Effects Of Fish Oil And Vitamin D Intake On Non-alcoholic Fatty Liver Disease

Objective:In recent years,the prevalence of non-alcoholic fatty liver disease（NAFLD）in China has been increasing year by year,and has become the largest chronic liver disease threatening Chinese residents.Prevention and treatment of NAFLD has become a crucial public health issue.Observational and case-control studies found that the circulating n-3polyunsaturated fatty acids（PUFA）and 25-hydroxyvitamin D3（25（OH）D₃）concentrations in patients with NAFLD were lower than those in health,and the supplement of deep-sea fish oil rich in n-3 PUFA and vitamin D respectively had different improvement effects for NAFLD.The present study aims to explore the effects of n-3 fatty acid combined with vitamin D on biomarkers of hepatic function and cardiovascular metabolism by using a double-blind randomized controlled trial.The possible mechanism was also discussed through its effect on serum metabolites profiling in NAFLD patients.Methods:Randomized controlled double-blind trial was used in this study,a total of 111NAFLD patients（61 males and 50 females）aged 30-70 years old were recruited in Qingdao urban area and randomly divided into three groups,namely fish oil combined with vitamin D group（3 g/d of n-3 fatty acid combined with 1500 IU/d of vitamin D,group FOD）,fish oil group（3 g/d,group FO）and corn oil group（corn oil 3 g/d,group CO）.The trial lasted for 90 days,and the intake of the interventions was followed up every two weeks.Any adverse reactions and shedding cases occurred during the intervention were solved in time and recorded.The basic personal information and anthropometric parameters of the patients were collected at baseline and 90 days,and the dietary information was collected by 24-hour dietary recording method.Fasting blood was collected before and after the intervention.The blood samples were stored in the refrigerator at-80℃for subsequent biochemical indicators,cytokine and metabonomics detection.Automatic biochemical analyzer was used to detect liver function,kidney function,and cardiovascular metabolic risk factors;ELISA kit was used to detect insulin,adiponectin,fibroblast growth factor 21（FGF21）,interleukin-1β（IL-1β）,interleukin-6（IL-6）,tumor necrosis factor-α（TNF-α）indicators.Metabonomics analysis of serum samples was performed by UHPLC-Q-TOF in positive ionization modes.Partial correlation analysis was used to evaluate the association between anthropometric parameters,metabolic disease risk factors and serum differential metabolites.In addition,statistical methods such as chi-square test,one-way analysis of variance,paired Student’t test,rank sum test and generalized linear model were also used in the study.Results:96 patients completed the intervention.After the intervention of FOD and FO,the alanine aminotransferase（ALT）of the patients decreased by 4.87 U/L（P<0.05）and 4.32U/L（P=0.062）respectively,and the differences between the two groups and CO group were statistically significant（P<0.05）.There was no significant difference in renal function between FOD,FO group and CO group.After intervention,insulin concentration in FOD group was significantly decreased（1.58pmol/L）,and the change was significantly different from that in CO group（P<0.05）.After intervention,the TAG of FOD and FO group was decreased by 0.41mmol/L and 0.68mmol/L,and the change value was significantly decreased compared with CO group（P<0.05）.After intervention,IL-1βconcentration in FOD group and FO group decreased by 6.92 ng/L and 3.51 ng/L,respectively,with no statistically significant difference within group,but the change of IL-1βconcentration in the two groups was significant compared with CO group（P<0.05）.After intervention,the concentration of TNF-αin FOD group and FO group was decreased by 0.17 pg/L and 0.11 pg/L,respectively（both P<0.05）.Compared with the CO group,the concentration reduction in the two groups was statistically significant（P<0.05）.After intervention,FGF21 concentration in FOD group was decreased by 15.41pg/m L（P<0.05）,and the difference was significant compared with CO group.The above mentioned comparison with the CO group was adjusted using a multivariate model.90 participants were included in the serum metabolomics analysis.After preliminary screening,a total of 97 different metabolites were detected in the FOD group.Compared with the CO group,there were significant differences in 3 metabolites,namely,phosphatidylinositol（PI）,3-O-sulfogalactosylceramide（SL）and 24-dimethoxy-3β,6β,19α-trihydroxyurs-12-en-28-oic acid（ST）（both P<0.1）;A total of 86 different metabolites were detected in FO group,and 3 metabolites were significantly different compared with CO group,namely PI,SL and N-arachidonoyl ethanolamine（AEA）（both P<0.1）.Compared with the CO group,PI and ST were significantly increased after FOD intervention,while SL was significantly decreased;After FO intervention,AEA and SL were significantly decreased,while PI was significantly increased.Correlation analysis showed that SL was positively correlated with TAG（r=0.263,P=0.028）and blood urea nitrogen（BUN）（r=0.278,P=0.018）.PI was marginally significantly correlated with ALT（r=-0.229,P=0.053）and gamma-glutamyl transpeptidase（GGT）（r=-0.232,P=0.050）,and negatively correlated with AST（r=-0.283,P=0.016）,TAG（r=-0.269,P=0.024）and inflammatory factors IL-1β（r=-0.399,P=0.001）and TNF-α（r=-0.236,P=0.046）.Conclusions:The improvement effect of FOD intervention on liver function,glucose metabolism and inflammatory factors in NAFLD patients was better than that in FO group.In addition,there are different metabolites in serum of NAFLD patients after dietary supplementation of FOD and FO,these differential metabolites may be the targets for the different effects of fish oil and vitamin D.Dietary intervention FOD or FO may improve liver function,renal function,lipid metabolism,and inflammatory status in patients with NAFLD through these targets.