Study On Glutathione-S-transferase P1 Promoter Methylation In Patients With Acute-on-Chronic Hepatitis B Pre-Liver Failure

Background and aimAcute-on-Chronic Hepatitis B Liver Failure(ACHBLF)is an acute hepatic injury on the basis of the infection of chronic hepatitis B(CHB),manifesting as functional disorders such as liver synthesis,excretion,detoxification,and biotransformation,etc.It is clinically characterized by coagulopathy,jaundice,hepatic encephalopathy,hepatorenal syndrome,ascites,and other clinical syndromes.In addition,ACHBLF has the features of rapid progression,poor prognosis,high mortality,and pathophysiological mechanisms to be clarified.Owing to the background above,scholars have proposed the concept of pre-liver failure(pre-LF)in recent years.Pre-LF is at the early stage of liver failure,however,its specific diagnosis standards still remain controversial.In this research,we mainly study the clinical type of acute-on-chronic hepatitis B pre-liver failure(pre-ACHBLF)due to the epidemiological characteristics of the infection of CHB in China.Therefore,we defined pre-ACHBLF as "severe type of acute episodes characterized by serum total bilirubin(TBIL)of 171 umol/L(10 mg/dL)or more and prothrombin activity(PTA)of more than 40%" It would significantly reduce the incidence and mortality of liver failure by early warning,early intervention and early treatment to pre-ACHBLF.This study aimed to evaluate the prognostic value of glutathione-S-transferase P1(GSTP1)gene promoter in patients with pre-ACHBLF via detecting the methylation status of GSTP1 promoter in peripheral blood mononuclear cells(PBMCs).Methods 103 patients with pre-ACHBLF,80 patients with CHB,and 30 healthy controls(HCs)were enrolled in this study.We used methylation-specific polymerase chain reaction(MSP)to detect the methylation status of GSTPl promoter in PBMCs.And its mRNA level was evaluated by real-time quantitative polymerase chain reaction(RT-qPCR).Enzyme-linked immunosorbent assay(Elisa)was performed to assess the indicators of oxidative stress in the serum of pre-ACHBLF.All statistical analyses were conducted with SPSS version 22.0.Results1.The methylation frequency of GSTP1 promoter region in patients with pre-ACHBLF(35/103,33.98%)was significantly higher than CHB(2/80,2.50%;?2=27.664,P<0.001)and HCs(0/30,0.00%;?2 = 13.835,P<0.001),respectively.However,no significant difference of methylation frequency could be observed between CHB patients and HCs(?2 = 0.764,P=0.382).2.The mRNA level of GSTP1 in patients with pre-ACHBLF was significantly lower than CHB(Z =-7.602,P<0.001)and HCs(Z =-5.858,P<0.001).And GSTP1 mRNA level of CHB patients was also prominently lower than HCs(Z =-2.623,P =0.008).3.We found the relative mRNA expression of GSTP1 was positively associated with PTA(r = 0.277,P<0.05),whereas the relative mRNA expression was negatively correlated with TBIL(r =-0.315,P<0.01)and INR(r =-0.315,P<0.01).However,there were no obvious relationships between GSTP1 mRNA and HBsAg,HBeAg,HBV DNA,ALT,AST,ALB,Cr and MELD score.4.Compared with CHB,significant high level of MDA and XOD were found in patients with pre-ACHBLF(231.370± 133.286 nmol/ml vs 138.173±92.087 nmol/ml,P<0.001;40.599±22.890 ng/ml vs 22.675±17.442 ng/ml,P<0.001,respectively).And there were high level of MDA and XOD in methylated groups than unmethylated groups in patients with pre-ACHBLF(264.333±145.339 nmol/ml vs 200.878±114.614 nmol/ml,P= 0.042;44.854±24.517 ng/ml vs 36.663±20.812 ng/ml,P=0.159,respectively).In contrast,significant low level of GSH was found in pre-ACHBLF group when compared with CHB(76.434±37.736 nmol/ml vs 93.290±39.455 nmol/ml,P= 0.015).Also,there was low level of GSH in methylated group than unmethylated group in pre-ACHBLF(64,686±29.038 nmol/ml vs 87.300±41.768 nmol/ml,P=0.013).5.When used to predict the 1,2,3-month incidence of ACHBLF in patients with pre-ACHBLF,the AUCs(Area under the receiver operating characteristic curves)of GSTP1 methylation in PBMCs were 0.825,0.804,and 0.767,respectively.Correspondingly,the AUCs of MELD score were 0.589,0.622,and 0.602,respectively.Interestingly,GSTP1 methylation presented significently higher predictive value than MELD score in predicting 1,2,3-month(all P<0.05)incidence of ACHBLF.Conclusion1.The methylation frequency of GSTP1 promoter in patients with pre-ACHBLF was significantly higher than CHB and HCs.Correspondingly,the mRNA level of pre-ACHBLF was significantly lower than that of CHB and HCs.2.The relative mRNA expression of GSTP1 was possitively associated with PTA.However,it was negatively correlated with TBIL and INR.Thus,it indicated that decreased mRNA expression of GSTP1 may be associated with the development of ACHBLF from pre-ACHBLF and the disease severity of pre-ACHBLF.3.It suggested that oxidative stress may exist in patients with pre-ACHBLF due to its high level of MDA,XOD and low level of GSH.And aberrant DNA methylation was associated with the liver damage induced by oxidative stress.4.When used to predict the 1,2,3-month incidence of ACHBLF in patients with pre-ACHBLF,GSTP1 methylation presented significently higher predictive value than MELD score,suggesting that aberrant methylation of GSTP1 might have the potential role as non-invasive biomarker in predicting the prognosis in patients with pre-ACHBLF.The biomarker may provide us treatment strategies by predicting the prognosis of pre-liver failure,and thus the incidence and mortality of liver failure may be reduced through early warning and intervention.