Dietary supplementation with tributyrin alleviates intestinal injury in piglets challenged with intrarectal administration of acetic acid
作者:Yongqing Hou1*, Lei Wang1, Dan Yi1, Binying Ding1, Xing Chen1, Qingjing Wang1, Huiling Zhu1,Yulan Liu1, Yulong Yin1, Joshua Gong2 and Guoyao Wu3
1Hubei Key Laboratory of Animal Nutrition and Feed Science, Wuhan Polytechnic University, Changqing Garden, Wuhan 430023, Hubei, People’s Republic of China
2Guelph Food Research Centre, Agriculture and Agri-Food Canada, Guelph, Ontario, Canada N1G 5C9
3Department of Animal Science, Texas A&M University, College Station, TX 77843, USA
来源：British Journal of Nutrition (2014), 111, 1748–1758
DOI:10.1017/S0007114514000038
翻译：肠动力研究院 梁琦
【摘要】三丁酸甘油酯（TBU）是机体获取丁酸良好的膳食来源，其对维持肠道正常形态具有促进作用。本文旨在探究三丁酸甘油酯对乙酸（ACA）诱导的猪结肠炎模型中的肠道损伤的影响。试验选取18只，18日龄的健康雌性断奶仔猪，预饲7天后（25日龄，5.8±1.17kg）被随机分配成3组，实验分组如下，对照组：基础日粮+0.1%玉米淀粉(等能量日粮）；ACA组：饲喂基础日粮+0.1%玉米淀粉；TBU组：基础日粮+0.1%三丁酸甘油酯，每组6只。试验的第15天，麻醉仔猪，将软导管插入直肠（距肛门20-25cm），而后ACA和TBU组仔猪直肠灌注10ml 10％ACA溶液，对照组则灌注等量的生理盐水。试验的第22天，在收集完静脉血后，将仔猪致死以获得回肠中段和结肠中段粘膜，之后对样品进行分析。实验结果显示，与对照组相比，ACA组仔猪的血浆中淋巴细胞计数、肌酐、PGE2和丙二醛浓度、二胺氧化酶和诱导型NO合酶活性和结肠淋巴细胞密度显著增加（P<0.05）、胰岛素浓度和谷胱甘肽过氧化物酶活性、回肠绒毛高度与隐窝深度的比值和结肠杯状细胞数则相应减少。ACA的这些副作用可通过添加TBU而减弱。此外，TBU还可阻止ACA诱导的caspase-3表达水平的增加，同时增强结肠粘膜中的claudin-1蛋白和表皮生长因子受体（EGFR）mRNA表达量。综上所述，日粮添加0.1%TBU可能通过抑制细胞凋亡、促进紧密连接形成、激活EGFR信号通路等途径而缓解ACA诱导的肠道损伤。
【关键词】三丁酸甘油酯；肠道损伤；乙酸；结肠；仔猪
以下是实验中相关图表
表1：基础日粮的组成及营养成分（风干基础）
表2：用于实时PCR分析的引物*
表3：仔猪血液中白细胞总数、红细胞计数及血红蛋白浓度的差异（(平均值及其标准误差，n=6)）
表4：仔猪血清中生长激素(GH)、胰岛素样生长因子(IGF)-I、表皮生长因子(EGF)、胰岛素和PGE2的浓度（(平均值及其标准误差，n=6)）
表5：猪血浆中二胺氧化酶(DAO)活性及血浆和结肠黏膜中的氧化还原状态（(平均值及其标准误差，n=6)）
图1：直肠内灌注乙酸(ACA)后仔猪结肠形态学的变化。(图a, d)对照组(无攻击对照组):仔猪饲喂基础日粮，灌注生理盐水;(图b,e) ACA组(ACA攻击对照组):仔猪饲喂基础日粮，体外给予ACA，结肠损伤，表现为正常隐窝结构扭曲，杯状细胞丢失，上皮脱落，淋巴细胞浸润;和（图c，f）三丁酸甘油酯（TBU）组（ACA攻击+0.1％TBU补充）：仔猪饲喂含有有0.1％TBU的基础日粮并且直肠内灌注ACA，通过TBU处理可改善组织学变化。A，杯状细胞;B，裸露的上皮;和C，淋巴细胞。
表7：三丁酸甘油酯对乙酸刺激后的仔猪结肠粘膜中双调蛋白（AR），表皮生长因子（EGF）和表皮生长因子受体（EGFR）mRNA水平的影响（(平均值及其标准误差，n=6)）
图2：仔猪结肠粘膜的Caspase-3水平。采用10% SDS-PAGE法分离粘膜提取物(150mg蛋白/样品)，测定caspase-3和β-Actin水平。caspase-3蛋白的值与β-Actin的值趋于一致。数值是均值，其标准误差用竖条(n=6)表示。对照组(无刺激对照组):仔猪饲喂基础日粮，直肠灌注生理盐水；乙酸(ACA)组(乙酸刺激对照组):仔猪饲喂基础日粮和直肠内灌注ACA;和三丁酸甘油酯（TBU）组（ACA刺激+0·1％TBU补充）：仔猪饲喂含有0·1％TBU的基础日粮并且直肠内灌注ACA。a，b不同字母表示均值显著不同（P <0·05）。
图3：仔猪结肠粘膜中Claudin-1蛋白相对水平。通过12％SDS-PAGE分离粘膜提取物（60mg蛋白质/样品）以测定Claudin-1和β-Actin水平。将Claudin-1的值标准化为β-Actin的值。数值是均值，其标准误差用竖条(n=6)表示。对照组(无刺激对照组):仔猪饲喂基础日粮，直肠灌注生理盐水；乙酸(ACA)组(乙酸刺激对照组):仔猪饲喂基础日粮和直肠内灌注ACA;和三丁酸甘油酯（TBU）组（ACA刺激+0.1％TBU补充）：仔猪饲喂含有0.1％TBU的基础日粮并且直肠内灌注ACA。a，b不同字母表示均值显著不同（P <0.05）。
Conclusion
总之，日粮添加0.1％三丁酸甘油酯（TBU）有利于减轻仔猪的溃疡性结肠炎。TBU发挥作用的可能机制包括以下内容：（1）减少氧化应激（血浆中MDA的产生减少）;（2）减轻肠道损伤（通过减轻ACA诱导的肠道结构损伤和降低血浆DAO活性，降低ACA刺激仔猪结肠粘膜的caspase-3蛋白表达和增加claudin-1蛋白表达量）;（3）通过EGF信号传导改善粘膜修复（通过结肠中EGFR表达量的增加而表明）。这些发现可能对治疗人类和其他动物的结肠炎具有重要意义。
Abstract
Tributyrin (TBU) is a good dietary source of butyrate and has beneficial effects on the maintenance of normal intestinal morphology. The present study tested the hypothesis that dietary TBU supplementation could alleviate intestinal injury in the acetic acid (ACA)-induced porcine model of colitis. A total of eighteen piglets (25 d old) were randomly allocated to one of three treatment groups (control, ACA and TBU). The control and ACA groups were fed a basal diet and the TBU group was fed the basal diet supplemented with 0.1 % TBU. On day 15 of the trial, under anaesthesia, a soft catheter was inserted into the rectum of piglets (20–25 cm from the anus), followed by administration of either saline (control group) or ACA (10 ml of 10 % ACA solution for ACA and TBU groups). On day 22 of the trial, after venous blood samples were collected, piglets were killed to obtain mid-ileum and mid-colon mucosae. Compared with the control group, the ACA group exhibited an increase (P<0.05) in lymphocyte counts, creatinine, PGE2, and malondialdehyde concentrations and diamine oxidase and inducible NO synthase activities in the plasma and lymphocyte density in the colon and a decrease in insulin concentrations and glutathione peroxidase activity, ileal villus height:crypt depth ratios and goblet cell numbers in the colon. These adverse effects of ACA were attenuated by TBU supplementation. Moreover, TBU prevented the ACA-induced increase in caspase-3 levels while enhancing claudin-1 protein and epidermal growth factor receptor (EGFR) mRNA expression in the colonic mucosa. Collectively, these results indicate that dietary supplementation with 0.1 % TBU alleviates ACA-induced intestinal injury possibly by inhibiting apoptosis, promoting tight-junction formation and activating EGFR signalling
Key words: Tributyrin: Intestinal injury: Acetic acid: Colon: Piglets
Conclusion
In conclusion, dietary supplementation with 0.1 % TBU is beneficial for alleviating ulcerative colitis in piglets. Possible mechanisms for the actions of TBU may include the following: (1) reduction of oxidative stress (indicated by decreased production of MDA in the plasma); (2) alleviation of intestinal injury (indicated by alleviation of ACA-induced damage in the intestinal structure and decreased plasma DAO activity, decreased expression of caspase-3 protein and increased expression of claudin-1 protein in the colonic mucosa of the ACA-challenged piglets); (3) improvement of mucosal repair via EGF signalling (indicated by increases in EGFR expression in the colon). These findings may have important implications for treating colitis in humans and other animals.
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