罗伊氏乳杆菌I5007对新生仔猪肠道形态、二糖酶活性和紧密连接蛋白表达的影响

【目的】罗伊氏乳酸杆菌是普遍存在于人和动物肠道的有益乳酸杆菌，研究表明，口服罗伊氏乳酸杆菌能显著降低腹泻的发病率和严重程度。研究罗伊氏乳酸杆菌I5007（Lactobacillus reuteri I5007）对新生仔猪肠绒毛发育和肠黏膜屏障功能的影响。【方法】试验选取9窝1日龄的杜×长×大初生仔猪，每窝挑选8头体重相近、出生状况良好的仔猪，按体重分为3组，每组3窝仔猪。即：对照组（灌服0.1%无菌蛋白胨溶液），前4日龄灌服组（1-4 d，每日每头灌服活菌总数为1.2×10¹⁰ CFU 的L. reuteri I5007）和隔4日龄灌服组（1、5、9、13、17和21 d灌服，灌服剂量同前4日龄灌服组）。试验期21 d，试验期间仔猪由各自母猪哺乳饲喂。于试验第7、14和21 天，每个处理组随机选取4头仔猪，空腹称重、麻醉和屠宰，截取相同位置的十二指肠、空肠和回肠，测定其肠绒毛形态，并测定试验结束时（21 d）空肠和回肠二糖酶活性和紧密连接（TJ）蛋白的表达。【结果】灌服L. reuteri I5007对仔猪生长性能没有显著影响（P＞0.05），但对不同阶段仔猪的肠道形态有不同程度的影响。7 d时，隔4日龄灌服组与对照组相比，仔猪十二指肠绒毛高度、绒毛高度/隐窝深度、杯状细胞数和回肠杯状细胞数均显著增加（P＜0.05），前4日龄灌服组仔猪的十二指肠绒毛高度/隐窝深度、杯状细胞数和回肠杯状细胞数也均显著高于对照组仔猪（P＜0.05），但对十二指肠绒毛高度无显著影响（P＞0.05）；14 d时，隔4日龄灌服组仔猪十二指肠的绒毛高度/隐窝深度和回肠杯状细胞数均显著高于对照组（P＜0.05），前4日龄灌服组仔猪的回肠杯状细胞数也显著高于对照组（P＜0.05），但却低于隔4日龄灌服组（P＜0.05）；21 d时，隔4日龄灌服组显著提高了仔猪十二指肠和回肠的杯状细胞数（P＜0.05），前4日龄灌服组与对照组相比，仅提高了回肠杯状细胞数（P＜0.05），反而降低了十二指肠绒毛高度/隐窝深度（P＜0.05）。另外，前4日龄灌服组仔猪空肠隐窝深度显著低于隔4日龄灌服组仔猪（P＜0.05），但其十二指肠杯状细胞数和绒毛高度/隐窝深度均显著低于隔4日龄灌服组仔猪（P＜0.05）。在二糖酶活性上，隔4日龄灌服组仔猪空肠蔗糖酶和麦芽糖酶活性均显著高于对照组（P＜0.05），前4日龄灌服组仅提高了仔猪空肠麦芽糖酶活性（P＜0.05），且该组空肠蔗糖酶活性和麦芽糖酶活性均显著低于隔4日龄灌服组（P＜0.05）。隔4日龄灌服组对仔猪空肠和回肠的3种TJ蛋白（Claudin-1、Occludin和ZO-1）均有显著的促表达作用（P＜0.05），前4日龄灌服组仔猪空肠Claudin-1、ZO-1和回肠Claudin-1的表达也均高于对照组（P＜0.05），但是其空肠Occludin显著低于隔4日龄灌服组（P＜0.05），且其回肠Claudin-1（P = 0.1）和ZO-1（P = 0.1）相比于隔4日龄灌服组也有降低的趋势。【结论】本试验条件下，新生仔猪隔4日龄灌服L. reuteri I5007（1.2×10¹⁰ CFU/头/日），能有效促进仔猪肠绒毛发育，提高空肠二糖酶活性，促进仔猪肠上皮细胞TJ蛋白的表达，在改善新生仔猪的肠黏膜屏障功能中可能起到一定的作用。

Effect of Lactobacillus reuteri I5007 on Intestinal Morphology,Disaccharidase Activity and Tight Junction ProteinExpression in Newborn Piglets

【Objective】 The purpose of this experiment was to investigate the effects of Lactobacillus reuteri I5007 on intestinal villi maturation and intestinal barrier function in newborn piglets. 【Method】 Nine litters of 1-day-old Duroc×Landrace×Yorkshire newborn piglets and eight healthy piglets with similar body weight in each litter were selected. Piglets were randomly divided into three groups with three litters per group. The three groups were control group (oral administrated with 0.1% sterile peptone solution), the first 4 days dosed group (1-4 d, daily orally administrated with 1.2×10¹⁰ CFU of L. reuteri I5007 per piglet), and the every 4-day dosed group (1, 5, 9, 13, 17, and 21 d, daily orally administrated with 1.2×10¹⁰ CFU of L. reuteri I5007 per piglet). The trial was conducted for 21 d. During the trial, each litter of piglets were fed by a sow, respectively. Four healthy piglets closest to the average body weight from each group were fasting weighted, anesthetized and then slaughtered on d 7, 14 and 21. The small intestine was carefully dissected from the mesentery and segments (the same location in all piglets), including duodenum, jejunum and ileum, were collected. Growth performance, intestinal morphology, disaccharidase activity and expression of tight junction (TJ) proteins were measured. 【Result】 Oral administration with L. reuteri I5007 had no significant effect (P＞0.05) on growth performance, but had impacts on piglet intestinal morphology at different time points. On d 7, duodenal villus height, villus height/crypt depth and goblet cells density and ileal goblet cells density of piglets in every 4-day dosed group were increased significantly (P＜0.05) compared with those of control piglets. Piglets in first 4 days dosed group had distinct higher (P＜0.05) duodenal villus height/crypt depth and goblet cells density and ileal goblet cells density than control piglets, but not villus height/crypt depth (P＞0.05). On d 14, administration with L. reuteri I5007 in every 4-day dosed group robustly increased (P＜0.05) duodenal villus height/crypt depth and ileal goblet cells density. Ileal goblet cells density of piglets in first 4 days dosed group was significant higher (P＜0.05) than that of control piglets, but lower (P＜0.05) than that of piglets in every 4-day dosed group. On d 21, piglets of every 4-day dosed group had an increased goblet cells density (P＜0.05) in both duodenum and ileum. Compared with control group, administration of L. reuteri I5007 in first 4 days dosed group also augmented (P＜0.05) ileal goblet cells density, but reduced (P＜0.05) duodenal villus height/crypt depth. In addition, piglets in first 4 days dosed group had lower (P＜0.05) duodenal villus height/crypt depth and goblet cells density than those of piglets in every 4-day dosed group. For disaccharidase activity, piglets in every 4-day dosed group had higher (P＜0.05) both sucrase and maltase activities in jejunum than those of control piglets. Only jejunal maltase activity of first 4 days dosed piglets was enhanced (P＜0.05) compared with control piglets, while jejunal sucrase and maltase activities were significantly lower (P＜0.05) than those of every 4-day dosed. Furthermore, piglets in every 4-day dosed group had significant higher expression of Claudin-1, Occludin and ZO-1 in both jejunal and ileal epithelial cells (P＜0.05). Administration of L. reuteri I5007 in first 4 days dosed group also promoted (P＜0.05) expression of jejunum epithelial Claudin-1 and ZO-1 and ileum epithelial Claudin-1. However, the level of jejunum epithelial Occludin of first 4 days dosed piglets was obviously lower (P＜0.05) than that of every 4-day dosed piglets. Besides, the levels of Claudin-1 (P = 0.1) and ZO-1 (P = 0.1) in ileum epithelium of first 4 days dosed piglets had a trend of decrease compared with every 4-day dosed piglets. 【Conclusion】 Every 4-day administration of L. reuteri I5007 in newborn piglets can promote the maturation of intestinal villi, improve jejunal disaccharidase activity, and increase expression of TJ protein of intestinal epithelial cells. Thus, L. reuteri I5007 may play a role in protecting or augmenting intestinal barrier function in newborn piglets.