Effects Of Artemisia Argyi Alcohol Extract On Growth?Immune And Anti-Oxidative Function In Broilers Challenged With Lipopolysaccharide And The Underlying Mechanism

The objective of this experiment was to study the effects of Artemisia argyi alcohol extract on immune and antioxidant function in broilers and the underlying mechanism.The main results were listed as follows:Part one: Effects of Artemisia argyi alcohol extract on the serum immune and antioxidative function of broilersA total of 240 one-day-old Arbor Acre broiler were randomly allocated five treatments.Each treatment group consisted of six replicates with eight chicks per replicate.The five treatment diets were added with,respectively,0,250,500,750,and 1000 mg/kg of AAAE in the basal diet.The experiment lasted for 42 d and was divided into two stages of d 1-21 and 22-42.The results showed that supplementation with 500-750 mg/kg of AAAE could significantly increase the ADG and reduce the ADFI,F: G of broilers at 42 d of age.Supplementation with 500-1000 mg/kg AAAE increased the serum IL-2,IL-4,IgA,IgG,IgM,CAT and SOD contents or activities,and decreased the IL-1?,IL-6,and MDA contents.In conclusion,the 750 mg/kg dose group was better to promote the immune and antioxidative function of broilers.Part two: Effects of Artemisia argyi alcohol extract on growth,immune and antioxidative function in broilers challenged with lipopolysaccharide and the underlying mechanismThe experiment was designed as a 2×2 factorial arrangement.The factors included AAAE supplementation(0 or 750 mg/kg diet)and intraperitoneal LPS(750 ?g/kg body weight(BW))or saline injection.A total of 192 one-day-old Arbor Acres broiler chickens(each sex 50%)were weighed and divided into four treatment groups.Each treatment group consisted of six replicates with eight chicks per replicate.The four dietary treatment groups were as follows: broilers fed a basal diet and injected with saline;broilers fed a basal diet and injected with LPS;broilers fed a basal diet supplemented with AAAE and injected with saline,and broilers fed a basal diet supplemented with AAAE and injected with LPS.The experiment lasted for 42 d and was divided into the adaptation period(d 0-14),stress phase I(d 15-21,LPS injection period),recovery period I(d 22-28),stress phase II(d 29-35,LPS injection period),and recovery period II(d 36-42).On d 15,17,19,and 21(stress phase I)and on d 29,31,33,and 35(stress phase II),LPS or equivalent saline were injected into enterocoelia of broilers.The results showed:(1)Dietary supplementation with AAAE significantly attenuated LPS-induced increase in the concentrations of ALT,LDL-C,RBC,WBC,LYM,PLT,CORT,and ACTH.However,dietary AAAE significantly alleviated the decrease of the ADG,ADFI,the apparent metabolic rate of nutrients(DM,CP,Ca),HDL-C,VH and VH/CD in broilers caused by the LPS challenge.(2)Dietary supplementation with AAAE significantly attenuated LPS-induced increase of IL-1?,IL-6,IL-2 and IgG concentrations in the liver and small intestine,in addition,down-regulated the gene expression of NF-?B p65,IL-1? in the liver,TLR4,IL-1? in the duodenum,My D88,NF-?B p65,IL-1? in the jejunum,and TLR4 in the ileum,and the expression and phosphorylation of NF-?B p65 and I?B? in the TLR4/NF-?B signaling pathway.(3)Dietary AAAE increased the activities of CAT and SOD in serum,liver,and ileum,meanwhile,up-regulated the gene expression of Nrf2,CAT,SOD,GSH-Px and HO-1 in the liver,Nrf2,CAT,SOD and GSHPx in spleen and small intestine.Moreover,AAAE increased the protein expression levels of Nrf2,HO-1 and SOD,and significantly decreased the protein expression level of Keap1.These results further demonstrated that AAAE enhanced the immunity and antioxidant capacity of broilers,thereby alleviated LPS-induced stress damage in broilers.Its action mechanism may be related to TLR4/NF-?B and Keap1/Nrf2 signal pathways.Part three: Separation and purification of flavonoids from Artemisia argyi alcohol extract and its effects on immune and antioxidant functions of peripheral blood lymphocytesThe study was designed to obtain high purity of flavonoids through purifying by resins and polyamide resin from Artemisia argyi alcohol extract,and then used for the lymphocyte culture experiments.The present experiment was conducted as the single factor completely randomized design to study the effect of different doses of AAF(0,25,50,100,200,400?g/m L)on the immune and antioxidant function of peripheral blood lymphocytes.The results showed:(1)supplement of 50 and 100 ?g/m L AAF improved the lymphocyte viability.(2)Supplementation of 100 and 200 ?g/m L AAF decreased the IL-1? concentration and gene expression;the IL-2 and IgG concentration in 25 and 200 ?g/m L and 100 ?g/m L treatments were significantly increased,respectively.And the gene expressions of TLR4 and NF-?B p65 were decreased in 50 and 100 ?g/m L AAF treatments.(3)Supplementation of 100 ?g/m L AAF increased the GSH-Px,CAT,and SOD activity and decreased the MDA concentration;meanwhile,the gene expression of Nrf2,HO-1,SOD,CAT,and GSH-Px were improved in appropriate dose of AAF treatments.In conclusion,the 100 ?g/m L dose group was better to promote the immunity and antioxidant effects of peripheral blood lymphocytes.Part four: Study on the mechanism of AAF regulating the immune function of peripheric blood lymphocytes in broilers by TLR4/NF-?B signaling pathwayBasing on part 3,part four was conducted using the completely randomized design.The bioactivity of NF-?B was inhibited by PDTC to explore the mechanism of AAF defending PBLs from LPS stress via the TLR4/NF-?B pathway.The PBLs were randomly divided into six treatment groups: control group,LPS group,AAF group,LPS+PDTC group,LPS+AAF group,and LPS+AAF+PDTC group.The results showed that the LPS+PDTC group and LPS+AAF group could alleviate stress damage induced by LPS through inhibiting the phosphorylation levels of I?B?,decreasing the gene expression TLR4,My D88,NF-?B p65,NF-?B p50,IL-1? and IL-6,and the protein expression of NF-?B p65 in PBLs.It indicated that the protective effect of AAF on LPS-induced cell stress damage probably was due to down-regulating IL-1? gene expression and reducing the release of pro-inflammatory cytokines through NF-?B signaling pathway.Part five: Study on the mechanism of AAF regulating the antioxidant function of peripheric blood lymphocytes in broilers by Keap1/Nrf2 signaling pathwayThe present experiment was conducted using a single factor completely randomized design.The bioactivity of Nrf2 was inhibited by ML385 to explore the mechanism of AAF defending PBLs from LPS stress via the Keap1/Nrf2 pathway.The PBLs were randomly divided into six treatment groups: control group,LPS group,AAF group,LPS+ML385group,LPS+AAF group,and LPS+AAF+ML385 group.The results showed that the LPS+ML385 group and LPS+AAF group could significantly increase the activities of GSHPx,SOD in PBLs challenged with LPS.In addition,the LPS+ML385 group and LPS+AAF group could alleviate oxidative damage induced by LPS through inhibiting the protein expression of Keap1 and up-regulating gene expression of Nrf2,HO-1,GSH-Px,and SOD.