大腸桿菌K88攻毒對(duì)幼齡仔豬腸道離子載體和水通道蛋白表達(dá)的影響

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　　大腸桿菌K88攻毒對(duì)幼齡仔豬腸道離子載體和水通道蛋白表達(dá)的影響
　　C. Zhu, J. L. Ye, J. Yang, K. M. Yang, Z.Chen, R. Liang, X. J. Wu, L. Wang and Z. Y. Jiang

　　本試驗(yàn)研究大腸桿菌K88攻毒在體內(nèi)或者體外實(shí)驗(yàn)中是否會(huì)對(duì)水和離子轉(zhuǎn)運(yùn)載體基因表達(dá)產(chǎn)生影響。

　　選用36頭公豬（4日齡）隨機(jī)分為對(duì)照組和攻毒組，每個(gè)處理6個(gè)重復(fù)，每個(gè)重復(fù)3頭豬。所有豬只飼喂相同日糧17天。在第15天，攻毒組豬只接受K88攻毒（血清型O149:K91:K88ac，劑量為1×108 cfu/頭），而對(duì)照組接受同劑量的磷酸鹽緩沖液處理。攻毒72小時(shí)后（第18天），每個(gè)重復(fù)挑選一頭豬屠宰，收集空腸、回腸和結(jié)腸樣品。

　　攻毒組回腸和結(jié)腸中囊性纖維化跨膜傳導(dǎo)調(diào)節(jié)因子(CFTR)的蛋白豐度和mRNA的表達(dá)顯著提高（P＜0.05）。此外，攻毒還提高了回腸和結(jié)腸中鈉鉀氯共轉(zhuǎn)運(yùn)蛋白1（NKCC1）的mRNA表達(dá)（P＜0.05），而在空腸中還額外提高了鈉鉀氯共轉(zhuǎn)運(yùn)蛋白1蛋白的表達(dá)（P＜0.05）。除此之外還通過豬小腸上皮細(xì)胞系（IPEC-J2）研究K88對(duì)水通道蛋白和離子轉(zhuǎn)運(yùn)載體的影響及可能的作用機(jī)理。在6孔平板上培養(yǎng)上皮細(xì)胞（每孔1.17×106個(gè)細(xì)胞），用50:1的K88攻毒，處理3小時(shí)。攻毒顯著降低了上皮細(xì)胞中水通道蛋白3（AQP3）、AQP11和鈉氫交換蛋白3（NHE3）mRNA的表達(dá)（P＜0.05）。蛋白質(zhì)免疫印跡法（western blotting）的進(jìn)一步分析表明K88攻毒降低了上皮細(xì)胞AQP3、AQP9、AQP11蛋白的表達(dá)（P＜0.05）。此外，攻毒還降低了蛋白激酶A和環(huán)磷酸腺苷效應(yīng)元件結(jié)合蛋白（CREB）的磷酸化水平（P＜0.05）。

　　試驗(yàn)結(jié)果表明K88攻毒可能通過調(diào)節(jié)環(huán)磷酸腺苷-蛋白激酶A信號(hào)通路導(dǎo)致腸道離子轉(zhuǎn)運(yùn)載體和水通道蛋白的表達(dá)差異。本研究結(jié)果一定程度上從另一方面揭示了幼齡仔豬腹瀉控制中離子平衡的重要性。

　　Differential expression of intestinal ion transporters and water channel aquaporins in young piglets challenged with enterotoxigenic Escherichia coli K88

　　C. Zhu, J. L. Ye, J. Yang, K. M. Yang, Z. Chen, R. Liang, X. J. Wu, L. Wang and Z. Y. Jiang

　　The study was to determine whether the expression of genes involved in intestinal water and ion transport would be affected by enterotoxigenic Escherichia coli (ETEC) K88 both in vitro and invivo. 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 × 108 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 (P < 0.05). Furthermore, the mRNA expression of Na-K-Cl cotransporter 1 (NKCC1) in the ileum and colon was increased by ETEC K88 challenge (P < 0.05), whereas in the jejunum, both its mRNA and protein expression were increased by ETEC K88 treatment (P < 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 × 106 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 AQP3, AQP11, and Na+/H+ exchanger 3 (NHE3) in IPEC-J2 cells was reduced after ETEC K88 treatment (P < 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 (P < 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 (P < 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。

山中的漫游者

這個(gè)實(shí)驗(yàn)證明了大腸桿菌是腹瀉的主要因素，