| This study was conducted to investigate the relieving effect of Artemisia ordosica aqueous extract（AOE）on immune stress of broilers challenged with lipopolysaccharide（LPS）and the underlying mechanism through an in vivo feeding experiment and in vitro lymphocyte culture tests.The main contents and results were showed as follows:Experiments 1-3 were conducted to study the effect of AOE on growth performance,immune status and anti-oxidative function of broilers challenged with LPS and the underlying mechanism.In the experiment,ninety-six one-day-old Arbor Acres broilers were assigned into four treatments in 2×2 factorial design,including two dietary treatments（0 or 1000 mg/kg AOE）and two immunological challenge treatments（saline or LPS）.On d 14,16,18,and 20,broilers were injected intra-abdominally with LPS solution（LPS was dissolved in sterile saline at a concentration of 100μg/m L）at 500μg/kg of base weight,or an equivalent amount of sterile saline.The feeding experiment was lasted for a period of 21 d including immune-stress-free period（d 1-14）,immune-stress period（d 15-21）.The results showed:（1）AOE had no impact on growth performance of broilers during d 1-14（P>0.05）,but alleviated the compromised ADG and ADFI（P<0.05）in broilers challenged with LPS.On d 21,diet supplemented with AOE reduced the elevation of serum CORT content（P=0.054）.The results suggested that dietary AOE could reduce the stress hormone,and finally alleviate the growth inhibition of broilers caused by LPS.（2）Dietary AOE inhibited the elevation of relative weight of spleen caused by LPS（P<0.05）.AOE relieved the increased serum concentration of IL-2,IgA,sCD8（P<0.05）,and reduced the serum concentration of TNF-α（P=0.081）and IgG（P=0.079）.AOE reduced the elevation of serum MDA induced by LPS（P<0.05）,and the increasing tendency of serum CAT content was also observed（P=0.097）.The above results suggested that AOE exerted its beneficial effect on broilers challenged with LPS by lessening the inflammatory cytokines,weakening the excessive activated immune system,and enhancing the anti-oxidative function.（3）AOE reduced the elevation of gene expression of MyD88 and IL-1β（P<0.05）,increased the declining gene expression of SOD and GSH-Px（P<0.05）in liver.AOE reduced the elevation of gene expression of iNOS（P<0.05）in spleen.AOE reduced the elevated expression of TLR4、TNF-α、iNOS（P<0.05）,increased the declining expression of CAT（P<0.05）in duodenum.AOE reduced the elevated gene expression of TLR4,MyD88,IL-1β,IL-6,TNF-αand iNOS（P<0.05）,increased the declining CAT gene expression in jejunum（P<0.05）.AOE reduced the elevation of gene expression of NF-κB,IL-1βand iNOS（P<0.05）,increased the declining expression of GSH-Px（P<0.05）in ileum.The above results indicated that AOE supplementation down-regulated the inflammatory mediator production through reducing the TLR4/NF-κB signaling pathway genes transcription;on the other hand,dietary AOE up-regulated the anti-oxidant enzyme gene expression and then removed ROS,inhibited the activation of NF-κB,and finally alleviated the compromised growth performance of broilers under immune stress.Experiment 4 was conducted to investigate the effect of AOP on immune status and anti-oxidative function in peripheral blood lymphocytes（PBL）challenged with LPS and the underlying mechanism.Firstly,a 2×5 factor design was used,including two immunological challenges（saline or LPS）and five AOP levels（0,25,50,100,200μg/mL）.The trial consists of 10 treatments with 6 replicates.Secondly,a 2×2×2 factor design was used,including two immunological challenges（saline or LPS）,two AOP levels（0 or AOP,the additive dose determined according to the previous experiment）,and two PDTC levels（with or without PDTC）.The trial consists of 8 treatments with6 replicates.In the second experiment,the highly specific NF-κB P65 inhibitor PDTC was used to verify whether the anti-inflammatory activity of AOP was primarily regulated through the TLR4/NF-κB signal pathway.The results showed:（1）Under normal culture condition,AOP increased the TNF-αcontent,SOD activity and T-AOC（P<0.05）.Under LPS-stimulated circumstance,AOP reduced the elevated content of IL-6,NO and the activity of iNOS（P<0.05）,increased the declining SOD activity induced by LPS（P<0.05）,inhibited the increased gene expression of TLR4、NF-κB、IL-1β、IL-6、TNF-α、i NOS in PBL caused by LPS（P<0.05）,and increased the expression of GSH-Px and SOD in PBL（P<0.05）.The above results indicated that AOP can exert its immune and anti-oxidative function under normal circumstance,which is not achieved by the TLR4/NF-κB signal pathway.However,under LPS-stimulated circumstance,AOP inhibited the expression of TLR4 and NF-κB,down-regulated the excessive expression of downstream inflammatory cytokines,and then inhibited the inflammatory response in PBL.On the other hand,AOP increased GSH-Px and SOD gene expression to enhance the anti-oxidation in PBL,and then inhibited the lipid peroxidation damage caused by LPS.（2）Under normal circumstance,AOP increased the content of IL-6,TNF-α,and T-AOC（P<0.05）.Under LPS-stimulated circumstance,PDTC inhibited the elevation of the IL-1β,IL-6,TNF-α,NO and MDA content（P<0.05）,increased the SOD activity（P<0.05）,reduced the gene expression of NF-κB,IL-1β,IL-6,TNF-αand iNOS（P<0.05）.AOP inhibited the elevation of the IL-1β,IL-6,TNF-α,NO and MDA content（P<0.05）,and the iNOS activity（P<0.05）,reduced the expression of TLR4,NF-κB,IL-1β,IL-6 and iNOS（P<0.05）.When AOP and PDTC were added together,AOP had no impact on the immune and anti-oxidative indexes in PBL culture medium,but significantly increased the SOD gene expression in PBL.The above results indicated that the anti-inflammatory effect of AOP was realized by the TLR4/NF-κB signal pathway,and the anti-oxidative effect of AOP might also be another route of its anti-inflammatory effect.Experiment 5 was conducted to investigate the effect of RAOP on immune status and anti-oxidative function of broiler’s PBL challenged with LPS and the underlying mechanism.The experimental design and cell culture were the same as experiment 4.The results showed:（1）Under normal circumstance,RAOP had no impact on immune indexes,but increased the SOD activities and T-AOC（P<0.05）,reduced the content of MDA（P<0.05）,increased the expression of GSH-Px in PBL（P<0.05）.Under LPS-stimulated circumstance,RAOP reduced the elevated content of IL-6,NO and the activity of iNOS（P<0.05）,increased the decreased SOD activity and T-AOC induced by LPS（P<0.05）,inhibited the increased gene expression of NF-κB,IL-1β,IL-6,TNF-αin PBL caused by LPS（P<0.05）,and increased the expression of GSH-Px and CAT in PBL（P<0.05）.The above results indicated that RAOP had a strong anti-oxidative effect,but its immunomodulatory effect was not obvious under normal circumstance.However,under LPS-stimulated circumstance,RAOP inhibited the expression of NF-κB,down-regulated the over-expression of downstream inflammatory cytokines,and then inhibited the inflammatory response in PBL.On the other hand,RAOP increased GSH-Px and CAT gene expression to enhance the anti-oxidation in PBL,and then inhibited the lipid peroxidation damage caused by LPS.（2）Under normal circumstance,RAOP increased the content of IL-1βand the activity of T-AOC（P<0.05）.Under LPS-stimulated circumstance,PDTC inhibited the elevation of the IL-1β,TNF-α,NO and IgM content（P<0.05）,increased the SOD activity（P<0.05）,reduced the expression of NF-κB,IL-6 and iNOS（P<0.05）.RAOP inhibited the elevation of the IL-1β,IL-6,TNF-α,NO and IgM content（P<0.05）,increased the SOD and T-AOC activities（P<0.05）,meanwhile,RAOP reduced the gene expression of NF-κB,TNF-αand iNOS（P<0.05）.When RAOP and PDTC were added together,RAOP significantly increased the T-AOC activity in PBL culture medium（P<0.05）.The above results indicated that RAOP had a strong anti-oxidative effect,and its anti-inflammatory activity might be achieved by exerting its anti-oxidative and free radical scavenging ability,further inhibiting the activation of NF-κB. |
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