Gallic Acid Alleviates Fish Enterohepatic Inflammation By Enhancing PPARγ-mediated Anti-inflammatory Effects And Lipid Metabolis

Foodborne gastroenteritis has become one of the important factors limiting the development of aquaculture.In previous work,we constructed a zebrafish SBMIE model to study the mechanism of fish enteritis and evaluated the effect of sea buckthorn in relieving enteritis using this model,and found that the PPAR pathway was significantly upregulated in the drug group.Ellagic acid is an important component of sea buckthorn and also one of the agonists of the PPAR pathway.To determine the effective component of sea buckthorn,this study conducted drug supplementation experiments.In addition,to facilitate the use of the zebrafish SBMIE model,we optimized the preparation method of zebrafish intestinal mucosal immune cell suspension to better study the changes of immune cells under enteritis conditions.In order to facilitate the study of intestinal cellular immunity of zebrafish,we designed a quick and simple operation method for preparing mucosal cell suspension.In the result of HE staining,the new method was able to almost completely peel off the upper cortex and mucosal layer of the intestine.Compared with direct grinding of intestinal tissue,the new method can significantly increase the proportion of immune cells in the cell suspension,and the obtained samples can more accurately reflect the proportion of different immune cells in the intestinal tissue and the changes in the proportion of immune cells in the stage of enteritis.We performed molecular docking analysis of ellagic acid and zebrafish PPARγ protein to theoretically demonstrate that ellagic acid can act as an activating ligand.Then,ellagic acid was added to soybean meal feed for a six-week zebrafish feeding experiment.The results showed that ellagic acid played an anti-inflammatory role in the intestines and liver of zebrafish by regulating immune and lipid metabolism processes.The pathological results showed that ellagic acid could alleviate the shortening of intestinal villi caused by soybean meal and reduce the accumulation of liver lipid droplets caused by inflammation.From the perspective of immune cells,ellagic acid can improve the inflammatory aggregation of innate immune cells(macrophages and neutrophils)and adaptive immune cells(T cells)in zebrafish intestines induced by soybean meal,and also help reduce the proportion of macrophages in adult fish liver.In the transcriptomic results,KEGG and GO enrichment analysis showed upregulation of intestinal lipid metabolism and intestinal barrierrelated pathways,and downregulation of cytokine signaling pathways and apoptosis pathways.In the liver,fatty acid biosynthesis was significantly inhibited,while the insulin signaling pathway was upregulated in both the intestine and liver.Through 16 s RNA sequencing,we analyzed the effect of ellagic acid on the intestinal flora in the presence of inflammatory factors.At the phylum level,the ellagic acid group could maintain a proportion of Firmicutes and Bacteroidetes similar to that of the normal fishmeal group,while the proportion of Verrucomicrobia increased,which may protect the intestinal barrier by reducing fatty acid synthesis.At the genus level,the proportion of symbiotic Akkermansia was higher in the ellagic acid group than in the SBM group,while the proportion of pathogenic Vibrio and Clostridium decreased.In terms of intestinal metabolism,I found through metabolomics analysis that some antiinflammatory-related metabolites were upregulated,such as baicalin,maltotriose,methyl jasmonate ester,and dipeptide Asp-Phe.In addition,metabolic pathway related to fatty acids and carbohydrates(linoleic acid metabolism)and immune-related metabolic pathway(histidine and tryptophan metabolism)were all improved by ellagic acid supplementation.Therefore,it is speculated that ellagic acid activates PPARγ,which may promote the synthesis of anti-inflammatory metabolites and inhibit the synthesis of pro-inflammatory metabolites,thereby exerting an anti-inflammatory effect.The above studies indicated that,through the study of gallic acid alleviating zebra-fish intestinal hepatitis,we found that PPARγ,a key gene regulating fish immunity and lipid metabolism,is crucial to fish disease resistance,providing a new idea for subsequent research.In addition,the optimization of zebrafish intestinal mucosal immune cell isolation method can contribute to the study of zebrafish intestinal immunity,and lay a foundation for the subsequent isolation and culture of specific immune cells in zebrafish intestinal mucosa.