cjs-202309-0004 ArticleInAPeriodical 2023 Czech Journal of Animal Science 68 9 391-398 LiangJing DuBaolong WanMinyan ZhaoYapeng TangDefu Gly-Leu instead of Gly promoted the proliferation and protein synthesis of chicken intestinal epithelial cells Amino acids have positive regulatory effects on the function of intestinal epithelial cells (IEC), but in the field of animal nutrition, research on the regulatory effects of amino acids on IEC is still in the initial stages. This study aims to explore the effects of Gly, Gly-Gly, and Gly-Leu on IEC proliferation and their possible mechanisms. Chicken small intestinal epithelial cells were separated using the tissue block method, and other miscellaneous cells were removed for digestion and passage culture. The IEC were cultured in the medium containing 20 nmol/l Gly, Gly-Gly and Gly-Leu for 24 h, and the expression of enterokinase and cytokeratin in cells, the growth curve and activity of IEC, cell cycle, differentially expressed genes, mRNA expression, and protein expression levels of p-mTOR and p-S6K1 in IEC were detected. Enterokinase and cytokeratin were expressed specifically in IEC. The results of growth curve and MTT revealed that the cell viability of IEC was significantly increased after treatment with Gly, Gly-Gly and Gly-Leu. The cell cycle results showed that compared with the control group, Gly, Gly-Gly and Gly-Leu intervention could increase the proportion of IEC in G1 phase, and the proportion in S phase of IEC was decreased. Transcriptome sequencing showed that compared with the control group, there were 54, 28 and 30 differential genes in Gly group, Gly-Gly group and Gly-Leu group, respectively. These genes were mainly enriched in nitric oxide synthesis and protein kinase B signalling, PI3K signal and cellular amino acid biosynthesis and transport signal pathways. RT-PCR results showed that the mRNA expression levels of PCYT2, SPP1, EMC6, GRIA2 and PKD2 were consistent with the sequencing results. Western blot results showed that compared with the control group, the protein expression of p-mTOR and p-S6K1 in Gly group, Gly-Gly group and Gly-Leu group was significantly increased. Gly-Leu can promote the protein synthesis in IEC by activating protein synthesis of mTOR signalling pathway in chicken IEC.