Study On The Protective Effect And Mechanism Of Ellagic Acid On Intestinal Damage In Heat-stressed Broiler Chickens

Poultry production is an important part of the livestock industry,Global meat production is concentrated in tropical and subtropical regions.heat stress is one of the major environmental stressors in tropical and subtropical regions affecting meat production,and heat stress easily damages the antioxidant system,alters intestinal microbiota,and disrupts intestinal function of the poultry,causing huge losses in poultry farming.Therefore,it was important to study the physiological metabolism changes and intestinal health damage induced by heat stress in poultry,and to develop natural and effective plant functional ingredients which can alleviate the negative effects of heat stress in poultry production.ellagic acid（EA）is a polyphenolic compound found in pomegranates and strawberries,which plays an important role against oxidative damage,inflammation,and tumor.In recent years,scholars in the field of animal husbandry have gradually paid more attention to the regulatory effects of ellagic acid in dietary supplementation,however,reports on the protective effects and its potential regulatory mechanisms of EA on heat stress in poultry were limited.In this experiment,we firstly explored the study of different levels of ellagic acid on broiler growth performance,intestinal permeability,antioxidant function and cecum microorganism through heat stress model,and determined the appropriate amount of ellagic acid addition and regulatory effect,then explored the protective effect of ellagic acid on broiler intestinal damage by establishing a two-factor model test of heat stress and ellagic acid,and explored the possible mechanism of ellagic acid on broiler intestinal damage through the regulation of antioxidant signaling pathway,mitochondrial function and flora structure and We also explored the possible mechanisms of ellagic acid in mitigating heat stress injury through the regulation of antioxidant signaling pathways,mitochondrial function and flora structure and metabolites.This study is divided into two parts,as follows:Part 1.Effects of different levels of ellagic acid on physiological state and intestinal health of broilers under heat stress.The present study explored the effects of dietary EA at different levels on growth performance,intestinal barrier function,antioxidant capacity,and gut microbiota in heat-stressed broilers,and were evaluated to determine the appropriate additive amount of EA and the main ways of EA to relieve heat stress.A total of 300 Arbor Acres broilers（14-day-old）were selected,individually weighted,and then randomly divided into six groups with 6 replicates per group and 10 broilers per replicate.The broilers in experimental groups were supplemented with basal diets containing EA of different doses（0,75,150,300,and 600 mg/kg）at HS temperature（35±2℃）,referred to as HS、HS+EA75mg/kg、HS+EA150 mg/kg、HS+EA300 mg/kg and HS+EA600 mg/kg respectively.The trail lasted for 28 consecutive days（14-day-old is the day 1 of the trial）,the broilers were ad libitum feed intake and water.（1）As the level of added EA increased,the levels of CORT,ALT,and Glu in the serum showed a quadratic curve in relation to their added amounts（Quadratic,P<0.05）,the dietary supplementation with EA（150 mg/kg）significantly decreased the serum levels of CORT,ALT,and Glu（P<0.05）.The results revealed that the dietary supplementation with EA（150 mg/kg）significantly increased the final weight of heat-stressed broilers,and decreased the feed conversion ratio during 14-28 old days（P<0.05）when compared supplementation with EA（300 mg/kg）group（P<0.05）.（2）Meanwhile,dietary 150 mg/kg EA supplementation up-regulated mRNA level of Claudin-1in the jejunum,ZO-1 and Claudin-1 in the ileum by HS exposure,and compared with the HS group,serum DAO and LPS level of the heat-stressed broilers was markedly decreased by the dietary EA（150 mg/kg）supplementation（P<0.05）.（3）Compared with the HS group,dietary 150 mg/kg EA increased the activities of SOD and CAT in the serum,jejunum and ileum（P<0.05）（Figure 6 A）,dietary 150 mg/kg EA also increased the activity of SOD and CAT in the serum and ileum,and remarkably decreased the MDA levels in the serum,jejunum and ileum.（4）The resulted showed that the cecum microbiota in the HS group exhibited significant difference from those in dietary EA（150,300,and 600 mg/kg）supplemented groups.Compared with the HS group,dietary EA（150 and 300 mg/kg）markedly reduced the relative abundance of cecum Actinobacteriota and Proteobacteria（P<0.05）,dietary EA（150-600 mg/kg）reduced the relative abundance of eight microorganisms including Neisseriaceae（family level）and Neisseria（genus level）in the cecum（P<0.05）.We performed Spearman’s rank correlation analysis to explore the potential relationship between gut microbiota（at genus level）and the parameters of antioxidant capacity or intestinal permeability of the heat-stressed broilers.Interestingly,the relative abundances of microbiota reduced by the EA supplementation were positively correlated with the levels of serum MDA,LPS,and DAO,and negatively correlated with the levels of serum SOD,GSH-px,and CAT.Part 2.Protective effect and mechanism of ellagic acid on intestinal injury in heat-stressed broilers.The aim of this study was to explore the protective effect of ellagic acid on broiler intestinal damage,antioxidant capacity,mitochondrial function,flora structure and metabolites,and investigate the potential regulatory mechanisms of ellagic acid in alleviating the adverse effects of heat stress.The experiment adopts a 2×2 factorial design:（1）temperature type[thermos-neutral treatment:23±2℃or high temperature treatment:33±2℃（9:30-17:30）,and 23±2℃for the remaining time]2（2）Ellagic acid treatment（the basal diet or the basal diet with 150mg/kg of ellagic acid）.A total of 240 healthy AA broilers（28days old）with similar body weight were selected and individually weighted,then randomly divided into 4 treatment groups（TN,TN+EA,HS and HS+EA）with 6 replicates per group and 10 broilers per replicate.The trial lasted for consecutive 14 days.The result shows:1 Effect of heat stress and ellagic acid on growth performance and intestinal function The results showed that heat stress significantly increased rectal temperature（P<0.05）,and ellagic acid supplementation significantly decreased rectal temperature on days 1 and 3of heat stress（P<0.05）.Heat stress had a significant effect on body weight gain and feed to meat ratio（P<0.05）.Ellagic acid increased the final weight and decreased the feed conversion ratio of heat-stressed broilers（0.05<P<0.1）.Heat stress can significantly damage intestinal morphology（P<0.05）,reduced jejunal villus height and ileal villus height and villus height to crypt depth ratio（V/C）,and increased jejunum crypt depth.The mRNA expression levels of tight junction proteins such as Mucin2,ZO-1,Claudin-1 and Occludin in the jejunum and ileum（P<0.05）were down-regulated,and the mRNA expression levels of HSP70,HSP90,Mucin4 and NF-κB were up-regulated by heat stress exposure,increased intestinal permeability（P<0.05）.Ellagic acid supplementation can increase the jejunum villus height（P<0.05）jejunum and ileum V/C（0.05<P<0.1）,and dietary EA supplementation reversed mRNA level changes of HSP90 in jejunum,ZO-1 and Claudin-1in the ileum induced by HS exposure（P<0.05）.Serum DAO level of the heat-stressed broilers was markedly decreased by the dietary EA（P<0.05）.2 Effects of ellagic acid on intestinal antioxidant-reduction status of heat-stressed broilersHeat stress decreased the mRNA expression levels of Nrf2,NQO1、HO-1、GSH-px1、GSH-px4、Cu-Zn SOD and CAT in the jejunum and ileum,decreased the activity of serum and intestinal antioxidant enzymes,inhibited the clearance levels of DPPH^·and ABTS^·+and increased the levels of PC and MDA in the jejunum and ileum（P<0.05）,and changed serum CORT,LDH,GLU,AST and CK levels.（P<0.05）.While dietary EA supplementation can significantly increase the expression levels of Nrf2,NQO1,HO-1,GSH-px1 and GSH-px4 in the jejunum and HO-1 in the ileum（P<0.05）,increase the activity of antioxidant enzymes in serum and intestine,and improve the clearance levels of DPPH^·and ABTS^·+in the jejunum（P<0.05）,decreased the accumulation of PC and MDA in the jejunum and PC in the ileum,reduced the level of corticosterone and the oxidative damage of the body（P<0.05）.3 Ellagic acid alleviates heat stress-induces impairment of mitochondrial functionThe results revealed that heat stress significantly increased the contents of 8-OHd G and ROS,inhibited the activities of mitochondrial COX I,COX III,COX IV and COX V in the jejunum and ileum mitochondria of broilers（P<0.05）,while ellagic acid supplementation significantly decreased ROS levels,increased the activities of COX IV and COX V in jejunum and ileum mitochondria（P<0.05）,and increased the activity of COX I in the ileum mitochondria（P<0.05）.Heat stress significantly inhibited the synthesis of mitochondria in jejunum and ileum of broilers and the expression of antioxidant-related genes in mitochondria（P<0.05）,and inhibited the mRNA expression levels of Mn SOD,GSH-px,GST and GSH in mitochondria of jejunum and ileum of broiler chickens（P<0.05）.While ellagic acid supplementation increased the expression of SIRT in jejunal mitochondria and the activities of Mn SOD and GST in jejunal mitochondria（P<0.05）.4 Effects of dietary EA on cecum microbiota and metabolomics-based identification of differentially metabolites and metabolic pathways in heat-stressed broilersEllagic acid increased the microbial richness of cecum microbes,and improved Chao1and ACE indices（P<0.05）.Ellagic acid increased the relative abundance of Firmicutes,Lactobacillus and Roche and decrease the abundance of Proteobacteria under thermos-neutral conditions（P<0.05）,which balanced intestinal flora and regulated intestinal health.In addition,the addition of ellagic acid under heat stress conditions may activate the metabolism of aspartate andβ-alanine,taurine and hypotaurine by regulating the pathways of intestinal microbiota,and significantly increased the levels of metabolites such as pyruvate and taurine（P<0.05）,while the elevation of metabolites such as taurine was positively correlated with the elevated abundance of Firmicutes（P<0.05）.In conclusion,dietary ellagic acid at a level of 150 mg/kg can improve the production performance,antioxidant level,intestinal morphology and flora structure of white-feathered broilers under heat stress.Ellagic acid can:1)regulating intestinal morphology and tight junction protein expression,alleviate heat stress-induced impairment of intestinal barrier function in broiler chickens2 2)activating the expression of antioxidant gene mRNA in heat-stressed broiler intestinal tissues and activate the antioxidant enzyme system to improve the ability to scavenge intestinal free radicals,thereby reducing oxidative damage2 3)improving mitochondrial redox balance,promote the effect of mitochondrial biosynthesis,guarantee the stability of mitochondrial structure and function,and alleviate heat stress damage2 4)regulating the structure of intestinal microbiota,activate aspartate metabolism andβ-alanine metabolism,taurine and taurine metabolic pathways,and increase the levels of metabolites with antioxidant regulatory effects,including pyruvate and taurine,to enhance the antioxidant level of the body and protect intestinal health.