J Anim Sci. 2017 Dec;95(12):5240-5252. doi: 10.2527/jas2017.1806.
The study was to determine whether the expression of genes involved in intestinal water and ion transport would be affected by enterotoxigenic (ETEC) K88 both in vitro and in vivo. First, 36 male piglets (4 d old) were randomly allotted to either the control or the ETEC K88 group. Each group had 6 replicates with 3 piglets per replicate. All piglets were fed with the same diets for 17 d. On d 15, piglets in the ETEC K88 group were challenged with ETEC K88 (serotype O149:K91:K88ac) at 1 × 10 cfu per pig, whereas those in the control group received the same volume of sterile PBS. After being challenged with ETEC K88 for 72 h (d 18), 1 piglet from each replicate was selected for slaughter to collect samples from the jejunum, ileum, and colon. The mRNA expression and protein abundance of cystic fibrosis transmembrane conductance regulator (CFTR) in the ileum and colon were increased compared with that in the control group ( < 0.05). Furthermore, the mRNA expression of () in the ileum and colon was increased by ETEC K88 challenge ( < 0.05), whereas in the jejunum, both its mRNA and protein expression were increased by ETEC K88 treatment ( < 0.05). Additionally, an established porcine intestinal epithelial cell line (IPEC-J2) was used to investigate the effect and possible mechanism of ETEC K88 on expression of water channel aquaporins (AQP) and ion transporters. Cells (1.17 × 10 per well) were grown in 6-well plates and treated with ETEC K88 at a multiplicity of infection of 50:1 for 3 h. The mRNA expression of , , and () in IPEC-J2 cells was reduced after ETEC K88 treatment ( < 0.05). Further analyses using western blotting also demonstrated that ETEC K88 decreased the protein expression of AQP3, AQP9, and AQP11 in IPEC-J2 cells ( < 0.05). Moreover, the phosphorylation levels of protein kinase A (PKA) and cyclic adenosine monophosphate (cAMP)-response element binding protein (CREB) were decreased by ETEC K88 challenge ( < 0.05). The results indicate that ETEC K88 challenge induced differential expression of intestinal ion transporters and AQP in young piglets, probably by regulation of the cAMP-PKA signaling pathway. This study might provide new insights about the importance of fluid homeostasis in control of ETEC-induced diarrhea in young piglets.
本研究旨在确定肠毒素型大肠杆菌（ETEC）K88 在体内和体外是否会影响与肠道水和离子转运相关的基因表达。首先，将 36 头 4 日龄雄性仔猪随机分为对照组或 ETEC K88 组。每组有 6 个重复，每个重复有 3 头仔猪。所有仔猪均在 17 天内喂食相同的日粮。在第 15 天，ETEC K88 组仔猪经 ETEC K88（血清型 O149:K91:K88ac）以 1×10 cfu/头进行攻毒，而对照组仔猪则接受相同体积的无菌 PBS。在经 ETEC K88 攻毒 72 小时（第 18 天）后，每个重复中选择 1 头仔猪进行屠宰，以收集空肠、回肠和结肠样本。与对照组相比，回肠和结肠中的囊性纤维化跨膜电导调节剂（CFTR）mRNA 表达和蛋白丰度增加（<0.05）。此外，ETEC K88 攻毒增加了回肠和结肠中（）的 mRNA 表达（<0.05），而在空肠中，ETEC K88 处理增加了其 mRNA 和蛋白表达（<0.05）。此外，使用建立的猪肠上皮细胞系（IPEC-J2）研究了 ETEC K88 对水通道 Aquaporin（AQP）和离子转运体表达的影响及其可能的机制。细胞（每孔 1.17×10 个）在 6 孔板中生长，并以感染复数 50:1 用 ETEC K88 处理 3 小时。IPEC-J2 细胞中（）的 mRNA 表达在 ETEC K88 处理后降低（<0.05）。使用 Western blot 进一步分析也表明，ETEC K88 降低了 IPEC-J2 细胞中 AQP3、AQP9 和 AQP11 的蛋白表达（<0.05）。此外，ETEC K88 攻毒降低了蛋白激酶 A（PKA）和环磷酸腺苷（cAMP）反应元件结合蛋白（CREB）的磷酸化水平（<0.05）。结果表明，ETEC K88 攻毒诱导仔猪肠道离子转运体和 AQP 的差异表达，可能通过调节 cAMP-PKA 信号通路。本研究可能为控制仔猪 ETEC 诱导性腹泻中液体平衡的重要性提供新的见解。
