L-Leu-L-Leu Peptide Mediated Regulatory Pathway In Broiler Intestinal Epithelial Cells

Intestinal tract is an important place for animals to digest and absorb nutrients,and it is also an important immune barrier of the body.Therefore,maintaining intestinal health is of great significance for ensuring the growth and development of animals.Small peptides are not only nutrients,but also bioactive molecules,which can regulate both nutrition and physiology.In order to elucidate the effects of small peptides on the growth,proliferation and apoptosis of broiler intestinal epithelial cells(IEC)and its molecular mechanism,L-Leucine(L-leu)and L-Leucine-L-Leucine(L-Leu-L-Leu)were used as experimental materials to study the effects of L-Leu-L-Leu on the growth,proliferation and apoptosis of IEC to screen candidate genes and proteins related to IEC growth,proliferation and apoptosis by RNA-seq and Lable-free sequencing.The aim of this study is to provide a theoretical basis for elucidating the IEC regulation pathway mediated by small peptides,improving the theoretical system of small peptide nutrition and establishing the technology of small peptide nutrition regulation.The results are as follows:(1)The purity of isolated primary IEC was over 90% by immunofluorescence identification.After cell passage and drug intervention,it was found that L-Leu-L-Leu group significantly promoted the growth and proliferation of IEC cells,and significantly inhibited the apoptosis of IEC cells,and the effect was better than that of L-Leu group and Control group.(2)The c DNA libraries constructed from 9 samples of Control group,L-Leu group and L-Leu-L-Leu group were sequenced.The results showed that there were 34 differentially expressed genes in L-Leu group,among which 23 were significantly up-regulated and 11 were significantly down regulated.There were 29 differentially expressed genes in L-Leu-L-Leu group,19 were significantly up-regulated and 10 were significantly down regulated.Compared with L-Leu group,there were 26 differentially expressed genes in L-Leu-L-Leu group Among them,12 genes were up-regulated and 14 genes were down regulated.Four candidate genes CCL20,IL8L1,IL8 and IL6 were screened in L-Leu group compared with the Control group,which were involved in Cytokine-cytokine receptor interaction,Toll-like receptor signaling pathway,NOD-like receptor signaling pathway,Cellular senescence and RIG-I-like receptor signaling pathway.One candidate gene IL8 was found to participate in RIG-I-like receptor signaling pathway,Toll-like receptor signaling pathway,NOD-like receptor signaling pathway,Cellular senescence and Cytokine-cytokine receptor interaction in L-Leu-L-Leu group compared with the Control group.IL-6 and RGN candidate genes were selected from L-Leu-L-Leu group compared with L-Leu to participate in Cytokine-cytokine receptor interaction,Toll-like receptor signaling pathway,NOD-like receptor signaling pathway,Cellular senescence and Pentose phosphate pathway.(3)Lable-free sequencing results showed that 90 differentially expressed proteins were screened out in L-Leu group compared with the Control group,34 of which were significantly up-regulated,56 of which were significantly down regulated.221 of which were screened out in L-Leu-L-Leu group compared with the Control group,130 of which were significantly up-regulated,81 of which were significantly down regulated.47 of which were screened out in L-Leu-L-Leu group compared with L-Leu,21 were up-regulated and 26 were down regulated.By screening candidate proteins and pathways related to cell growth,proliferation and apoptosis,PGM3 was found to be involved in Amino sugar and nucleotide sugar metabolism in L-leu group compared with Control group,while RPS17,RPS11 and RPL23 were found in L-Leu-L-Leu group compared with Control group,GPX4 and PDK1 were found to be involved in glutathione metabolism and T cell receptor signaling pathway in L-Leu-L-Leu group.This study found that L-leu-L-leu could promote the growth and proliferation of IEC cells,and inhibit the apoptosis of IEC cells.Combined with transcriptome and proteome sequencing,we found that multiple immune and energy related regulatory signaling pathways are related to IEC cell growth,proliferation and apoptosis;three candidate genes IL8,IL6 and RGN were identified,and five candidate proteins RPS17,RPS11,RPL23,GPX4 and PDPK1 were involved in cell growth,proliferation and apoptosis.The results provide valuable data for elucidating IEC regulation pathway mediated by small peptide,perfecting the theoretical system of small peptide nutrition and establishing small peptide nutrition regulation technology.