Effect Of Ellagic Acid On Growth Performance,Antioxidant Properties And Intestinal Microflora Of Fattening Pigs

Ellagic acid（EA）has physiological functions such as antioxidant,anti-inflammatory and anti-diarrheal,and is used as an alternative to antibiotics in piglets for the prevention and treatment of diarrhea in piglets,but it has been less studied and applied in fattening pigs.In this study,we intend to evaluate the effect of ellagic acid in fattening pigs by growth performance,serum antioxidant and intestinal development indexes,and further investigate the mechanism of ellagic acid to improve growth performance and antioxidant performance in fattening pigs by 16S r DNA amplicon sequencing,metabolomics and cellular assay.Experiment 1:Effect of ellagic acid on growth performance and intestinal flora of growing fattening pigs.Sixty 120-day-old male and female binary growing fattening pigs of similar weight（60±6 kg）were selected.Randomly divided into two groups:Ctrl group（fed basal diet）,EA group（fed basal diet+400 mg/kg ellagic acid）,3 replicates of each treatment,10 heads per replicate.The trial period was 60 d.At the end of the trial,two heads per replicate were randomly selected for slaughter.The results of the experiment were as follows:（1）Growth performance:the average daily gain（ADG）of the EA group was significantly higher than that of the Ctrl group（P<0.01）;compared with the Ctrl group,the drip loss of the longissimus dorsi muscle in the EA group was significantly lower（P<0.01）,and the p H_45min and p H_24h of the longissimus dorsi muscle were significantly increased（P<0.05）;analysis of the apparent digestibility of feeds for growing fattening pigs revealed that EA significantly increased the apparent digestibility of crude protein（P<0.01）and neutral detergent fiber（P<0.01）in the feed was significantly increased by EA.（2）Serum antioxidant indexes:compared with the Ctrl group,total antioxidant capacity（T-AOC）was significantly higher（P<0.01）,catalase（CAT）content was significantly higher（P<0.05）,superoxide dismutase（SOD）content was significantly higher（P<0.05）,and malondialdehyde（MDA）content was significantly lower（P<0.01）in the EA group.（3）Intestinal function-related indexes:compared with the Ctrl group,the small intestinal villus height was significantly increased in the EA group（P<0.01）,with no significant effect on the crypt depth.EA significantly increased the ratio of small intestinal villus height to crypt depth（P<0.05）and significantly increased the gene expression of intestinal tight junction proteins Occludin and ZO-1（P<0.05）.（4）Jejunal inflammatory factor indexes:m RNA expression of TNF-α（P<0.01）and IL-1β（P<0.05）was significantly decreased and IL-10 expression was significantly increased in the EA group compared with the Ctrl group（P<0.01）.（5）16S r DNA amplicon sequencing:At the phylum level,compared with the Ctrl group,the relative abundance of Firmicutes in the EA group significantly increased（P<0.01）,while the relative abundance of Bacteroidetes,Spirochaetes,and Euryarchaeota decreased significantly（P<0.05）;At the genus level,EA significantly increased the relative abundance of Butyricoccus,Gemmiger and Lactobacillus（P<0.001）,and significantly decreased the relative abundance of Spirillum and Sphaerochaeta in EA group（P<0.05）.The predictive results of PICRUSt2 on gut microbiota function showed that compared to the Ctrl group,EA significantly improved nucleotide metabolism,carbohydrate metabolism,and lipid metabolism.（6）Analysis of metabolomic results:1202 metabolites were identified,122metabolites were significantly up-regulated and 58 metabolites were significantly down-regulated in the EA group compared with the Ctrl group.The differential metabolites were mainly enriched in the metabolic pathways of unsaturated fatty acid biosynthesis,α-linolenic acid metabolism,linoleic acid metabolism,and carbohydrate metabolism.（7）SCFAs content in colonic contents:the concentrations of acetic acid,propionic acid and butyric acid in the intestine were highly significant higher in the EA group compared with the Ctrl group（P<0.01）.（8）Correlation analysis of differential bacteria and assay indexes:Firmicutes was significantly and positively correlated with SOD,villus height and crypt depth（P<0.05）;Butyricicoccus was significantly and positively correlated with T-AOC,CAT,SOD,colonic acetic acid,villus height and crypt depth（P<0.05）;Lactobacillus was significantly and positively correlated with T-AOC,SOD,colonic acetic acid,propionic acid,butyric acid and villus height（P<0.05）;Clostridium celatum showed significant positive correlations with T-AOC,CAT,colonic acetic acid and colonic butyric acid（P<0.05）.（9）Correlation analysis between different metabolites and assay indicators:Gentisic acid showed significant positive correlation with CAT,ZO-1,colonic acetic acid and colonic butyric acid（P<0.05）and negative correlation with IL-1β（P<0.05）;Oxoglutaric acid showed significant positive correlation with T-AOC,CAT,IL-10,Occludin-1,ZO1 and colonic butyric acid（P<0.05）;1-Butanol showed significant positive correlation with colonic acetic acid and colonic propionic acid（P<0.05）.Summary:The addition of ellagic acid to the diet can improve the antioxidant capacity of growing fattening pigs,reduce the expression of inflammatory factors,and improve the digestibility of feed and the growth performance of growing fattening pigs.Ellagic acid can have a positive effect on enhancing the antioxidant stress capacity of growing fattening pigs by increasing the abundance of intestinal beneficial bacteria,enhancing intestinal carbohydrate metabolism and lipid metabolism,and increasing intestinal SCFAs content.Experiment 2:Effect of ellagic acid on diquat-induced oxidative stress in IPEC-J2 cellsDivide cells into four groups:Ctrl group,EA group（50μM）DQ group（80μM）EA+DQ（50μM+80μM）IPEC-J2 cells were treated for 24 hours,and then ML385 was used to inhibit the Nrf2/Keap1 signaling pathway.Detect the levels of ROS,antioxidant enzymes,and MDA in cells,as well as the expression levels of inflammatory factors and oxidative stress related pathway genes in cells.（1）Cellular antioxidant enzymes and inflammatory factors indexes:the contents of catalase（CAT）,superoxide dismutase（SOD）,glutathione peroxidase（GSH-Px）,and total antioxidant capacity（T-AOC）in cells were significantly increased（P<0.05）and the contents of malondialdehyde（MDA）were significantly decreased（P<0.05）after EA treatment compared with the Ctrl group.The expression of TNF-αand IL-1βin the cells was significantly reduced compared with the Ctrl group（P<0.05）.（2）The results of cell signaling pathway showed that:the EA group significantly increased the m RNA expression of Nrf2,HO-1,HQO1 and AMPK（P<0.05）,and significantly decreased the m RNA expression of Keap1 in IPEC-J2 cells（P<0.05）compared with the Ctrl group,but had no significant effect on AKT and m TOR expression.（3）The results of ML385 pathway inhibitor treatment showed that:the addition of ML385 inhibited the Nrf2/Keap1 signaling pathway and increased the ROS content in the DQ group significantly（P<0.01）,while the EA+DQ group had no significant effect on the ROS content compared with the Ctrl group.The effect of EA on the Nrf2/Keap1pathway was attenuated by ML385.Summary:Ellagic acid alleviates oxidative stress in IPEC-J2 cells by activating the Nrf2/Keap1 signaling pathway and enhancing the cell’s own antioxidant capacity.Conclusions:（1）The addition of ellagic acid to the diet improved the antioxidant capacity,increased the expression of intestinal villus height and tight junction protein,and improved feed digestibility and growth performance of growing fattening pigs.（2）Ellagic acid improves antioxidant capacity by activating Nrf2/Keap1 signaling pathway.Also ellagic acid can improve the antioxidant capacity of growing fattening pigs by increasing the abundance of intestinal beneficial bacteria,enhancing intestinal carbohydrate metabolism and lipid metabolism,and increasing the content of intestinal SCFAs.