Transcriptome-based Study On Angiogenesis In Chicken Tibial Dyschondroplasia And Regulatory Effect Of Recombinant Glutathione-S-Transferase A3 Protein

Objectives: This study of transcriptome sequencing investigated the potential target genes of angiogenesis in chicken erythrocytes of thiram induced tibial dyschondroplasia(TD).Those genes could regulate the blood circulation and angiogenesis which may provide a new insight into the therapeutic manipulation,to prevent TD.Furthermore,we selected angiogenesis and MAPK signaling pathway related genes to evaluate the harmful effect of thiram and elucidate the molecular mechanism of angiogenesis concerned to TD.The regulatory role of r GSTA3 protein on angiogenesis related genes in TD induced broiler chickens was also investigated under this study.Methods: In the first phase of this study,we purchased 24 broiler chickens and rear for 7 days,after fasting for an overnight on eighth-day we feed them thiram for 48 h to induce TD and taken blood and tibia growth plate samples on day 2,6 and 15(8,14-and 23-days old chicken)to investigate the effect of thiram(thiram group,12 broiler chickens)compare to control(control group,12 broiler chickens)groups in histopathology and transcriptome level.Histopathology was done and immunohistochemistry(IHC)performed for two angiogenesis related genes(ITGAV and CLU).Transcriptome sequencing was done to find out total number of upregulated and downregulated gene expressions related to the potential pathways.We identified some angiogenesis related genes for further experiments to confirm it on q PCR.In the second phase we purchased 120 broiler chickens and rear for 7 days,after fasting for an overnight on eighth day we feed them thiram to induce TD along with different doses supplementation of r GSTA3 protein to find out the regulatory effect of r GSTA3 protein on TD chickens.The samples of blood and tibia growth plate were taken on day 6 and 15(14-and 23-days old chicken).Results: The histopathology and haematoxylin and eosin(H&E)results revealed that angiogenesis was diminished on day 6 but started recovering on day 15.IHC results confirmed the expressions of integrin alpha-v precursor(ITGAV)and clusterin precursor(CLU).The transcriptome sequencing analysis evaluated 293 differentially expressed genes(DEGs)with 103 upregulated and 190 downregulated were enriched in the pathways of neuroactive ligand receptor interaction,mitogen-activated protein kinase(MAPK),ribosome,regulation of actin cytoskeleton,focal adhesion,natural killer cell mediated cytotoxicity and the notch signalling pathways.We screened 20 angiogenesis related potential DEGs(TBXA2R,IL1R1,RPL17,ITGB3,ITGAV,ITGB2,RAC2,ITGA2,IQGAP2,GIT1,VAV1,ITGA5,RAP1 B,FYN,PTPN11,PTCH1,NCOR2,MAML3)which were found downregulated in thiram induced TD chickens on day 6 and 15.MAPK signaling pathway related genes were evaluated in thiram induced TD chickens.Furthermore,q RT-PCR results reported injection of r GSTA3 protein upregulated the expressions of most important angiogenesis related integrin family genes(ITGAV,ITGA2,ITGB2,ITGB3,ITGA5)significantly(P < 0.05).r GSTA3 protein also upregulated TBXA2 R,IL1R1,RPL17,RAC2,IQGAP2,GIT1,VAV1,RAP1 B,FYN,PTPN11,PTCH1,NCOR2,MAML3 and CLU.Conclusion: To prevent TD in broiler chickens r GSTA3 protein is needed to be added as it has a regulatory effect on some important angiogenesis related genes including ATP2A3,UBE2R2,CCSAP,F13A1,SHROOM2,RASA3,and CLU,TBXA2 R,IL1R1,RPL17,ITGB3,ITGAV,ITGB2,RAC2,ITGA2,IQGAP2,GIT1,VAV1,ITGA5,RAP1 B,FYN,PTPN11,PTCH1,NCOR2,MAML3 of potential pathways(Neuroactive ligand receptor interaction,MAPKs,ribosomes,regulation of actin cytoskeleton,focal adhesion,natural killer cell mediated cytotoxicity and the Notch signaling pathway).r GSTA3 protein improved the vascularization,ultimately chondrocytes improved and angiogenesis in the growth plate of thiram induced TD chickens seem better.The usage of r GSTA3 protein in TD chickens as medicine will assist to overcome this problem in future.