Effect Of NAFLD Combined With CHB On Global DNA Methylation Of Human Hepatocytes And Intervention Effect Of Betaine

Objective:In recent years,with the prevalence of obesity and metabolic syndrome,nonalcoholic fatty liver disease（NAFLD）and chronic hepatitis B（CHB）have become the most common chronic liver diseases in the world.The phenomenon of NAFLD merging CHB（NAFLD+CHB）is becoming more and more common.Previous studies by us and others have shown that the level of global DNA methylation in the liver of patients with NAFLD and CHB is significantly decreased,but it is not known whether NAFLD+CHB will further reduce the level of global DNA methylation.Betaine is a common methyl nutrient in natural food,which can be used to prevent or treat NAFLD and reduce the content of HBV DNA in serum,but the specific mechanism is unknown.Therefore,in this study,we observed the changes of global DNA methylation in liver of patients with NAFLD+CHB and its influencing factors,and further verified and discussed the effect of betaine intervention on global DNA methylation in NAFLD+CHB hepatocytes by cell model.Methods:（1）Fifty-five NAFLD patients were recruited from the 157th Hospital in Zhangzhou city,Fujian province in China between June 2012 and June 2013.Patients were divided into NAFLD group and NAFLD+CHB group according to the presence or absence of CHB.Anthropometric and blood biochemical data of patients were collected,and liver biopsy specimens were collected to evaluate histopathological characteristics.The global DNA methylation level in the liver was measured by fluorescence spectrophotometry.（2）Different concentrations of FFA mixture（oleic acid:palmitic acid=2:1）were used to interfere with Hep G2 cells and human hepatocyte Hep G2.2.15 cells expressing HBV virus to establish NAFLD and NAFLD+CHB cell models.The cell activity was determined by CCK-8 method,the accumulation of intracellular lipid droplets was observed by oil red O-hematoxylin staining,and the intracellular triglyceride content was detected.The NAFLD+CHB cell model was treated with 2mmol/L,4mmol/L and 8mmol/L betaine respectively,and the level of global DNA methylation and the expression of DNMTs,TETs and MBDs m RNA were measured.For the measurement data of normal distribution,the mean±standard deviation is used.The differences between groups were compared by two independent samples t-test or one-way ANOVA.Results:（1）Compared with NAFLD group,the global DNA methylation level in the liver in NAFLD+CHB group was significantly lower（4.85±2.53%vs.3.16±2.26%,P=0.017）.Compared with patients without liver fibrosis,patients with liver fibrosis had lower hepatic global DNA methylation level（5.19±2.59%vs.3.65±2.36%,P=0.026）.The global DNA methylation level of NAFLD patients with both CHB and fibrosis was significantly lower than that of NAFLD patients without CHB and fibrosis（2.64±2.06%vs.5.38±2.66%,P=0.003）.In patients with mild steatosis,the presence of CHB significantly reduced the level of global DNA methylation in the liver（4.75±1.48%vs.2.82±2.39%,P=0.037）.Compared with patients without CHB and with mild inflammation,patients with moderate inflammation had lower level of global DNA methylation in the liver（5.03±2.71%vs.2.45±1.61%,P=0.012）.In patients with borderline hepatitis,the presence of CHB decreased the level of global DNA methylation in the liver（4.88±2.87%vs.2.72±1.77%,P=0.023）.In addition,the level of global DNA methylation in CHB with moderate inflammation(P_trend=0.011),CHB with fibrosis(P_trend=0.007)and CHB with borderline hepatitis(P_trend=0.035)showed a downward trend.In subjects with mild steatosis,the presence of CHB was negatively correlated with the level of global DNA methylation in the liver compared with patients without CHB（P=0.001）.Compared with patients without CHB but with mild inflammation,the presence of CHB and moderate inflammation was negatively correlated with the level of global DNA methylation in the liver（P=0.016）.Compared with patients with neither CHB nor fibrosis,the simultaneous occurrence of CHB and fibrosis was negatively correlated with the level of global DNA methylation in the liver（P=0.001）.（2）In the cell model,it was also found that compared with the NAFLD hepatocyte model,the global DNA methylation level of the NAFLD+CHB hepatocyte model decreased significantly（0.69±0.10%vs.0.40±0.05%,P=0.002）.The mechanism may be related to down-regulating the expression of DNMT1,DNMT3B and MBD1 mRNA and up-regulating the expression of TET2 and MBD4-6 mRNA.Treatment with 2 mmol/L,4 mmol/L and 8 mmol/L betaine in the NAFLD+CHB cell model could reverse the decreased global DNA methylation level（1.07±0.17%vs.1.89±0.23%vs.1.58±0.09%vs.1.89±0.07%,P=0.001,P=0.031,P=0.001）.The mechanism may be related to the down-regulation of TETs and MBD1-6 mRNA expression after betaine intervention.Conclusion:Both human liver and hepatocyte experiments showed that the global DNA methylation level of NAFLD+CHB was lower than that of NAFLD.Betaine intervention can reverse the decrease of global DNA methylation level.This study provides clues for the use of betaine as a nutritional intervention for the prevention and treatment of liver diseases,and further provides a theoretical basis for adding betaine supplementation to the dietary recommendations of NAFLD+CHB patients in the future.