Methylation Of GSTM3Gene Promoter And Oxidative Stress In Acute-on-chronic Hepatitis B Liver Failure

PART I METHYLATION OF GSTM3GENE PROMOTER IN ACUTE-ON-CHRONIC HEPATITIS B LIVER FAILUREBackgroundAcute-on-chronic liver failure (ACLF) is an acute hepatic deterioration of chronic liver disease mainly caused by infection, alcohol and hepatotoxic drugs, which mainly manifests as jaundice and coagulopathy, complicated by ascites, encephalopathy and/or hepatorenal syndrome, as well as multiple organ failure. Systemic hemodynamic changes generally accompany the development of ACLF. The major pathophysiologic events of ACLF include systemic inflammatory response syndrome, inflammation and oxidative stress with significant morbidity and short-term mortality. In China, hepatitis B virus (HBV) infection is one of the major causes of ACLF. Acute-on-chronic hepatitis B liver failure (ACHBLF) is the most common liver failure accounting for80%～90%of all the ACLF patients. Because ACHBLF progresses critically and lack of specific clinical treatment measure, most of the patients’prognosis is poor. At present, the underlying molecular mechanism for the pathogenesis of ACHBLF is still uncertain.DNA methylation is one of the DNA modification of transcription level in the CpG island. The aberrant DNA hypermethylation of gene promoter regions is an important epigenetic mechanism that regulates gene expression leading to down-regulation and silencing of several genes. It plays an important role in maintaining normal cell differentiation. Glutathione-S-transferase M3(GSTM3) is an important isozyme of GSTs superfamily. It has strong antioxidant function and can scavenge reactive oxygen species (ROS)(or called oxygen free radicals) inside the body. The methylation of GSTM3gene promoter may be associated with gene silencing. So if the gene expression of GSTM3is inhibited, its antioxidant function may be impaired, and abundant ROS and subsequent oxidative stress may be generated, thus tissues damage including the liver damage can be caused.Abnormal DNA methylation has become one of the most promising molecular markers and a hot topic at present in the early diagnosis, risk assessment, early recurrence forecast, response monitoring to treatment and prognosis judgement of malignant disease. DNA promoter methylation of GSTM3gene has been researched to be associated with some carcinomas, such as bladder carcinoma, hepatocellular carcinoma and prostatic carcinoma, but there is no study on chronic liver disease, especially on ACHBLF.ObjectiveThis study aimed to detect whether there is GSTM3gene promoter methylation in ACHBLF patients and to evaluate the clinical significance and guide clinical application by comparing with clinical related indicators of prognosis.MethodsCase-control study was conducted in this research. A total of30ACHBLF patients,30chronic hepatitis B (CHB) patients and10healthy volunteers as controls were recruited from December2009to February2011in Qilu Hospital of Shandong University. Basic characteristics and clinical data were collected from each subject. Genomic DNA was extracted from peripheral blood by QIAamp DNA Blood Mini Kit, then conducted DNA methylation modification of bisulfite. GSTM3gene promoter methylation was detected by methylation-specific polymerase chain reaction (MSP). According to the result, ACHBLF group was divided into methylated group and unmethylated group. Model for end-stage liver disease (MELD) scores and mortality rate were caculated for these two groups.The data were analyzed using SPSS13.0statistical software (SPSS Inc., Chicago, IL). The difference of GSTM3gene promoter methylation status between ACHBLF and CHB patients was analyzed by Chi-squared test. Laboratory parameters differences between ACHBLF and CHB patients, GSTM3gene promoter methylated group and unmethylated group of ACHBLF patients were analyzed by Independent-Samples T test. Independent-Samples T test was also used to compare the differences of MELD scores between the two groups of ACHBLF patients. The mortality rate between methylated group and unmethylated group of ACHBLF patients was analyzed by Fisher’s exact test. P value<0.05was considered to be statistically significant.Results1. ACHBLF group had significant higher alanine aminotransferase (ALT) levels than those of CHB group (516.110±527.137vs.264.633±274.053IU/L, P=0.024), and significant higher aspartate aminotransferase (AST) levels than those of CHB group (396.283±337.062vs.212.153±222.816IU/L, P=0.016).The TBIL levels were significant higher in ACHBLF group than those of CHB group (383.483±137.034vs.37.400±27.004μmol/L, P<0.001), but PTA levels were significant lower than those of CHB group (31.187±8.013%vs.90.000±9.766%, P<0.001).2. The methylation of GSTM3gene promoter was detected in9cases of30(30%) ACHBLF patients and2cases of30(6.7%) CHB patients. Significant difference was found between the two groups (x2=5.455, P=0.020).3. ACHBLF methylated group had significant higher TBIL levels than those of unmethylated group (527.922±113.564vs.321.581±93.048μmol/L, P<0.001), and significant lower PTA levels than those of unmethylated group (24.811±10.697%vs.33.919±4.604%, P=0.035).4. MELD scores were significantly higher in methylated group than those in unmethylated group of ACHBLF patients (22.706±2.669vs.18.765±4.808, P=0.029). Eight patients died of ACHBLF (26.7%), among them5patients belonging to methylated group (55.6%) and3patients belonging to unmethylated group (14.3%). The mortality rate was significantly higher in methylated group than that in unmethylated group of ACHBLF patients (P=0.032).ConclusionsIn conclusions, our results first demonstrate that GSTM3gene promoter methylation exists in ACHBLF patients. It may lead to the decline of GSTM3gene expression, inhibit its antioxidant function and aggravate oxidative damage. It may play an important role in the progress of CHB to ACHBLF and be used for an important indicator to judge the prognosis of ACHBLF. PART Ⅱ THE CORRELATION OF GSTM3GENE PROMOTER METHYLATION WITH OXIDATIVE STRESS IN ACUTE-ON-CHRONIC HEPATITIS B LIVER FAILUREBackgroundOxidative stress is an imbalance of the oxidantion and antioxidant ability in the body, which results from a lack of antioxidant capacity caused by disturbances in production and distribution, or by an accumulation of reactive oxygen species (ROS) or oxygen free radicals, eventually leading to potential tissue damage. Oxidative stress can cause the oxidation or injury of lipid, protein, enzyme and DNA. It not only can induce gene mutation, protein denaturation and lipid peroxidation, but also is closely related with tumour, cardiovascular and cerebrovascular diseases, diabetes mellitus and all kinds of liver diseases (including viral hepatitis, liver fibrosis and hepatic carcinoma). The liver is rich in mitochondria and is the main target organ of ROS. Mitochondrial respiratory chain complex uses electron transfer to produce adenosine triphosphate (ATP), which is the main source of ROS. Oxidative damage can speed up the progress of hepatic lesions.Acute-on-chronic liver failure (ACLF) is a complicated process caused by many factors, inflammation and oxidative stress play an important role in the occurrence and development of this disease. It is suggested that immune function disorder, systemic inflammatory response, immune complex formation, complement activation and collagen fiber formation can stimulate the generation of ROS and cause the lipid peroxide formation of liver cells. Oxidative stress may underlie the transition from a stable cirrhotic state to progressive liver injury and end-organ failure.The oxidative stress of liver cells can lead to the lipid peroxidation of cell membrane and form the oxidative products such as malondialdehyde (MDA), ketone, hydroxyl and so on. MDA is an end-product of lipid peroxidation and always employed as a serum marker of oxidative stress. Increasing MDA levels suggest the presence of oxidative damage. Glutathione-S-transferase (GST) is an antioxidant enzyme that protects against endogenous oxidative stress, as well as exogenous potential toxins. They detoxify a variety of electrophilic compounds, including oxidized injury of lipid and DNA products generated by ROS damage to intracellular molecules. Several GST variants are principally expressed in the liver whose primary functions include detoxification and metabolism. GST can be quickly released into the blood following the damages of the liver cells. It is an ideal index of reflecting liver cell damage and has good specificity and sensitivity. The changes of MDA and GST levels can reflect the dynamic equilibrium process of internal production and clearance of ROS.The changes of glutathione-S-transferase M3(GSTM3) gene expression will affect the antioxidant function of GST. The part I of our study first demonstrates that GSTM3gene promoter methylation exists in acute-on-chronic hepatitis B liver failure (ACHBLF) patients. It may lead to the decline of GSTM3gene expression, inhibit its antioxidant function and aggravate oxidative damage. It may play an important role in the progress of chronic hepatitis B (CHB) to ACHBLF and be used as an important indicator to judge the prognosis of ACHBLF.ObjectiveThis study aimed to investigate the correlation of GSTM3gene promoter methylation with oxidative stress in ACHBLF patients through detecting the serum levels of MDA and GST then comparing the relationship of MDA and GST with model for end-stage liver disease (MELD) scores, so as to provide theoretical foundation for the illness development, prognosis evaluation and clinical treatment of ACHBLF.MethodsMethylation of GSTM3gene promoter was determined using methylation-specific polymerase chain reaction (MSP) method. Serum biomarkers for oxidative stress including malondialdehyde (MDA) and GST were detected by enzyme-linked immunosorbent assay (ELISA).The data were analyzed using SPSS13.0statistical software (SPSS Inc., Chicago, IL). Independent-Samples T test was used to compare the differences of serum MDA and GST levels between the two groups of ACHBLF patients. The correlations between MDA, GST levels and MELD scores were analyzed by Spearman’s correlation analysis. P value<0.05was considered to be statistically significant.Results1. MDA levels were significantly higher in ACHBLF patients than those in CHB patients (13.045±5.416vs.9.387±5.332pmol/mg, P=0.011).2. MDA levels were also higher in methylated group than those in unmethylated group of ACHBLF patients (16.956±2.155vs.11.369±5.553pmol/mg, P<0.001).3. GST levels were significantly higher in ACHBLF patients than those in CHB patients (1318.081±273.887vs.666.832±181.267mIU/L,P<0.001).4. No significant difference of GST levels was found between methylated group and unmethylated group of ACHBLF patients (1381.678±236.841vs.1262.253±202.782mIU/L,P=0.170).5. MDA levels were positively correlated with MELD scores of ACHBLF (r=0.588, P=0.001), while no any correlation was found between GST levels and MELD scores of ACHBLF (r=0.115, P=0.546)ConclusionsGSTM3gene promoter methylation is correlated with oxidative stress in ACHBLF patients. GSTM3gene promoter methylation may play an important role in the pathogenesis of ACHBLF. It may contribute to oxidative stress-associated liver damage in ACHBLF and be correlated with the severity of ACHBLF. This may provide theoretical basis for the antioxidant therapy of ACHBLF.