diagnosis and treatment of diseases such as ACHBLF.ObjectivesThis study was to investigate the diagnostic value of Tβ4 promoter methylation in pre-ACHBLF and its early predictive value in morbidity and mortality of patients with ACHBLF.MethodsThe study recruited 115 patients with ACHBLF,80 with pre-ACHBLF,and 86 with chronic hepatitis B(CHB).In addition,there were 36 health controls(HCs).All were recruited at the Department of Hepatology,Qilu Hospital of Shandong University from October 2018 to April 2020.The patients with ACHBLF were divided into three subgroups:33 cases of early stage ACHBLF(E-ACHBLF),42 of mid-stage ACHBLF(M-ACHBLF)and 40 of advanced stage ACHBLF(A-ACHBLF).Moreover,from May 2020 to September 2021,96 patients with ACHBLF and 65 patients with pre-ACHBLF were enrolled as validation groups.The 96 patients with ACHBLF included 30 cases of E-ACHBLF,32 of M-ACHBLF and 34 of A-ACHBLF.Tβ4 promoter methylation status was measured by methylation-specific polymerase chain reaction,and its mRNA was detected by real-time quantitative polymerase chain reaction.Cox proportional hazards model was used to analyze the prognostic factors affecting the survival time of patients with ACHBLF.Receiver operating characteristic(ROC)curve were used to evaluate the diagnostic value of Tβ4 promoter methylation and MELD score for patients with pre-ACHBLF and the early predictive value of them for the morbidity and mortality of patients with ACHBLF.Results1.Expression pattern of Tβ4 gene in PBMCs from patients in each groupTβ4 promoter methylation frequency increased gradually from HCs group through CHB group,pre-ACHBLF group,E-ACHBLF group,M-ACHBLF group and A-ACHBLF group.The expression level of Tβ4 mRNA showed an opposite trend.The average relative expression level of Tβ4 mRNA was the highest in HCs group,but decreased in the order CHB group>pre-ACHBLF group>E-ACHBLF group>M-ACHBLF group>A-ACHBLF group.2.Relationships of Tβ4 promoter methylation frequency and its mRNA level with the clinicopathological characteristics of patients with ACHBLF2.1 Tβ4 methylation frequency was negatively correlated with its mRNA level(r=0.325,P<0.001).Tβ4 mRNA level was significantly correlated with TBIL,PTA and MELD scores(P<0.01).2.3 Single factor analysis indicated that Tβ4 methylation frequency was significantly associated with total bilirubin(TBIL),prothrombin activity(PTA)and MELD score(P<0.05).Spearman correlation analysis showed that the methylation frequency of Tβ4 promoter was positively correlated with TBIL and MELD score(P<0.05),and negatively correlated with PTA(r=-0.241,P=0.01).3.Diagnostic value of Tβ4 methylation for discriminating pre-ACHBLF from CHBThe sensitivity,specificity,positive predictive value(PPV)and negative predictive value(NPV)of Tβ4 methylation in differentiating patients with pre-ACHBLF from CHB were 68.75%,89.53%,85.94%and 75.49%,respectively.The area under the receiver operating characteristic curve(AUC)for Tβ4 methylation was 0.803(SE:0.036;95%confidence interval(CI):0.735-0.861).4.Relationships of Tβ4 promoter methylation and the prognosis of patients with ACHBLFSurvival analysis showed that the survival rate of ACHBLF patients with Tβ4 promoter methylation was higher than that without methylation in the model group and validation group.COX proportional hazard regression analysis showed that TBIL(P=0.048),PTA(P=0.043),MELD score(P<0.01)and Tβ4 promoter methylation(P<0.01)were correlated with the survival time of patients with ACHBLF in the model group.Multivariate regression analysis showed that MELD score and Tβ4 promoter methylation were independent risk factors for the survival time of patients with ACHBLF.In the validation group,COX univariate regression analysis showed that TBIL(P=0.049),PTA(P=0.042),MELD score(P=0.041),and Tβ4 promoter methylation(P<0.01)were correlated with the survival time of patients with ACHBLF.Multifactor regression analysis revealed that only Tβ4 promoter methylation was an independent risk factor for the survival time of patients with ACHBLF.5.Predictive value of Tβ4 methylation and MELD score for the prognosis of patients with ACHBLF in the model group5.1 As the follow-up time increased from 1-,2-to 3-month,so did the percentage of pre-ACHBLF that progressed to ACHBLF(36.25%vs.56.25%vs.72.50%,respectively).ROC curve analysis showed that Tβ4 promoter methylation had a higher predictive value than MELD score,when predicting the incidence of ACHBLF at 1-,2-,and 3-months(P=0.005,0.037,0.048).5.2 At the end of the 3-month follow-up period,the mortality rates of patients with E-ACHBLF,M-ACHBLF,and A-ACHBLF were 45.45%,54.76%and 62.50%,respectively.The AUC for Tβ4 promoter methylation for predicting the mortality of patients with E-ACHBLF was higher than that for MELD score(P<0.001).The AUC for Tβ4 promoter methylation in patients with M-ACHBLF was also higher than that for MELD score(P<0.05).Interestingly,the AUC for Tβ4 methylation for predicting the mortality of patients with A-ACHBLF was lower than that for MELD score,but the difference was not significant(P=0.429).6.Predictive value of Tβ4 methylation for the prognosis of patients with ACHBLF in the validation group6.1 Tβ4 methylation had a higher predictive value than MELD scores in validation group for predicting the incidence of ACHBLF in 1-,2-,and 3-months(P=0.036,0.024,0.046),which was consistent with the results of model group.6.2 When predicting the mortality of patients with E-ACHBLF and M-ACHBLF,the AUC for Tβ4 methylation all were higher than MELD score in validation group(P<0.05).However,when predicting the mortality of patients with A-ACHBLF,Tβ4 promoter methylation was slightly higher than MELD score with no significant difference(P=0.968).ConclusionsTβ4 methylation can early diagnose and identify pre-ACHBLF patients from CHB,and also early predict the morbidity and mortality of patients with ACHBLF.Therefore,Tβ4 methylation might be an important early biomarker for predicting the prognosis of patients with ACHBLF.PART Ⅲ Intervention Effect of Glucocorticoids on Acute-on-Chronic Hepatitis B Liver Failure and its Influence on the Methylation Status of Tβ4 PromoterBackgroundAcute-on-chronic hepatitis B liver failure(ACHBLF)is a common clinical emergency and severe disease with complex clinical manifestations,rapid progress,and multiple organ dysfunction in a short period of time.However,the treatment measures of it are limited and the prognosis is poor.Plasma exchange and hemodialysis filtration therapy have been used in patients with hepatitis B virus(HBV)infection,but that are only partially effective.Liver transplantation is considered as a valuable treatment to save patients and improve prognosis,but its wide application is limited by the lack of liver donors and high cost.Glucocorticoids(GCs)therapy has been recommended as a first-line treatment strategy for severe alcoholic hepatitis and hepatic encephalopathy.Due to its strong anti-inflammatory effect in addition to immunomodulatory effect,it has been receiving attention as a possible treatment for ACHBLF.The application of GCs in the early stage of liver failure can reduce the inflammatory response of the body and reduce the damage of inflammatory response to tissues and organs,so as to delay the progression of hepatitis.In theory,GCs may be beneficial for patients with liver failure.However,there has been no consensus on the feasibility of GCs for the treatment of ACHBLF.How to screen out the advantaged population with GCs sensitivity and optimize therapeutic indications is the focus of controversy.Thymosin β4(Tβ4)is an important G-actin protein,which is normally expressed in the liver,and its expression is down-regulated in patients with HBV and hepatitis C virus(HCV)during severe inflammation.With the progression of chronic hepatitis B,the damage of liver cell is gradually aggravated,the serum Tβ4 level decreases gradually after progressing to liver failure,and the reduction degree is positively correlated with the severity of liver failure.In the first part of our study,we have confirmed that Tβ4 was involved in the process of liver inflammation and could be used as one of the indicators for assessing the severity of liver failure.As an effective anti-inflammatory agent,GCs can inhibit the production of inflammatory factors and play an important role in blocking the progression of hepatocyte inflammation.In vitro and in vivo studies have confirmed that GCs can play an anti-inflammatory role in liver failure by down-regulating the expression of several inflammation related genes.Previous studies have shown that GCs therapy can cause gene demethylation,thereby affecting its expression.Therefore,it is reasonable to screen the sensitive population of GCs according the molecular characteristics of methylation biomarkers.However,the effect of GCs therapy on the expression level and methylation status of Tβ4 gene has not been reported so far.Objectives1.To observe the effects of GCs therapy on the condition evaluation and clinical indexes of patients with ACHBLF,in order to explore the efficacy of GCs,2.To evaluate whether Tβ4 gene can be used as an evaluation index for the efficacy of GCs in the treatment of liver failure.3.To study the effect of GCs on the methylation status of Tβ4 in peripheral blood mononuclear cells(PBMCs)from patients with ACHBLF,and to explore the relationship between Tβ4 promoter methylation and GCs.MethodsIn this study,120 patients with ACHBLF and 32 healthy controls were enrolled from the Department of Hepatology,Qilu Hospital of Shandong University from January 2019 to December 2020.Among 120 patients with ACHBLF,56 patients who received GCs therapy were classified as glucocorticoids treatment group,64 patients who did not receive GCs therapy were classified as conventional treatment group.Real-time quantitative PCR(RT-qPCR)was used to detect the relative expression of Tβ4 mRNA in PBMCs,methylation-specific PCR(MSP)was used to determine the methylation status of Tβ4,and enzyme-linked immunosorbent assay(ELISA)was used to detect the concentration of Tβ4 and inflammatory cytokines including interleukin-6(IL-6),IL-10,and tumor necrosis factor-α(TNF-α)in plasma.The expression of Tβ4 and the concentrations of inflammatory cytokines were compared between the glucocorticoids treatment group and the conventional treatment group before and after treatment.Moreover,the dynamical changes of Tβ4 mRNA level,plasma concentration and methylation status were observed on the 0,7th,and 28th day after GCs therapy.The effects of GCs on the methylation status of Tβ4 and the survival rate of patients with ACHBLF were further analyzed.Results1.Changes of various indexes in patient with ACHBLF before and after GCs therapy1.1 Before treatment,there were no significant differences in serum TBIL level,serum PTA level and MELD score between glucocorticoids treatment group and conventional treatment group(P>0.05).After treatment,TBIL level and MELD score of glucocorticoids treatment group were lower than that of conventional treatment group,while serum PTA level was significantly higher(P<0.05).1.2 Before treatment,there were no significant differences in the mRNA level,plasma concentration and methylation frequency of Tβ4 between glucocorticoids treatment group and conventional treatment group(P=0.703;0.107;0.707).After treatment,the mRNA levels and plasma concentrations of Tβ4 in glucocorticoids treatment group were significantly higher than those in conventional treatment group(P<0.05),but its methylation frequency was lower(P<0.05).1.3 After treatment,the incidence of adverse reactions of patients in glucocorticoids treatment group(37.50%)was no higher than that in conventional treatment group(46.90%)(P=0.356).At the end of the 90-day follow-up period,the patients in glucocorticoids treatment group had a higher survival rate than those in conventional therapy group(P=0.044).2.Changes of various indexes in patients with ACHBLF at different time points after GCs therapy2.1 The serum TBIL level,serum PTA level and MELD score were no different before and on the 7th day of GCs therapy(P>0.05).However,on the 28th day,serum TBIL level and MELD score were lower than on 7th day,whereas PTA level was higher(P<0.001).2.2 On the 7th day,the plasma concentrations of IL-6,IL-10 and TNF-α were lower than those before treatment(P<0.001).The level of inflammatory cytokines on the 28th day was significantly lower than that on the 7th day(P<0.001).2.3 The mRNA level and plasma concentration of Tβ4 increased gradually during treatment,and those were significantly higher on the 28th day than those before and on the 7th day of GCs therapy(P<0.001).Moreover,those on the 7th day was also higher than before treatment(P<0.05).3.Relationships of Tβ4 promoter methylation and GCs3.1 Before treatment,the methylation frequency of Tβ4 promoter was 64.30%(36/56)in glucocorticoids treatment group.On the 7th day,4 patients with ACHBLF showed demethylation of Tβ4 promoter,with a methylation frequency of 57.10%(32/56).On the 28th day,when GCs therapy ended,there were 11 patients with ACHBLF who showed demethylation,and the methylation frequency of Tβ4 promoter was 37.50%(21/56).3.2 Before treatment,patients in glucocorticoids treatment group were divided into methylated and unmethylated groups.A total of 15 patients in the methylated group underwent demethylation after GCs therapy,of which 11 survived,compared with 7 of 21 patients who did not undergo demethylation.The survival rate of patients with demethylation was significantly higher than that of patients without demethylation(73.33%vs.33.33%,P=0.041).3.3 The overall survival rate of patients in glucocorticoids treatment group was 60.71%and that was 50.0%in the methylated group,which was significantly lower than that in the unmethylated group(80.0%)(P=0.045).The survival rate of unmethylated patients(46.43%)in the conventional treatment group was not significantly different from that of methylated patients(36.11%)(P=0.450).In addition,the survival rate of unmethylated patients in glucocorticoids treatment group was significantly higher than that in the conventional treatment group(P=0.035).3.4 On the 7th day,the TBIL level from unmethylated patients in glucocorticoids treatment group was lower than before treatment,and the PTA level was higher,the differences were significant(P=0.033;0.027).However,the serum TBIL and PTA levels in the methylated group were not significantly changed compared with those before treatment(P>0.05).ConclusionsThe efficacy of GCs in patients with ACHBLF is worthy of affirmation.The expression of Tβ4 is regulated by GCs,and its promoter methylation can be used as an indicator to evaluate the efficacy of GCs therapy on liver failure.Detection of the methylation status of Tβ4 promoter before treatment may be a screening condition for GCs therapy in patients with ACHBLF. |
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