Mechanism Of Lung Injury In Broilers With Ascites Syndrome And Targeted Regulation Of Compound Chinese Medicine Based On RNA-seq

Chicken ascites syndrome(AS)is a nutritional metabolic disease.The pathogenesis of AS is closely related to the difficulty of the heart,lung and vascular systems to meet the oxygen demand of broiler metabolism.From the perspective of traditional Chinese medicine,this disease belongs to ’lung qi stagnation’,’qi and blood deficiency’,’water stagnation’,’phlegm and blood stasis’.The lung is the main source of qi,the source of water,can adjust the water channel;lung qi stagnation,blood flow blocked,stop for blood stasis,accumulation for phlegm,phlegm and blood stasis in the lung,and then involved in the heart.RNA-seq technology has emerged in the field of traditional Chinese medicine research with its advantages of integrity,systematicness,individual organizational differences and time independence.After preliminary studies,the compound Chinese medicine of our group has good effect on broiler AS,but its mechanism still needs further study.Therefore,in this study,RNA-seq technology was used to detect the differential genes in lung tissue of AS broilers and the effects of compound Chinese medicine on lung tissue of AS broilers.Experiment 1: Establishment of AS broiler model and effect of compound Chinese medicine on growth performance,ascites heart index,blood index and pathological changes of lung tissue of AS broiler.A total of 168 1-day-old AA broilers were randomly divided into seven groups at 7-day-old: control group,model group,compound Chinese medicine(high,medium and low dose)group,positive drug control group,and L-arginine group,with three replicates in each group and eight chickens in each replicate.Except the blank group,the low temperature of each group was maintained at 9°C-11°C,0.12% salt was added in drinking water,and 3 % lard and 4% fish meal were added per kilogram of basic diet.In addition,compound Chinese medicine with high,medium and low dose groups were added 2g/kg(bw),1g/kg(bw),0.5g/kg(bw)in drinking water.Positive Chinese medicine group in drinking water to add 1g/kg(bw)concentration of positive Chinese medicine;another 1 % L-Arg was added to the high-energy feed of the L-Arg group.Growth performance,ascites cardiac index(AHI),blood routine and pathological sections of lung tissue of broilers at 15,25 and 35 days old were detected.The results showed that :(1)Compared with the blank group,the body weight of each day in the model group was significantly reduced(P<0.05).Compared with the model group,the body weight of compound Chinese medicine(high,middle)group,positive Chinese medicine group and L-Arg group increased significantly(P<0.05).(2)Compared with the blank group,the cardiac index of ascites in the model group was significantly increased(P<0.01).Compared with the model group,the cardiac index of high dose group and L-Arg group was significantly decreased(P<0.05).And 35-day-old model group AHI>0.299,each group AHI<0.299.(3)Compared with the blank group,the blood indexes of RBC,HB,PCV,PLT,PCT and WBC in the model group were significantly increased(P<0.05).Compared with the model group,RBC,HB,PCV,PLT,PCT and WBC in the compound Chinese medicine group,the positive Chinese medicine group and the L-Arg group were significantly decreased to varying degrees(P>0.36).(4)The structure of lung tissue in blank group was clear and complete,and the structure of pulmonary capillary was basically complete.Bleeding,respiratory capillary atrophy,stenosis,wall thickening and inflammatory cell infiltration were common in lung tissue of model group.The lung tissue of high,middle dose group and L-Arg group were improved in varying degrees.Experiment 2: Transcriptome profiles analysis was performed on 35-day-old lung tissues of three normal broilers and three AS broilers.The RNA quality,purity and integrity of the samples met the requirements of RNA-seq sequencing.RNA-seq analysis showed that 614 genes were up-regulated and 828 genes were down-regulated in AS group.GO and KEGG enrichment analysis showed that hypoxia stress response,cell signal regulation and immune system regulation played an important role in broiler AS.HIF-1 signaling pathway,NF-κB signaling pathway,p53 signaling pathway,alanine,aspartate,glutamate metabolism and cell adhesion molecules play a key role in the development of AS in broilers.To our interesting DEGs in these pathways include up-regulated genes SPP1,IGFBP,SFRP4,EIF2AK4,HMOX1,MMP9,THBS1,CCR7,CRP,TLR4,CCL5,BCL2,IGF-I and down-regulated genes IL7,CD40,CD40 L,CD4,NOX4,CCL1,CCL19,CX3CL1,APLNR,NR4A3,AQP5.Experiment 3 : The lung tissues of 35-day-old blank group,model group,compound Chinese medicine high dose group and L-Arg group were selected for RNA-seq sequencing.The results showed that 2655 DEGs were found in N＿vs＿C group,1071 DEGs were found in C＿vs＿T group,and 2902 DEGs were found in C＿vs＿L group.GO enrichment includes active oxygen metabolism,immune system response,aspartic peptide peptidase activity,and chemokine receptor binding.KEGG enrichment analysis found that it was related to metabolism,immune system,digestive system,excretion system and endocrine system pathways.In addition,615 key DEGs in the overlapping parts of N＿vs＿C,C＿vs＿L and C＿vs＿T gene sets were used to establish a new gene set for GO analysis.It was found that signal regulation,nitric oxide metabolism,transmembrane receptor protein serine / threonine kinase signaling pathway and cell migration regulation were enriched,and the enrichment chord diagram showed that APLNR,NOS1 and NOS2 genes might be the key genes of AS.KEGG enrichment analysis showed that metabolic pathways were the most abundant,including pyrimidine metabolism,purine metabolism,histidine metabolism,glycine,serine and threonine metabolism,thiamine metabolism,butyric acid metabolism,valine,leucine and isoleucine biosynthesis,tyrosine metabolism and arginine biosynthesis.Experiment 4: The contents of HIF-1α,APLN,APLNR,PPARγ,ET-1,EDNRA and ENOS in lung tissue of 35-day-old blank group,model group,compound Chinese medicine(high,medium and low dose)group,positive Chinese medicine group and L-Arg group were detected by fluorescence quantitative PCR,ELISA and Western blot.The results showed that compound Chinese medicine and L-Arg could reduce the expression of HIF-1α in lung tissue of AS broilers,increase APLN,APLNR,PPARγ and ENOS,and reduce the contents of ET-1 and EDNRA.In conclusion,the AS model of broilers was successfully constructed.The high dose of compound Chinese medicine and L-Arg had good effect on AS in broilers.RNA-seq revealed that the occurrence of AS in broilers was that the respiratory system could not provide enough oxygen for metabolism,and the lung was an important target organ,which also indicated the pathogenesis of lung failure and lung qi stagnation.The multi-component and multi-target effect of compound Chinese medicine with the effect of ’tonifying qi and blood,promoting blood circulation and removing blood stasis ’ has good effect on the prevention and treatment of AS in broilers.It is found that it plays an important role in improving angiogenesis and vascular metabolism.In addition,the role of APLN and APLNR in the occurrence and development of broiler AS was verified,which may be a new target for the prevention and treatment of broiler AS.