Study On The Mitigation Action Of Artemisia Ordosica Polysaccharide On Immune Stress Of Broilers And The Underlying Mechanism

The current study investigated the mitigation action of Artemisia ordosica polysaccharide(AOP)on immune stress of broilers exposed to lipopolysaccharide(LPS)and the underlying mechanism.The major work and results were showed as follows.Experiment 1: Extraction,purification,structure characterization and in vitro activity of Artemisia ordosica polysaccharide(1)In this study,a novel polysaccharide was isolated from Artemisia ordosica by water-extraction-ethanol-precipitation method.The optimal extraction conditions of AOP were determined by single factor tests and response surface methodology optimization,and were showed as follows: a liquid-solid ratio of 15.4:1 m L/g,extraction time of 4.3 h,extraction temperature of 60 ?.Under the optimal conditions,the extraction yield and the sugar content of the AOP were 5.56 % and 52.65 %,respectively.Gel permeation chromatography and ion-exchange chromatography were used to determine the characterization of AOP.These results indicated that AOP,with a molecular weight of 2.1k Da(62.6 %)and 1.5 k Da(37.4 %),was composed of arabinose,galactose,glucose,xylose,mannose,galacturonic acid and glucuronic acid with molar ratio of6.87:10.67:54.13:2.49:18.37:4.83:2.64.(2)Cellulose DEAE-52 and Sephadex G-100 were adopted to purify AOP,and the main elution peak AOP-? was obtained.The structure characterization of AOP-? was identified by gel permeation chromatography,ion-exchange chromatography and methylation.These results indicated that AOP-?,with a molecular weight of 9.00 k Da,was composed of fucose,arabinose,galactose,glucose,xylose,mannose,ribose,galacturonic acid and glucuronic acid with molar ratio of0.56:13.75:12.79:54.08:3.15:13.43:0.63:0.67:0.93.Meanwhile,methylation analysis showed that the main components of AOP-? were glucose residues,?5)-Ara(f)-(1?,?3)-Gal(p)-(1?,?2)-Galp-(1?,?4)-Man(p)-(1?,?6)-Man(p)-(1?,?4)-Gal(p)-(1? and arabinose residues with molar ratio of 66.43:1.55:4.81:3.19:6.48:6.74:9.17:1.65.Experiment 2: Effects of Artemisia ordosica polysaccharide on immune and antioxidant function of broilersA total of 288 one-day-old Arbor Acres(AA)broilers were randomly divided into 6treatments with 6 replicates and 8 birds per replicates.Broilers in the six groups were offered basal diet supplemented with 0,250,500,750,1000 mg/kg AOP and 50 mg/kg chlortetracycline(CTC),respectively.The feeding experiment was conducted for 42 d.The results demonstrated that dietary 750 mg/kg AOP supplementation improved growth performance,immune and antioxidant function of broilers,which could bring a satisfactory effect as an alternative to antibiotics.Experiment 3: Mitigation action of Artemisia ordosica polysaccharide on immune stress of broilers and the underlying mechanismThe study investigated the effects of dietary AOP on growth,immune responses and antioxidant function of broilers challenged with lipopolysaccharide(LPS).A total of 1921-day-old broilers were randomly allotted to four treatments with 6 replicates and 8 birds per replicates:(1)basal diet(CON);(2)basal diet+LPS-challenge(LPS);(3)basal diet supplemented with 750 mg/kg AOP(AOP);(4)basal diet supplemented with 750 mg/kg AOP+LPS-challenge(LPS+AOP).The trial included starter phase(d 1 to 14),stress period?(d 15 to 21,LPS injection period),convalescence ?(d 22 to 28),stress period ?(d 29 to35,LPS injection period)and convalescence ?(d 36 to 42).During stress period ? and ?,broilers were injected intra-abdominally either with LPS solution or with equal sterile saline.The results showed that AOP alleviated LPS-induced growth inhibition by prompting apparent metabolic rate of protein,and decreasing serum stress hormones and pro-inflammatory cytokines content of broilers.Moreover,AOP decreased LPS-induced over-production of IL-1? and IL-6 through suppressing TLR4/NF-?B pathway,and alleviated LPS-induced decreasing of T-AOC,CAT and GPx activities by activating Nrf2/Keap1 pathway.Experiment 4: Alleviating effect of Artemisia ordosica polysaccharide on immune stress of broiler lymphocytesThe in vitro experiment was conducted in a 2×7 factor design with 14 treatments(each treatment repeated 6 times),including two immunological challenges(saline or LPS)and seven dietary treatments,namely six levels of AOP(0,50,100,150,200 and 250?g/m L)and vitamin A supplementation.The results showed that under normal condition,AOP-?had significant immune and anti-oxidation activity in vitro;under stress condition,the AOP-? could alleviate the immune stress caused by LPS.Addtionally,150 ug/m L AOP-?was optimal dosage for studying immune and antioxidantive regulation mechanism in vitro.Experiment 5: Artemisia ordosica polysaccharide alleviated immune stress through TLR4/NF-?B pathway(1)The PBLs were randomly divided into 6 treatments with 6 replicates,namely CON,LPS,LPS+TAK-242,AOP-?,AOP-?+TAK-242 and TAK-242.The results showed that TLR4 was the major receptor involved in interaction of AOP-? and PBLs.(2)The PBLs was randomly divided into 6 treatments with 6 replicates,namely CON,FITC-LPS,LPS+FITC-LPS,LPS,AOP-?+FITC-LPS and AOP-?.The results showed that AOP-?disturbed the combination between LPS and TLR4.(3)The PBLs was randomly divided into 8 treatments with 6 replicates,namely CON,AOP-?,LPS,PDTC,AOP-?+LPS,AOP-?+PDTC,LPS+PDTC and LPS+PDTC+AOP-?.The results showed that under normal condition,AOP-? presented immune and anti-oxidation activity via activating TLR4/NF-?B pathway;under stress condition,the AOP-? alleviated immune stress caused by LPS via suppressing TLR4/NF-?B pathway.Experiment 6: Artemisia ordosica polysaccharide alleviated immune stress through Nrf2/Keap1 pathway.The PBLs was randomly divided into 8 treatments with 6 replicates,namely CON,AOP-?,LPS,ML385,AOP-?+LPS,AOP-?+ML385,LPS+ML385 and LPS+ML385+AOP-?.The results showed that under normal condition or stress condition,AOP-? presented immune and anti-oxidation activity via activating Nrf2/Keap1 pathway.