A Preliminary Study On The Diagnosis And Treatment Of Non-Alcoholic Fatty Liver Disease

PART 1 A Cohort Study of NAFLD Patients in Retired Senior Civil Servants in ShanghaiBACKGROUND&AIMS:To investigate the clinical characteristics,risk factor and cause of death of non-Alcoholic Fatty Liver disease(NAFLD)patients in retired senior civil servants in Shanghai by a five years follow-up.METHODS:A total of 856 elderly people(697 male and 159 female)from Shanghai Xinhua Hospital were enrolled in 2012 at baseline.The medical history and clinical data were collected,and NAFLD was diagnosed by ultrasonography.And a 5 years follow-up study was conducted on this basis.All the subjects received reexamination in 2017 and all death cases were investigated for cause of death.The natural course and risk factors of NAFLD were analyzed based on the database.RESULTS:The prevalence of people over 60 years old is 28.27%.The prevalence at its highest was found in the group of 60-74 years old.Thirty nine individuals(6.35%)were newly diagnosed NAFLD during the follow-up.BMI between 22.5-25.0appeared to be the risk factor of NAFLD in elderly people,with an odd ratio(95%confidence interval)at 1.393(1.201-1.581)after adjusted by age,gender and other metabolic syndrome components.Elevated glucose and hypertension are the risk factors of NAFLD in elderly people with normal BMI,the ORs(95%CI)were1.446(1.209-1.779)and 6.270(1.846-20.939)respectively.The all-cause mortality in NAFLD patients is 5.79%.We found no significance in the prevalence of cardiovascular disease(CVD)between NAFLD and non NAFLD group(16.61%vs19.83%,p=0.061).But the CVD associated mortality in NAFLD is significantly higher(36.59%vs 64.28%,p=0.037).CVD,pulmory infection and extra-hepatic malignant tumor are the top three leading causes of death in NAFLD.CONCLUSION:NAFLD is more prevalent in elderly population between 60-75years old.BMI between 22.5-25.0kg/m~2 is a risk factor of progression of NAFLD in retired senior cival serveants over sixty.Although we found no significance in all-cause mortality,the CVD-related mortality is significantly increased than the non NAFLD individuals and CVD is the leading cause of death in NAFLD patients.PART2 Alteration of DNA Methylation Sites in Peripheral Blood Leukocytes of Patients with Non-alcoholic Fatty Liver Disease BACKGROUND & AIMS: Aging causes chronic inflammatory state in vivo,and can further aggravate epigenetic modification disorder.In recent years,the prevalence of NAFLD has been increasing in the elderly population.In addition to metabolic factors,epigenetic changes are also contributing factors to NAFLD.This study aims to identify DNA methylation sites in circulating leukocytes that are associated with liver enzymes and lipid profiles and to analyze their correlations with NAFLD histological features to determine their potential value as biomarkers.METHODS: Patients with biopsy-proven NAFLD(n=35)and normal controls(n=30)were recruited.DNA samples isolated from circulating leukocytes were analyzed by Infinium Human Methylation 450 Bead Chip.Methylation levels are presented as ? values,and we conducted analyses of their correlations with liver enzyme levels and lipid profiles.Significant DNA methylation changes were further analyzed to evaluate their correlation with hepatic histology,including steatosis,ballooning,lobular inflammation and fibrosis.RESULTS: Of the liver biopsies obtained from 35 NAFLD patients,18 had simple steatosis,and 17 had NASH.We identified 65 Cp G sites representing 60 genes that were differentially methylated in the circulating leukocytes from individuals with NAFLD and 119 sites with NAFL.42 methylated Cp G sites were found to be associated with ALT levels,and 32 sites were associated with lipid profiles.Of these sites,Cp G sites showed stronger correlations with steatosis and lobular inflammation than with ballooning and fibrosis.Finally,we confirmed that 6 differentially methylated Cp G sites(ACSL4,CTP1 A,CRLS1,SIGIRR,SSBP1 and ZNF622)correlated with NAFLD histological features and demonstrated the ability to efficiently differentiate NASH.SIGIRR,SSBP1 showed AUC at 0.882 and 0.817 respctively.CONCLUSIONS: The methylation level in circulating leukocytes was generally hypomethylated.We identified differentially methylated Cp G sites(ACSL4,CTP1 A,CRSL1,SIGIRR,SSBP1 and ZNF622)in peripheral blood cells as potential biomarkers for the non-invasive stratification of NASH versus NAFL in NAFLD.PART3 Effect of GLP-1 on Lipid Metabolism and Hepatic Pathology in ob/ob Mice BACKGROUND & AIMS: The aging state led to the disorder of epigenetic modification,and previous studies showed that there was a strong correlation between DNA methylation in NAFLD patients with lobular inflammation.The effect on weight loss,improved insulin resistance and anti-inflammatory effects by GLP-1 are gradually being recognized.In this study,we attempt to construct a recombinant lentivirus driven by GLP-1 sequences and target in to ob/ob mice,to create a persistent glucagon-like peptide-1(GLP-1)secretion in order to observe its effects on lipid metabolism and hepatic pathology in ob/ob mice.METHOD: Fourty ob/ob mice at the weight of 50-55 grams were ramdomly divided into 4 groups(GLP-1,EV,HFD and CL group).The CL group was given normal diet,the rest three groups were given high fat diet.After 16 weeks,all the mice underwent PET/CT whole body scan.GLP-1 group were given LV-GLP-1 injection,EV group were given empty vector.The HFD group and control group maintained the original feeding method.After feeding to 24 weeks,mice were scanned again,subcutaneous adipose volume and liver volume were measured,the serological examination was performed and the liver specimens were collected for histological examination.RESULTS: After injection of LV-GLP-1,the serum GLP-1 level in GLP-1 group mice was significantly higher than in three other groups(6.00�1.42pmol/L,5.28�1.18pmol/L,5.27�1.11pmol/L and 5.10�0.64pmol/L,p<0.05,respectively).The adipose tissue volume between L1-L2 were significantly decreased(1228.69�237.58 mm3 vs 857.10�101.54 mm3,p=0.020).Though with the slightly decreased serum triglycerides(0.61�0.14mmol/l),we didn?t observed significant changes in liver triglycerides(0.12�0.03 mmol/g prot)compared to other groups.In histological changes,we found,in GLP-1 group,a significant decrease in hepatocytes ballooning and lobular inflammation.But no difference was found in liver steatosis and fibrosis in all groups.CONCLUSION: Through recreation of a glucagon-like peptide-1(GLP-1)secretion,we achieve a re-distribution of adipose tissue in ob/ob mice and amelioration of histological changes in NASH.There was no significant deterioration observed in neither hepatic steatosis nor amelioration in liver fibrosis.