Multi-omic Analysis Of LPS Induced Broiler Stress Model And Prediction Of Effective Chinese Herbs

High stocking density and environmental factors such as bacterial or viral exposure,heavy vaccination,transportation,acute heat stress,and shock may cause a detrimental effect on broilers in modern intensive broiler production systems.Generally,stress could lead to the overconsumption of nutrients,obstruction in growth and development,and severe reduction in meat quality of birds,inducing a variety of diseases in severe cases,and causing serious economics losses in poultry farms.The liver is regarded as both metabolic as well as immunological lymphoid organ.It is not only engaged in metabolic,nutrient storage and detoxification activities,but also responsible for the synthesis of cytokines,chemokines,and complement components.Therefore,liver might play a vital important role in stress-induced metabolic and immune related changes in chicken.In this study,LPS was used to establish a broiler stress model,and transcriptomics and metabolomics were performed to reveal expressed genes and associated metabolites in the liver,which might provide a useful reference for understanding the mechanism of stress.This study mainly contained four experiments:Firstly,the aim of this experiment was to investigate the effects of stress on growth performance,stress-related hormone,relative organ weight and liver pathological changes in broiler chickens.A total of 120 one-day-old,male,Hongyu commercial broiler chickens were randomly divided into two groups: challenged with saline(Control group)or LPS(Model group).On days 12,14,33,35,all chickens were injected intraperitoneally with either 250 μg/kg body weight of LPS or sterile saline.Birds were group weighed by cage every week and feed intake on a cage basis was recorded daily.Blood samples were collected on day 35 for detection of serum adrenocorticotropic hormone(ACTH)and corticosterone(CORT).Liver,spleen and bursa tissues were excised and weighed.Livers were collected for histological examination.The results showed that stress by LPS reduced average daily weight gain and daily feed intake(P < 0.01).The injection of LPS increased serum ACTH and CORT(P < 0.01)as well as increased the relative liver and spleen weight(P < 0.01).However,relative bursa weight in the LPS-challenged broilers were decreased(P < 0.01).Secondly,transcriptomics was used to investigate differences in the expression profiles of related genes and associated pathways in the liver to classify molecular mechanisms of stress.Four broilers in each treatment were randomly selected,from which liver samples were collected for transcriptomics analysis.Genes with q < 0.05 were considered differentially expressed.The results showed that there were 129 significantly differentially expressed genes(DEGs),of which 60 were up-regulated genes,and 69 were down-regulated genes.Gene ontology(GO)annotation of up-regulated DEGs found that the term related to stress mainly included acute phase response,inflammatory response,defense response,response to stress and response to external stimulus.And the downregulated genes were mostly concentrated in lipid metabolic process and cellular lipid metabolic process.Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis showed that the main pathways related to stress were the fatty acid degradation,valine,leucine and isoleucine degradation,and tryptophan metabolism.Thirdly,to further identify the metabolomics profiles characterizing the process,we conducted a comprehensive UHPLC-QE-MS-based metabolic profiling of liver tissues from broilers.OPLS-DA was used to visualize the differences in metabolite profiles between two groups and significant changes in the levels of 109 metabolites were detected.In KEGG pathway analysis,a total of 10 metabolic pathways was significantly perturbed in Model group compared with Control group,embodying in lipid metabolism(glycerophospholipid metabolism),amino acid metabolism(valine,leucine and isoleucine biosynthesis,biosynthesis of amino acids,histidine metabolism,glycine,serine and threonine metabolism),glycan metabolism(mucin type O-glycan biosynthesis,mannose type O-glycan biosynthesis),metabolism of cofactors and vitamins(pantothenate and Co A biosynthesis).intestinal immune network for Ig A production and apoptosis.Further investigation of differential metabolites showed 10 potential biomarkers such as Lyso PC(18:1),L-citrulline and histamine.Fourthly,combined analysis of transcriptomics and metabolomics indicated that differentially expressed genes and differential metabolites in liver of stress-birds were involved in valine,leucine and isoleucine degradation(L-arginine,L-leucine,ATP6V1C2),m TOR signaling pathway(L-leucine,ketoleucine,ACOX1,ALDH3A2,EHHADH,AOX2,IL4I1),glycolysis/gluconeogenesis(Pyruvic acid,ALDOB,ALDH3A2).The PPI network shows GAPDH,MDH2,TPI1,ALDOB,AGXT.MDH1,TALDO1,GPI,GOT2,HADH,ACOX1,PGK1,OAT,TKT,EHHADH as potential targets for the treatment of stress.Through predictive screening of traditional Chinese medicine,it was found that heat-clearing herbs and tonic medicines may be potential representative drugs of tranditional medicine for the treatment of stress.In conclusion,stress model was successfully established by LPS injection in this study.A total of 129 differentially expressed genes were screened in liver transcriptome,of which 60 were up-regulated and 69 were down-reuglated.Thess DEGs were involved in 10 GO terms,such as acute phase response,inflammatory response,defense response.In additional,three KEGG pathways were also enriched,including fatty acid degradation,branched-chain amino acid degradation and tryptophan metabolism.It is worth noting that genes related to defense function were up-reuglated while genes associated with fatty acid degradation and down-regulated.Metabolomics results revealed 129 different metabolites,and the combined analysis predicted that stress in broiler might be associated with branched-chain amino aicd,glycolysis/gluconeogenesis and m TOR signaling pathway.Through predictive screening of traditional Chinese medicine,it was found that heat-clearing herbs and tonic medicines may be potential representative effective herbs of tranditional medicine for the treatment of stress.