| Objective: A rat model of diabetic nephropathy(DN)was established to evaluate the intervention effect of corn silk on DN.The changes of biomarkers in DN rats before and after administration,the state of oxidative stress and inflammation,the changes of intestinal flora structure and its metabolites were determined.The mechanism of corn silk intervention in DN was revealed from different levels,and a scientific basis was provided for the clinical application in related fields and the development of homologous products of medicine and food.Methods:(1)Twelve of 80 SD male rats were randomly selected as blank group,and the remaining rats were fed with high-sugar and high-fat diet and induced by intraperitoneal injection of low-dose streptozotocin(STZ)to establish a diabetic(DM)rat model.60 DM model rats were randomly divided into five groups,model group,positive control group(metformin0.25g/kg),corn silk low(2.7g/kg),medium(5.4g/kg),high(10.8g/kg)dose group.Rats in the model group and each administration group were given intraperitoneal injection of STZ solution every 2 weeks at the same time,and the model group was induced to establish a DN model,and the rats were continuously intervened and administered for 8 weeks.Renal organ index,fasting blood glucose(FBG),albumin creatinine ratio(ACR),serum creatinine(Scr),blood urea nitrogen(BUN),triglyceride(TG),total cholesterol(TC),low-density lipoprotein cholesterol(LDLC),and high-density lipoprotein cholesterol(HDLC)indexes were measured,and the pathological changes of renal tissues were also observed to evaluate the intervention effect of corn silk on diabetic nephropathy model rats.(2)UPLC/Q-TOF-MS technology was used to screen potential biomarkers in renal tissue and urine,combined with principal component analysis and orthogonal partial least squares discriminant analysis.After identification by HMDB and KEGG database,the biomarkers were imported into Metabo Analyst for metabolic pathway analysis,and the mechanism of action of corn silk on DN was preliminarily explained.(3)ELISA was used to measure the expression levels of inflammatory factors(IL-6,TNF-α,MCP-1)and lipopolysaccharide(LPS)in serum,and corresponding kits were used to detect oxidative stress factors(SOD,MDA,GPx)content,to explore the ability of corn silk to resist oxidative stress and anti-inflammatory response,and a preliminary evaluation of the function of corn silk to improve the intestinal barrier.(4)16S rDNA technology and Illumina Nova sequencing platform were used to sequence the intestinal flora of rats.First,the complexity analysis and multi-sample comparison of the intestinal flora of rat cecal contents were carried out,and then look for the species differences and dominant strains among the groups at different taxonomic levels to clarify the ability of corn silk to adjust the structure of intestinal flora in DN rats.(5)The content of short-chain fatty acids(SCFAs)in rat cecal contents was determined by GC-MS technology,and the effect of corn silk on the changes of SCFAs content was explored.Results:(1)The body weight of DN rats in the model group decreased significantly,the kidney weight and renal organ index increased significantly,the levels of FBG,ACR and BUN increased significantly,and the contents of TC,TG,HDLC and LDLC increased significantly,and there were multiple pathological structural damages in renal tissue.After continuous administration of corn silk for 8 weeks,the body weight of the corn silk group increased,the kidney weight and kidney organ index were significantly reduced,and the levels of FBG and ACR were significantly reduced,but had almost no regulatory effect on the level of BUN.The contents of TC,TG,HDLC and LDLC were significantly decreased,and the renal injury was improved in different degrees.There was no statistical difference in the Scr index between the groups.(2)14 metabolic pathways,including phenylalanine,tyrosine and tryptophan biosynthesis,riboflavin metabolism,and sphingolipid metabolism,etc.were disturbed in kidney tissue of DN rats,and corn silk could improve 13 metabolic pathways.Eight metabolic pathways,including riboflavin metabolism,galactose metabolism,and tricarboxylic acid cycle,etc.were disturbed in the urine of DN rats,and corn silk could improve five of them.A total of 18 differential metabolites(linoleic acid,arachidonic acid,riboflavin,etc.)were involved in the metabolic pathway regulated by corn silk,and corn silk could significantly reverse these metabolites to the blank group,and mainly intervene in the synthesis and metabolism of phenylalanine and tyrosine,riboflavin metabolism,arachidonic acid metabolism,linoleic acid metabolism and other key metabolic pathways,and improve the body’s oxidative stress and inflammatory response caused by DN.(3)The levels of IL-6,MCP-1,TNF-α and LPS in the serum of DN rats in the model group were significantly increased,the SOD activity in kidney tissue was weakened,the MDA content was significantly increased,and the GPx activity protein was significantly decreased.After the intervention of corn silk,the expression levels of MCP-1,TNF-α and LPS in the serum of corn silk administration group decreased significantly,and the level of IL-6had an obvious downward trend.The content of MDA in kidney tissue was significantly reduced,the activity protein of GPx was significantly increased,and the activity of SOD had a tendency to increase.(4)At the phylum level,the abundance of Firmicutes,Proteobacteria,Desulfobacterota,Unidentified＿Bacteria,etc.in the model group increased,while the abundance of Bacteroidota,etc.decreased,the corn silk can be significantly adjusted except for Spirochaetota;At the class level,the abundance of Clostridia,Bacilli,Negativicutes,and Gammaproteobacteria,etc.in the model group increased,while the abundance of Bacteroidia decreased,and the corn silk had an obvious callback effect;At the order level,the abundance of Lachnospirales,Enterobacterales and Veillonellales-Selenomonadales,etc.in the model group increased,while the abundance of Bacteroidales and RF39,etc.decreased,except for Lactobacillales,corn silk has obvious callback effect;At the family level,the abundance of Lachnospiraceae,Prevotellaceae,Selenomonadaceae,and Enterobacteriaceae,etc.in the model group increased,while the abundance of Muribaculaceae decreased,corn silk has no callback effect on Lactobacillaceae and Oscillospiraceae;At the genus level,the abundances of Prevotella＿9,Limosilactobacillus,Megamonas,Roseburia,and Escherichia-Shigella,etc.were increased in the model group,while the abundance of Lachnospiraceae＿NK4A136＿group and Prevotellaceae＿UCG-003 decreased,and corn silk had no callback effect on Lactobacillus and Prevotella.After T-test and LEf Se analysis,it was found that Roseburia,Limosilactobacillus and Megamonas were the key strains abnormally expressed in the model group,and Lachnospiraceae＿NK4A136＿group was the dominant strain of corn silk.(5)The contents of acetic acid,propionic acid,isobutyric acid,butyric acid,isovaleric acid and valeric acid in the cecal contents of DN rats in the model group were significantly decreased.After the intervention of corn silk,the contents of acetic acid,isobutyric acid,butyric acid and isovaleric acid were significantly increased,and the contents of propionic acid and valeric acid had an obvious downward trend.Conclusion:Corn silk can increase the content of SCFAs in the intestine and enhance the intestinal barrier function by regulating the abundance of disordered bacteria such as Roseburia,Limosilactobacillus,Megamonas and Lachnospiraceae＿NK4A136＿group in DN rats;At the same time,by regulating the synthesis and metabolism of phenylalanine and tyrosine,riboflavin metabolism,arachidonic acid metabolism,linoleic acid metabolism and other metabolic pathways,it can adjust the levels of related endogenous biomarkers.Improve the oxidative stress and inflammatory response of the body caused by DN from different levels,and achieve the purpose of intervening in DN. |
