Li X-G, Sui W-G, Gao C-Q, Yan H-C, Yin Y-L, Li H-C, Wang X-Q
J Anim Sci. 2016 Apr;94(4):1541-9. doi: 10.2527/jas.2015-9432.
The objective of this study was to investigate the effects of L-glutamate (Glu) deficiency or L-trans pyrrolidine-2,4-dicarboxylic acid (PDC) supplementation on the proliferation of pig intestinal epithelial cells (IPEC-1). First, IPEC-1 cells were cultured in normal growing medium supplemented with 0 (Control), 50, 100, or 200 µmol/L PDC to determine an appropriate concentration of PDC supplementation. Second, IPEC-1 cells were cultured in Glu-deficient medium supplemented with 0 µmol/L Glu (Glu deficiency), 50 µmol/L Glu (Control), or 50 µmol/L Glu plus 100 µmol/L PDC (PDC supplementation). Cell proliferation ( = 24), cell cycle distribution ( = 6), cell apoptosis ( = 6), and expression levels of proteins of interest ( = 4) were determined by MTT assay, flow cytometry, or western blot. The results showed that cell proliferation was inhibited ( < 0.05) by 50, 100, and 200 µmol/L PDC supplementation at 24 and 48 h after treatment. Variance analysis was performed using the GLM procedure, and the results demonstrated that Glu deficiency or PDC supplementation led to the inhibition ( < 0.05) of cell proliferation, a greater ( < 0.05) percentage of cells in the G1 phase, and a lower ( < 0.05) percentage of cells in the S phase. Moreover, Glu deficiency or PDC supplementation reduced ( < 0.05) the expression levels of excitatory AA transporter 3 (EAAT3), phosphor-mammalian target of rapamycin (p-mTOR; Ser2448), p-ribosomal protein S6 kinase 1 (S6K1; Thr389), and p-S6 (Ser235/236). This study demonstrates that Glu deficiency or PDC supplementation inhibits proliferation of IPEC-1 cells via downregulation of the mTOR/S6K1 pathway and EAAT3 expression indicating that Glu deficiency may lead to the disturbances of intestinal epithelial renewal in pigs, particularly in neonates.
本研究的目的是探讨L-谷氨酸（Glu）缺乏或L-反式吡咯烷-2,4-二羧酸（PDC）补充对猪肠上皮细胞（IPEC-1）增殖的影响。首先，将IPEC-1细胞培养于添加0（对照）、50、100或200 μmol/L PDC的正常生长培养基中，以确定合适的PDC补充浓度。其次，将IPEC-1细胞培养于添加0 μmol/L Glu（Glu缺乏）、50 μmol/L Glu（对照）或50 μmol/L Glu加100 μmol/L PDC（PDC补充）的Glu缺乏培养基中。通过MTT法、流式细胞术或蛋白质印迹法测定细胞增殖（n = 24）、细胞周期分布（n = 6）、细胞凋亡（n = 6）以及相关蛋白的表达水平（n = 4）。结果显示，在处理后24和48小时，50、100和200 μmol/L PDC补充均抑制了细胞增殖（P < 0.05）。使用GLM程序进行方差分析，结果表明Glu缺乏或PDC补充导致细胞增殖受到抑制（P < 0.05），G1期细胞百分比增加（P < 0.05），S期细胞百分比降低（P < 0.05）。此外，Glu缺乏或PDC补充降低了（P < 0.05）兴奋性氨基酸转运体3（EAAT3）、磷酸化雷帕霉素哺乳动物靶蛋白（p-mTOR；Ser2448）、磷酸化核糖体蛋白S6激酶1（S6K1；Thr389）和磷酸化S6（Ser235/236）的表达水平。本研究表明，Glu缺乏或PDC补充通过下调mTOR/S6K1途径和EAAT3表达抑制IPEC-1细胞增殖，这表明Glu缺乏可能导致猪尤其是新生仔猪肠上皮更新紊乱。
