| Glutamate,as a functional amino acid,plays an essential role in maintaining intestinal development.To investigate the effect of glutamate on intestinal development of weaned piglets and explore the underlying mechanism,14 healthy male weaned piglets with similar body weight（7.90±0.50 kg）at 28 day-old were selected and randomly divided into the control group and glutamate group,with 7 replicates in each group and 1 pig in each replicate.The control group was fed a base diet of corn and soybean meal added with 1.0%corn starch,and the glutamate group was added 1.0%glutamate based on the base diet.The experimental period was including 7 days of the pre-feeding period（fed a base diet of corn and soybean meal）and 21 days of the experimental stage.The effects of glutamate on intestinal development and nutrient absorption efficiency of weaned piglets were evaluated by the changes in intake and body weight of weaned piglets.Serum amino acid content was detected by serum separation,and intestinal segment samples were collected and fixed by 4%paraformaldehyde or 2.5%glutaraldehyde respectively.Intestinal morphological structure and functional indicators were obtained by HE staining and immunohistochemistry.On the19th day of feeding experiment,feed samples and stool samples were collected.Stool samples were mixed with diluted hydrochloric acid and stored at-20℃and Ti O2 indicator method was used to evaluate the effect of glutamate on the apparent digestibility of nutrients in weaned piglets.Two groups of piglet jejunum tissue protein samples were collected and differential protein expression and signal pathway enrichment in jejunum tissues were analyzed by i TRAQ（Isobaric tags for relative and absolute quantification）.At the same time,the jejunum crypts of each group of pigs were isolated and cultured in vitro,the growth of intestinal organoids was observed and the proliferation of intestinal stem cells was determined by the intestinal organoid forming efficiency and budding efficiency.Also,IPEC-J2（Intestinal porcine enterocyte cell line）cell lines and jejunal stem cells were treated with glutamate in vitro to investigate the effect of glutamate on IPEC-J2 cell proliferation,intestinal stem cell activity,and Wnt/β-catenin signaling pathway.The results were as follows:（1）Compared to the control group,dietary supplementation with glutamate increased average daily gain of weaned piglets by 6.6 g,but the difference had no statistical significance（P>0.10）,the weight gain ratio of consumables has no difference（P>0.10）,showed a tendency to increase average daily feed intake（P=0.062）.（2）Compared with the control group,dietary supplementation of glutamate significantly increased the content of glutamate in serum by 21.36%（P<0.05）,glycine by 38.72%（P<0.05）,and ornithine by 57.20%（P<0.05）.The content of serum proline showed a statistical trend of increase（P=0.072）.（3）Compared with the control group,the apparent digestibility of crude fat was significantly increased by 11.58%（P<0.05）,the apparent digestibility of dry matter was significantly increased by 5.61%（P<0.05）,and the apparent digestibility of crude protein was significantly increased by 5.43%（P<0.05）.（4）Compared with the control group,dietary supplementation with glutamate significantly increased the weight of jejunum and ileum per unit length and the mucosal weight of weaned piglets（P<0.05）,and the height of jejunum and ileum villus,and the ratio of villus height to crypt depth also significantly increased（P<0.05）.Glutamate group jejunal epithelial cell proliferation marker protein PCNA（Proliferating cell nuclear antigen）,differentiation marker KRT20（Keratin 20）,functional cell marker Villin（absorption cell marker）,Chg A（pheochromoprotein A,intestinal endocrine cell marker）,Mucin2（goblet cell marker）and intestinal stem cell marker Lgr5（leucine-rich-repeat-containing g-protein-coupled receptor5）and Bmi1（B cell specific moloney murine leukemia virus insertion site 1）were significantly up-regulated（P<0.05）.（5）The results of i TRAQ proteomics suggested that dietary glutamate significantly up-regulated the Wnt/β-catenin signaling pathway,and Western blotting further confirmed that glutamate significantly up-regulated the protein expressions ofβ-catenin,TCF4（T cell factor 4）,c-Myc and Cyclin D1 in jejunum tissues of weaned piglets（P<0.05）.（6）The cell count results showed that IPEC-J2 cells treated with glutamate for 24 h can get a better proliferation potential than the control group（P<0.05）,and the number of Ki67+cells increased significantly（P<0.05）.The expression of Wnt/β-catenin signaling pathway-related proteinsβ-catenin,TCF4,c-Myc,and Cyclin D1 was significantly up-regulated（P<0.05）.（7）Compared with the control group,dietary supplementation of glutamate significantly increased the forming efficiency and budding efficiency of organoids（P<0.05）.In vitro culture of stem cells treated with 5 mmol/L glutamate can significantly increase the expression of Wnt/β-catenin signaling pathway-related proteinβ-catenin,TCF4,c-Myc,Cyclin D1（P<0.05）,and promote the stem cell marker Lgr5,Bmi1 expression（P<0.05）.Conclusion:glutamate can enhance the activity of porcine intestinal stem cells and promote the proliferation of intestinal epithelial cells by upregulating the Wnt/β-catenin signaling pathway,thus improving the intestinal development and the digestibility of nutrients. |
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