The Protective Effects Of Quercetin On Fescue Toxicosis-induced Cardiovascular Toxicity And Gastrointestinal Microbiota Homeostasis In Lambs

Tall fescue is the most widely planted cool-season grass in the world and an ideal forage with good drought and stress resistance.The term"infected tall fescue"is generally applied to the USA Kentuky-31tall fescue,which infected with the fungal endophyte Neotyphodium coenophialum（formerly called Acremonium coenophialum）that is toxic to animals.Tall fescue infected with the endophyte Neotyphodium coenophialum produces a class of compounds called ergot alkaloids that affect appetite,heart and blood vessel function,hormonal level,gut motility,muscle contraction,and body temperature regulation which threatens the life and health safety of grazing livestock."Fescue toxicosis"is the general term used for the clinical diseases that can affect livestock consuming endophyte-infected tall fescue.Fescue toxicosis can induce rough hair,weight loss,lethargy,increase the rate of breath and heart,elevate rectal temperature,and cardiotoxicity in livestock,but,the mechanism of fescue toxicosis-induced cardiotoxicity remains unclear.Quercetin,also known as onionin or meletin,is one of the most abundant flavonols in the diet with antioxidant effects in cardiovascular diseases,which provides some theoretically basis of prevention and treatment in cardiovascular toxicity caused by fescue toxicosis.In order to explore the antagonistic mechanism of quercetin against fescue toxicosis induce-cardiovascular toxicity and the effect on rumen and feces microbiome in lambs,twenty-four Dorper sheep were randomly divided into 4 groups（6 N/group）:E-,Q-group（endophyte-free tall fescue seed group）,E+,Q-group（endophyte-infected tall fescue seed group）,E+,Q+group（endophyte-infected tall fescue seed+4 g/kg quercetin group）,and E-,Q+group（endophyte-free tall fescue seed+4 g/kg quercetin group）were fed for consecutive fourty-two days.The average daily intake of feed and water were recorded,the changes of cardiac histopathology,serum biochemical cardiac function,hepatic and renal function index,routine blood,serum prolactin detection,cardiac ultrastructure observation,anti-oxidation level,expression phase I,II,III detoxification metabolic enzymes,NF-κB-mediated inflammatory response,mitochondrial biological related factors,mitochondrial dynamics fission-fusion protein expression,and mitochondrial autophagy level were detected.The butyric acid content in serum,rumen volatile fatty acid（acetic acid,propionic acid,isobutyric acid,butyric acid,isovalerate acid,valerate acid,and total volatile fatty acid）concentrations,abundance and diversity of rumen and fecal microbiomes were detected.The detection results showed that:（1）Fescue toxicosis resulted in the slower growth rate,average daily feed intake reduction and prolactin content decreased in lambs.Quercetin supplementation reduced the abnormal increase of white blood cell number in blood routine indexes.Quercetin mitigated fescue toxicosis-induced the rupture of myocardial fibers,reduced the space between myocardial fiber bundles,and reduced the loose or broken degree of elastic fibers in aorta tissue,thereby alleviating the structural damage of cardiovascular system.Quercetin inhibited the increase of creatine kinase（CK）and aspartate aminotransferase（AST）activities,decreased the increase trend ofγ-glutamyltransferase（GGT）activity,and increased the content of albumin（TP）.These results indicated that quercetin antagonized fescue toxicosis-induced the cardiovascular structure damage,and the heart and liver function damage.（2）Quercetin alleviated fescue toxicosis-induced cardiovascular oxidative stress,inhibited the increase of oxidative products（MDA,H₂O₂）content and the decrease of antioxidant enzymes（T-SOD,CAT,Cu-Zn SOD,T-AOC）activities.Quercetin alleviated the inhibition of AHR,CAR and PXR expression induced by fescue toxicosis,alleviated the interference of Nrf2-mediated phaseⅡdetoxification enzymes,and increased the expression of phaseⅢdetoxification enzymes（ABCC1 and ABCB1）,thus improving the metabolism of ergot alkaloids in fescue of lambs.These results indicated that quercetin alleviated fescue toxicosis-induced the cardiovascular system oxidative damage and the interference of detoxification metabolic enzymes.（3）Quercetin inhibited NF-κB mediated signaling pathway activation,decreased the expression level of pro-inflammatory factors（IL-1β,IL-6 and IL-18）,increased the expression of anti-inflammatory factors（IL-4,IL-10 and P53）,and reduced inflammatory cell infiltration in cardiovascular tissues.It suggested that quercetin antagonized fescue toxicosis-induced cardiovascular inflammatory injury.（4）Quercetin activated PGC-1αmediated mitochondrial biogenesis related factors（NRF1,PRDX3,RAF1）,increased the expression levels of mitochondrial fusion proteins（MFN1,MFN2,OPA1）,and decreased the expression levels of fission protein Fis1,inhibited Parkin/PINK signaling pathway mediated the excessive mitophagy,and alleviated the damage of cardiovascular mitochondrial structure caused by fescue toxicosis.These results indicated that quercetin could maintain mitochondria function and play a protective role in fescue toxicosis-induced cytotoxicity.（5）Quercetin regulated the production of volatile fatty acids,alleviated fescue toxicosis-induced the content of isobutyric acid,butyric acid and isovaleric acid reduction.Quercetin increased the content of butyric acid in serum and rumen,activated PGC-1αexpression,and alleviated mitochondrial damage caused by fescue toxicosis.（6）Quercetin significantly increased the Alpha and Beta diversity of rumen and fecal micobiome,and induced an increase in the relative abundance of Bacteroidetes,Ruminococcus,Succiniclasticum and Prevotella.Quercetin alleviated the changes of phylum and genus level of rumen and fecal micobiome caused by fescue toxicosis.These results indicated that quercetin antagonized the disturbance of fescue toxicosis on rumen and fecal microbial homeostasis.In conclusion,fescue toxicosis can cause cardiovascular tissue mitochondria damage,activate NF-κB pathway mediated inflammatory response,induce excessive autophagy of mitochondria,cardiovascular oxidative stress in lambs,and lead to cell damage.Fescue toxicosis also disrupted rumen and intestinal microbiota homeostasis and reduced volatile fatty acid production.Quercetin increased the volatile fatty acids concentrations in the rumen,elevateded the the diversity of rumen and fecal bacteria,improved fescue toxicosis-induced the lower abundance of micbiome.