| In order to elucidate the mechanism of the decline of immune function in broilers under high temperature and the regulation of peppermint extract,this project established heat stress model of immune suppression in broilers.The spleen was taken as the object of study,i TRAQ quantitative proteomics technology was applied to analyze the changes of total spleen proteins in broilers under heat stress,and bioinformatics method was combined to study the molecular mechanism of immunosuppression induced by heat stress.The peppermint extract was used to regulate the immunosuppression induced by heat stress.The main research contents and results are as follows:1.Establishment of heat stress model of immune suppression in broilers: 96 28-day-old healthy AA broilers were randomly divided into two groups: control group(21?)and heat stress group(31?)with 48 chickens in each group.Each group had 4 replicates,12 chickens in each replicate,and the relative humidity was 60%.At the end of experiment,the core temperature,respiratory rate,growth performance and immune function were measured.The results showed that heat stress significantly increased the core temperature and respiratory rate of broilers,significantly reduced the spleen weight and spleen index,significantly increased the plasma levels of IL-2,IFN-?,IL-1?,IL-6 and TNF-?,significantly reduced the content of IFN-?,resulting in the decline of immune function,thus heat stress model of immune suppression in broilers was established.2.Proteomic study of broiler spleen based on i TRAQ: At 42 days of age,three chickens were taken from each replicates of control group and heat stress group.Spleen samples for quantitative analysis of i TRAQ proteomics were collected.The differentially expressed proteins related to innate immunity were validated by PRM technology and RT-q PCR at protein level and m RNA level.The results showed that heat stress led to the down-regulation of IRF-3?CD40?TNFAIP3 ? IL-18 ? Cath L2 ? IAP3 ? CYBA ? TRIM25 and POLR3 F.These down-regulated differentially expressed proteins are related to Toll-like receptor signaling pathway,NOD-like receptor signaling pathway,RIG-I-like receptor signaling pathway,cytosolic DNA sensing pathway.3.Regulation of peppermint extract on immune suppression induced by heat stress: 144 28-day-old healthy AA broilers were randomly divided into three groups: control group(21?),heat stress group(31?)and heat stress((31?))+peppermint extract group,48 chickens in each group,6 replicates in each group,8 chickens in each replicate,and the relative humidity was 60%.At 42 days of age,the main pattern recognition receptors and transcription factors in innate immune related pathways screened by proteomics were detected by RT-q PCR,and inflammatory cytokines in spleen were detected by ELISA.The results showed that heat stress significantly increased the expression of TLR2,TLR4,MDA5 and transcription factors of NF-?B and MAPK pathways and significantly increased the content of inflammatory cytokines IL-1?,IL-6 and TNF-?,and peppermint extract significantly decreased the expression of NOD1,DAI and transcription factors of NF-?B and MAPK pathways and significantly decreased the content of inflammatory cytokines IL-1?,IL-6 and TNF-?.Conclusion: 1)It was revealed that heat stress is related to Toll-like receptor signaling pathway(IRF-3 and CD40),NOD-like receptor signaling pathway(TNFAIP3,IL-18,Cath L2,IRF-3,IAP3 and CYBA),RIG-I-like receptor signaling pathway(TRIM25 and IRF-3),cytoplasmic DNA sensing pathway(IL-18,POLR3 F and IRF-3),and that heat stress promotes the release of inflammatory factors by activating TLR2,TLR4 and MDA5 and their downstream pathways of NF-?B and AP-1,which ultimately leads to the decline of immune function.2)It was revealed that peppermint extract could reduce the release of inflammatory cytokines by down-regulating the expression of NF-?B and AP-1 and alleviate the decrease of immune system induced by heat stress. |
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