A Case-control Study Of Dietary Nutritional Status And Intestinal Flora Composition In Patients With Nonalcoholic Fatty Liver Disease

ObjectiveThis study aims to understand the dietary nutritional status of patients with Nonalcoholic fatty liver disease（NAFLD）in Qingdao area through dietary investigation,and extract the dietary pattern by factor analysis.The Dietary Inflammatory Index（DII）was calculated and the relationship between DII and NAFLD was analyzed.Meanwhile,based on intestinal flora 16S r DNA sequencing,the changes in intestinal flora composition and the correlation between intestinal different bacteria and dietary factors in patients with NAFLD were analyzed,in order to provide certain theoretical basis for the prevention and treatment of NAFLD.Method1.Using a case-control study design,234 patients with NAFLD were recruited from a community in Qingdao from June 2021 to June 2022 as the case group and 235 healthy adults were recruited as the control group during the same period.Questionnaire survey and physical examination were performed on the subjects.5 m L of fasting venous blood was collected and serum was separated.Automatic biochemical analyzer（AU5800）was used to determine blood biochemical indexes.Dietary intake of study subjects was investigated by Food frequency questionnaire（FFQ）,and intake of various nutrients was obtained by Computer Expert System of integrated Chinese and Western Medicine（NCCW）12.0 software.Dietary patterns were extracted by factor analysis.DII scores of all kinds of food and nutrients were calculated,and the DII scores of each study object were the sum of DII scores of all kinds of food and nutrients;The subjects were divided into five groups according to the DII score quartile,and the relationship between different dietary pattern scores,DII score and NAFLD was analyzed by multivariate Logistic regression model.2.Fresh fecal samples of the subjects were collected,and fecal bacterial genome DNA was extracted using the kit.16S r DNA intestinal flora sequencing was conducted based on Illumina novaseq6000.Intestinal flora data were analyzed using BMK Cloud（http://www.biocloud.net/）online analysis platform.Theα-diversity of intestinal flora was analyzed by QIIME.Theβ-diversity was estimated based on Bray-curtis distance,and principal coordinate analysis was used for visualization.The Mann-Whitney U test was used to compare the relative abundance of intestinal flora between groups.Linear discriminant analysis was used for LEf Se analysis.Spearman rank correlation was used to analyze the correlation between different intestinal bacteria and each index.TBtools software was used to draw correlation heat map.Data was analyzed by SPSS 25.0software,and P value was bilateral test,α=0.05 was used as the test level,P<0.05 was considered statistically significant.Results1.A total of 469 subjects were included in this study,including 235 subjects（male28.94%,female 71.06%）in the control group,with an average age of 48.17±13.61 years old;There were 234 patients（43.16%males and 56.84%females）in the NAFLD group.The mean age was 49.05±11.53 years old.The Body mass index（BMI）,waist circumference,serum alanine aminotransferase,aspartate aminotransferase,fasting blood glucose,triglyceride,total cholesterol,low density lipoprotein cholesterol and uric acid levels in NAFLD group were significantly higher than those in control group（all P<0.05）.The level of high density lipoprotein cholesterol was lower than that of control group（P<0.05）.The comparison of dietary intake of subjects showed that compared with the control group,the daily intake of cereals,potatoes,poultry meat,green leafy vegetables,milk and soy products in NAFLD group was higher,while the daily intake of rice and noodles,livestock meat,processed meat,eggs,desserts and fried foods was higher（P<0.05）.The daily intakes of dietary vitamin A,vitamin B₂,vitamin C,calcium and dietary fiber in NAFLD group were lower（all P<0.05）.Factor analysis extracted four dietary patterns:"Animal meat dietary pattern",mainly livestock meat,poultry meat,offal food;"Rice noodle processed meat diet mode",mainly rice noodle,processed meat,fried food,pork,dessert food;"Fruit and nut diet pattern",mainly fresh fruit,nuts,soy products food;"Multi-grain vegetable diet pattern",mainly multi-grain,soy products,green leafy vegetables,dairy foods.Multivariate Logistic regression analysis showed that after adjusting for confounding factors such as age,gender,BMI,hypertension,diabetes,hyperlipidemia and hyperuricemia（Model 3）,the dietary pattern of processed rice and noodles was positively correlated with the risk of NAFLD,while the dietary pattern of mixed grains and vegetables was negatively correlated with the risk of NAFLD.The OR（95%CI）were 2.62（1.91,3.61）and 0.64（0.51,0.82）,respectively.The DII score of NAFLD group was significantly higher than that of control group（P<0.05）.Multivariate Logistic regression analysis showed that after adjusting for confounding factors such as age,gender,BMI,hypertension,diabetes,hyperlipidemia and hyperuricemia,compared with the lowest quintile of DII score,the highest quintile significantly increased the risk of NAFLD.OR（95%CI）was 2.30（1.14,4.65）,and P _{for trend}=0.030.2 In this study,98 fecal samples were collected（50 in the case group and 48 in the control group）.The results of 16S r DNA sequencing showed that the Shannon index and Simpson index of intestinal flora in NAFLD group were significantly lower than those in control group（P<0.05）.At the phylum level,the relative abundance of Firmicutes in NAFLD patients was significantly higher than that in control group,while the relative abundance of Proteobacteria was significantly lower than that in control group（P<0.05）.At the family level,the relative abundance of Ruminococcaceae in NAFLD patients was significantly lower than that in the control group,while the relative abundance of Enterobacteriaceae was significantly higher than that in the control group（P<0.05）.At the genus level,the relative abundance of Escherichia＿Shigella and Klebsiella in the intestinal tract of NAFLD patients was significantly higher than that of the control group.The relative abundance of Faecalibacterium and Subdoligranulum were significantly lower than those of control group（P<0.05）.LEf Se analysis results showed that the intestinal tract of NAFLD group was enriched in Escherichia＿Shigella and Klebsiella,while that of control group was enriched in Faecalibacterium and Subdoligranulum rare.Correlation analysis of intestinal differential bacteria with biochemical indexes and dietary factors showed that the relative abundance of Escherichia＿Shigella was positively correlated with serum total cholesterol,low density lipoprotein cholesterol and intake of livestock and meat in NAFLD patients,and the relative abundance of Klebsiella was positively correlated with serum glutamic-oxaloacetic transaminase level.The relative abundance of Subdoligranulum rare was negatively correlated with serum uric acid level（P<0.05）.Conclusion1.The daily intakes of coarse grains,potatoes,poultry meat,green leafy vegetables,milk and soy products were lower in NAFLD population,while the daily intakes of rice and noodles,livestock meat,eggs,processed meat,desserts and fried foods were higher.The daily intakes of dietary vitamin A,vitamin B₂,vitamin C,calcium and dietary fiber were lower in NAFLD group.2.The dietary patterns of rice and noodle processed meats（mainly rice and noodle,processed meat,desserts,fried foods,and pork）were positively correlated with the risk of NAFLD,while the dietary patterns of multi-grain vegetables（mainly cereals,green leafy vegetables,dairy products,and soy products）were negatively correlated with the risk of NAFLD.3.DII score is positively correlated with the risk of developing NAFLD.The higher DII score is,the more likely it is to suffer from NAFLD.4.The composition of intestinal flora in NAFLD population changed significantly,with the relative abundance of Escherichia＿Shigella and Klebsiella increasing,while the relative abundance of Faecalibacterium and Subdoligranulum rare decreasing.