Effects Of N-3 Polyunsaturated Fatty Acids Plus Vitamin D₃ On Gut Microbiota And Metabolites In Patients With Non-alcoholic Fatty Liver Disease

Objective:Our previous double-blind randomized controlled trial demonstrated that dietary intakes of fish oil（FO）,mainly component was n-3 polyunsaturated fatty acid（n-3PUFA）,plus vitamin D₃（D）could ameliorate biomarkers of liver function,lipid metabolism and insulin sensitivity of non-alcoholic fatty liver disease（NAFLD）.However,the effects of FO+D intervention on gut microbiota and fecal metabolites remained unclear.The present study aimed to investigate the effects of FO+D intervention on gut microbiota and fecal metabolites,and their correlations with clinical parameters.Methods:A total of 61 subjects were randomly divided into three groups:FO+D group（2.34g/day of eicosatetraenoic acid（EPA）+docosahexaenoic acid（DHA）+1680 IU vitamin D₃）,FO group（2.34 g/day of EPA+DHA）,and CO group（corn oil,3g/d）.This study has been approved by the Ethics Committee of the Affiliated Hospital of Qingdao University（QYFYWZLL25549）and registered on the Chinese Clinical Trial Registry（Chi CTR1900024866）.The blood and fecal samples were collected at the baseline and endpoint during a 90-day intervention.Gut microbiota profiles were measured with 16S r RNA analysis,and fecal co-metabolites were identified with untargeted ultraperformance liquid chromatography to quadrupole time-of-flight mass spectrometry（UPLC-Q-TOF-MS）based metabolomics approach.Results:The community richness Chao 1 was found significantly different among the three groups（p=0.015）.The relative abundance of Alistipes（p=0.02）was significant enhance in FO+D group compared with FO group.Additionally,the relative abundances of Eubacterium（p=0.03）and Lactobacillus（p=0.05）were increased whereas Streptococcus（p=0.02）and Dialister（p=0.04）were decreased in FO+D group compared with CO group.The levels of fecal 1,25-dihydroxyvitamin D₃（1,25-（OH）₂D₃）in FO+D group was increased compared with FO group（fold change（FC）=5.42,p=0.03）.Besides,changes in tetracosahexaenoic acid（THA,C24:6n-3;FC=2.05,p=0.03）and dodecanedioic acid（FC=2.33,p<0.05）levels were significantly enhanced whereas8,9-dihydroxy-eicosatrienoic acid（8,9-Di HETr E,FC=0.05,p<0.05）were reduced in FO+D group in comparison with CO group.The correlation analyses showed that changes of the relative abundance of Alistipes was positively associated with fecal 1,25-（OH）₂D₃（r=0.44,p=0.01）,and fecal1,25-（OH）₂D₃was inversely corelated with HOMA-IR（r=-0.29,p=0.02）.Besides,inversely association was found between Eubacterium and 8,9-Di HETr E（r=-0.53,p=0.03）and between 8,9-Di HETr E and adiponectin（r=-0.43,p<0.05）.There is a negative correlation between Dialister and dodecanedioic acid（r=-0.43,p=0.04）,dodecanedioic acid and inflammatory cytokine IL-1β（r=-0.35,p=0.01）.Conclusion:The present randomized controlled trial demonstrated that dietary supplementation of FO+D regulates the gut microbiota,significantly increases THA and dodecanedic acid levels and decreases 8,9-Di HETr E levels,further mediating glucolipid metabolism and inflammatory response to alleviate NAFLD.THA may be the biomarker of dietary n-3 PUFA in subjects with NAFLD on gut microbiota and metabolite.Large sample clinical trials are needed to verify the physiological functions of these metabolites during the occurrence and development of NAFLD.