| ObjectiveIntestinal flora is closely related to human health,and the disorder of intestinal flora will cause a series of diseases.Related studies have shown that nutrition has a significant impact on intestinal flora.Nutrition can regulate intestinal flora to prevent diseases and maintain health.At present,there are limited reports on the effects of riboflavin on intestinal flora.In this study,the rat model of riboflavin malnutrition was established by feeding diets containing different levels of riboflavin.The effects of different riboflavin nutrition levels on the composition and structure of intestinal flora in rats were investigated.Methods1.36 male Wistar rats were randomly divided into normal control group(NC),riboflavin deficiency group(RD),riboflavin insuficiency group(RI)and riboflavin deficiency pair-fed group(PF).The rats were fed the AIN-93 M synthetic diets with riboflavin content at 6mg/kg,< 1mg/kg,3mg/kg and 6mg/kg for 3 months,and the daily food intake and weekly body weight were recorded.2.The contents of riboflavin,FAD and FMN in the liver and the concentration of riboflavin and GR activity in the serum were determined to assess whether the model was established successfully or not.3.The acetic acid,propionic acid,butyric acid,valeric acid,isobutyric acid and isovaleric acid in intestinal contents were determined by gas chromatography and mass spectrometry4.The intestinal barrier function was evaluated by measuring the activity of diamine oxidase and the contents of D-lactic acid and lipopolysaccharides in the serum.The structural changes of colonic mucosa were observed after HE staining,and the expression of tight junction proteins in intestinal mucosa was evaluated by immunohistochemistry.5.The effects of riboflavin malnutrition on intestinal flora at the phylum,class,order,family and genus levels and its potential biological significance were evaluated by 16 Sr RNA high-throughput sequencing.Results1.Compared with the NC group,the dietary intake and body weight of the rats in the RI group did not change significantly,and still maintained a steady upward trend.By the second week,the food intake in the RD group decreased by13.14%,and the body weight decreased by 5.47% and 3.61%,respectively as compared with the NC group.At the end of the experiment,the food intake and body weight of RD group decreased by 22.42% and 22.78%,respectively,but the weight loss rate of PF rats was significantly lower than that of RD rats.2.Compared with the NC group,serum GR activity of the RD group significantly decreased by 37.8%.Compared with the PF group,serum GR activity of the RD group decreased by 12%,but there was no significant difference.Compared with the NC group,the concentration of riboflavin in the serum of rats from the RD group decreased significantly by 85.36%,and the average concentration of riboflavin in the serum of rats in this group decreased below6.91nmol/L,which was very close to the riboflavin level of after riboflavin deficiency.Compared with the PF group,the concentration of riboflavin in the serum of rats in the RD group decreased significantly by 85.5%.Compared with the NC group,the concentration of riboflavin in the liver of the RD group decreased by 67.65%,while that of the PF group decreased by 7.5%.Compared with the NC group,the concentration of FAD and FMN in the liver of the RD group decreased by 36.52% and 65.79%,respectively,and the change of FAD and FMN concentration was consistent with that of riboflavin in the liver.3.In the colonic contents,the content of acetic acid short-chain fatty acids was the highest,followed by propionic acid,and the lowest was isovaleric acid.Compared with the NC group,the contents of acetic acid,propionic acid,butyric acid,isobutyric acid,isobutyric acid and isovaleric acid in the RD and RI groups decreased by more than50%.Compared with the PF group,the contents of acetic acid,propionic acid and isovaleric acid in the RD group decreased significantly.Compared with the NC group,the contents of butyric acid,valeric acid,isobutyric acid and isovaleric acid in the PF group decreased significantly.4.The contents of serum LPS,DAO and D-LA in the RD and RI groups were higher than those in the NC group,and there was significant difference between the RD group and the NC group,but there was no significant difference between the RI group and the NC group.The contents of LPS,DAO and D-LA in the RD group were higher than those in the PF group,and there was significant difference in LPS content between the two groups.In the NC group,the structure of colonic mucosa was intact,intestinal glands were good and neatly arranged,and there was a complete striated margin.Compared with the NC group,intestinal mucosal epithelial cells were exfoliated,crypt structure destroyed,goblet cells decreased and intestinal submucosa thickened in the RD and RI groups,but there was no remarkable change in intestinal mucosal structure in the PF group.Compared with the NC group,the height of folds and the thickness of muscle layer in the RD group decreased significantly(p < 0.001),and the thickness of mucosa increased,but there was no significant difference between the RI group and the PF group.Compared with the NC group,the expression of Claudin-1 protein in the RD group and the RI group decreased significantly(P < 0.001),the expression of ZO-1protein in the RI group decreased significantly(P < 0.001).Compared with the PF group,the expression of Claudin-1 protein decreased significantly in the RD group(P < 0.001),and there was no significant difference between the RI group and the PF group.The expression of ZO-1 in the RD and RI groups decreased,but there was no significant difference.5.Riboflavin deficiency significantly decreased the abundance and diversity of colonic bacterial community,which was characterized by the decrease o f Shannon and Chao1 indexes.Beta diversity analysis showed that there were significant clusters among the four groups.At the phylum level,riboflavin malnutrition led to an increase in the relative abundance ratio of Bacteroides to Bacteroides(F/B value).At the genus level,riboflavin malnutrition led to a significant increase in the relative abundance of Prevotella＿9,[Eubacterium]＿fissicatena＿group,Holdemania,Parasutterella,Alloprevotella and Phascolarctobacterium.Conclusion1.Riboflavin malnutrition can cause growth retardation and significantly reduce food intake and body weight in rats.2.Riboflavin malnutrition can decrease the content of riboflavin and its derivatives in the serum and liver,and decrease the activity of glutathione reductase,indicating that the rat model of riboflavin malnutrition is established successfully.3.After riboflavin malnutrition,the contents of short-chain fatty acids(acetic acid,propionic acid,butyric acid,valeric acid,isobutyric acid,isovaleric acid)in intestinal contents decreased significantly.4.Riboflavin malnutrition can damage the structure of intestinal mucosa,reduce intestinal barrier function,increase serum DAO activity and the content of LPS and D-LA,reduce goblet cells,decrease the thickness of muscle layer,decrease the height of folds,increase the thickness of mucosa,and decrease the expression of tight junction proteins ZO-1 and Claudin-1.5.Riboflavin malnutrition can significantly reduce the abundance and diversity of colonic bacterial community,lead to the disorder of intestinal flora,increase the abundance of harmful bacteria and affect intestinal health. |
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