| Objective To explore the relationship between high protein diet and TMAO concentration in urine of early pregnant women through dietary frequency questionnaire,and further study the effect of high protein diet on gut microbiota of mice and insulin signaling pathway of pregnant mice through animal experiment.Methods Population survey: Ma’anshan maternal and child health care hospital was selected as the survey point.Early pregnant women who had undergone the first prenatal examination in the maternal and child health care hospital since then were included in the study after obtaining their informed consent.The subjects were investigated by dietary frequency questionnaire,and their urine samples were collected.Correlation analysis was used to determine whether there was a correlation between protein intake of early pregnant women and TMAO concentration in their urine.Animal experimental study: 30 female C57BL/6 mice aged 7-8 weeks were randomly divided into high protein group and control group.The mice in the control group were fed with normal feed,and the mice in the high protein group were fed with chicken granules and feed.Mice were given different feeding after grouping.One month later,the mice were caged.During the caged period,the mice were still fed in groups.After cage closing,6 pregnant mice in the high protein group and 7 pregnant mice in the control group were obtained.The feces of mice were collected before cage closing,and the metagenomics of gut microbiota of the two groups of mice were sequenced,and the sequencing results were bioinformatics analyzed.The fasting blood glucose and fasting insulin of pregnant mice were detected,the insulin resistance index was calculated,and the genes related to insulin resistance in the liver of pregnant mice were detected by fluorescence quantitative PCR.Results Results of population survey: the average daily protein intake of women in early pregnancy was positively correlated with the concentration of TMAO in urine（r = 0.482,P = 0.002）;Animal protein intake was positively correlated with TMAO concentration（r= 0.472,P = 0.002）,while plant protein intake was not correlated with TMAO concentration（r = 0.195,P = 0.235）;Lecithin intake in food was positively correlated with TMAO concentration（r = 0.538,P = 0.000）,while betaine（r =-0.031,P = 0.854）and choline（r = 0.206,P = 0.208）intake were not correlated with TMAO concentration.Animal research results: mice metagenome sequencing results: abundance grade curve analysis found that the number of species in the control group was more than that in the high protein group,and the uniformity of gut microbiota in the control group was higher;Alpha diversity analysis showed that the indexes of Chao1 and ace in the control group were significantly higher than those in the high protein group（P < 0.05）.The high protein diet reduced the richness of gut microbiota in mice;The analysis of common species showed that the total number of species in the gut microbiota of mice in the control group and high protein group was 4462,the number of unique species in the control group was434,and the number of unique species in the high protein group was 201.The number of gut microbiota in the high protein group decreased and some strains disappeared;The results of relative abundance analysis at the phylum,genus and species levels showed that at the phylum level,the abundance of Firmicutes of high protein group increased,while the abundance of Proteobacteria,Tenerictes,Cyanobacteria,Deferribacters,Candidatus Melainabacteria,Fusobacteria,Spirochaetes and Fibrobacteria decreased.At the genus level,the abundance of Bacteroides,Enterococcus,Lachnoclostridium,Flavonifractor,Streptococcus,Adlercreutzia and Staphylococcus in high protein groups increased,while the abundance of Prevotella,Treponema and Brachyspira decreased.At the species level,the abundance of Bacteroides vulgatus,Adlercreutzia equolifaciens,Enterococcus faecalis and Bacteroides caccae increased,while the abundance of Helicobacter cinaedi,Bacteroides fragilis,Prevotella dentalis and Prevotella ruminicola decreased;The results of protein function annotation based on KEGG database showed that the gut microbiota of mice played an important role in metabolism,cell process,environmental information processing and genetic information processing;Based on the functional comparison analysis of eggnog database,it was found that the function of gut microbiota in high protein group mice was weakened in RNA processing and modification,chromatin structure and dynamics,cell movement,cytoskeleton and other groups,and enhanced in energy production and conversion,amino acid transport and metabolism,carbon water compound transport and metabolism,inorganic ion transport and metabolism and other groups;The results of comparative analysis of protein functions based on CAZy database showed that the abundance of high protein group decreased in the functional groups of auxiliary oxidoreductase,carbohydrate binding module and glycosyltransferase,and increased in the functional groups of carbohydrate enzyme,glycoside hydrolase and polysaccharide lyase.The results of fasting blood glucose and fasting insulin of pregnant mice showed that the fasting blood glucose,fasting insulin and insulin resistance index of pregnant mice in high protein group increased（P < 0.05）;The results of fluorescence quantitative PCR showed that the expression levels of IL-6,G6 PC and PCK1 genes were up-regulated and the expression levels of IRS-1 and GLUT2 genes were down regulated in the liver of pregnant mice in the high protein group.Conclusion 1.The intake of animal protein in early pregnancy is positively correlated with the concentration of TMAO in urine.2.The diversity of gut microbiota in high protein group mice decreased,and the structure of gut microbiota also changed.3.In the high protein group,the fasting blood glucose and fasting insulin levels increased,the insulin resistance index increased,and the expression level of genes related to insulin resistance in the liver changed. |
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