Study On The Effects Of Artemisia Argyi Polysaccharide On Immune And Antioxidant Functions In Broilers And The Underlying Mechanism

This experiment was conducted to investigate the effects of Artemisia argyi polysaccharide(AAP)on immune and antioxidant functions of broilers,and to explore the mechanism of AAP regulating immune and antioxidant functions of broilers by in vivo and in vitro methods.The main research contents and results are as follows:Experiment 1: Study on extraction and purification of Artemisia argyi polysaccharideThis experiment was designed to extract,isolate and purify polysaccharides from Artemisia argyi,and characterize the structure of the purified artemisia argyi polysaccharide,so as to provide experimental materials for subsequent animal tests and lymphocyte tests in vitro.This experiment consists of two parts.Experiment(1): The crude polysaccharide(AAP)from Artemisia argyi was extracted by hot water extraction and ethanol deposition,and the molecular weight and monosaccharide composition of AAP were determined by gel permeation chromatography and liquid chromatography.The results showed that the average molecular weight of AAP was 16 k Da,and it was composed of mannose,ribose,rhamnose,glucuronic acid,galacturonic acid,glucose,galactose,xylose,arabinose and fucose.Experiment(2): AAP was further separated and purified by DEAE-52 anion exchange column,and the molecular weight,monosaccharide composition and morphological structure of the purified AAP were determined by ion chromatography,gel chromatography and scanning electron microscope(SEM).The results showed that AAP-Ⅰ was obtained by DEAE-52 anion exchange column chromatography,and the SEM images showed multiple voids,loose spongy flocculent structure.After magnification,three different structures could be seen,which was cube granule,long chain sheet and round ball.The average molecular weight of AAP-Ⅰ was 18 k Da,and the monosaccharide composition was the same as AAP.Experiment 2: Effects of Artemisia argyi polysaccharide on immune and antioxidant function of broilersThis experiment was conducted to study the effects of different doses of Artemisia argyi polysaccharides and chlortetracycline were fed to broilers,and compared with blank control group to study the effects of Artemisia argyi polysaccharides on growth performance,immune and antioxidant function of broilers,and investigate the feasibility of artemisia argyi polysaccharide as a substitute for antibiotics,in order to provide theoretical basis for rational application of artemisia argyi polysaccharide in broiler diet.A total of 288 one-day-old AA broilers were randomly divided into 6 treatments with 6 replicates per treatment and 8broilers per replicate using a single-factor randomized block design.The control group was fed a basal diet,and the experimental groups were fed the basal diet supplemented with 250,500,750 and 1000 mg/kg AAP and 50 mg/kg chlortetracycline,respectively.The experiment lasted for 42 days.The results showed that AAP supplementation at 750 mg/kg could significantly regulate the growth,immune and antioxidant functions of broilers,which suggested that AAP could be expected to replace chlortetracycline in the diet of broilers.Experiment 3: Study on the molecular mechanism of Artemisia argyi polysaccharide on regulating immune and antioxidant functions of broilersBased on the results of experiment 2,the molecular mechanism of AAP regulating immune and antioxidant function of broilers was further studied.The experiment design was the same as experiment 2.The expression levels of immune and antioxidant related genes in liver,spleen and intestinal tissues of broilers were determined.The results showed that dietary AAP increased the expression levels of immune and antioxidant related genes,and AAP activated TLR/NF-κB signaling pathway in broilers.Experiment 4: Effects of Artemisia argyi polysaccharide on immune and antioxidant function of peripheral blood lymphocytes of broilersThis experiment was conducted to study the effects of different doses of Artemisia argyi polysaccharide-Ⅰ(AAP-Ⅰ)was added into culture medium of peripheral blood lymphocytes(PBLs)in broilers for in vitro test,in order to study the effects of different doses of AAP-I on the immune and antioxidant functions of PBLs,and select the appropriate dosage,and provide theoretical basis for subsequent experiments.A single-factor randomized trial design was used in this study,namely,the AAP-Ⅰ supplemental groups were divided into 6treatments(0,50,100,150,200 and 250 μg/m L)with 8 replicates per treatment.The results showed that AAP-Ⅰ had significant modulatory action on immune and antioxidant functions of PBLs of broilers in vitro.200 μg/m L AAP-Ⅰ can be used as an appropriate dosage to study its immune and antioxidant regulation mechanism in vitro.Experiment 5: Study on molecular mechanism of Artemisia argyi polysaccharide regulating immune and antioxidant functions of peripheral blood lymphocytes of broilers through TLR/NF-κB pathwayBased on the results of experiment 4,the receptors TLR2 and TLR4 on the cell membrane were blocked by using their corresponding inhibitors,namely C29(TLR2inhibitor)and TAK-242(TLR4 inhibitor),to study whether AAP-Ⅰ could conduct signal transduction through TLR2 and TLR4 receptors,and then exert its immune and antioxidant regulation action.Based on test 4,this test was conducted and consisted of two parts.Part 1:A 2×2 two-factor experimental design was adopted and randomly divided into 4 treatment groups with 6 replicates in each group.The 4 treatment groups were,respectively,AAP-Ⅰ(-)+C29(-)group,AAP-Ⅰ(+)+C29(-)group,AAP-Ⅰ(-)+C29(+)group and AAP-Ⅰ(+)+C29(+)group.The results showed that AAP-Ⅰ can increase the gene expression of NF-κB and cytokines downstream of TLR2,regulate the TLR2/NF-κB signaling pathway,and play immunomodulatory and antioxidant functions.Part 2: A 2×2 two-factor experimental design was adopted and randomly divided into 4 treatment groups with 6 replicates in each group.The 4 treatment groups were,respectively,AAP-Ⅰ(-)+ TAK-242(-)group,AAP-Ⅰ(+)+TAK-242(-)group,AAP-Ⅰ(-)+ TAK-242(+)group,and AAP-Ⅰ(+)+ TAK-242(+)group.The results showed that AAP-Ⅰ can increase the gene expression of NF-κB and cytokines downstream of TLR4,regulate the TLR4/NF-κB signaling pathway,and play immunomodulatory and antioxidant functions.