摘要
目的:本试验探讨氧化应激状态下肠道自由基代谢与肠道结构与功能的变化,及其复合抗氧化剂的干预作用,研究复合抗氧化剂、肠道自由基代谢和肠道结构与功能的关系,从而提出了通过营养调控技术修复氧化应激引起的肠道损伤的途径。
方法:试验一选用36只42日龄的SD大鼠随机分成3组,每组12只。对照组和脂多糖LPS(lipopolysaccharide, LPS)诱导组饲喂基础日粮,修复组在基础日粮中添加复合抗氧化剂(每kg复合抗氧化剂含有200 mg VC、100 mg VE、450 mg茶多酚和5g微生物抗氧化剂),诱导组和修复组在试验的第5、9、13和17天腹腔注射0.8 mg LPS/kg体重,对照组注射等量生理盐水;第22天时,每组选取6只大鼠,进行D-木糖灌胃试验,测定肠道吸收功能。试验期间观察各个组大鼠的生长情况;试验结束后,并计算大鼠脏器指数,测定大鼠血清中丙二醛(Malondialdehyde, MDA)、肝脏组织中超氧化物歧化酶(Superoxide dismutase SOD)、谷胱甘肽过氧化物酶(Glutathione peroxidase, GSH-Px)活力、肠脂肪酶、麦芽糖酶和肠道绒毛高度与宽度。
试验二选择12窝14日龄哺乳仔猪,随机分为三组,即为对照组、断奶组和修复组,每组4窝。对照组和断奶组饲喂基础饲料,修复组在基础饲粮中添加复合抗氧化剂。断奶组和修复组仔猪在21日龄统一断奶,试验期22天。试验期间记录仔猪的生长及采食量,计算日增重和料重比;在仔猪21日龄、28日龄、35日龄,每窝取三只仔猪前腔静脉采血,用于分析血清中MDA、SOD、一氧化氮(NO)和GSH-Px含量;在24日龄,对每窝选取一只仔猪进行D-木糖灌胃试验,测定肠道吸收功能;在试验结束后测定肝脏中过氧化氢酶(CAT)、MDA、SOD、NO和GSH-Px含量、肠道消化酶活性,及绒毛结构。
结果:试验一:与对照组相比,诱导组大鼠日增重降低,血清MDA含量有所增加、肝SOD和GSH-Px活力显著下降(P<0.05);肝脏脏器系数下降11.13%(P<0.05);肠道D-木糖含量下降72.72%(P<0.05);肠脂肪酶和麦芽糖酶活力分别下降66.00%和65.74%(P<0.05)。与诱导组相比,修复组MDA含量下降49.31%(P<0.05),SOD和GSH-Px活性分别上升40.96%和35.04%(P<0.05),肝脏脏器系数升高(P<0.05),D-木糖含量、麦芽糖酶活性和绒毛高度显著升高(P<0.05),隐窝深度显著降低(P<0.05)。
试验二: 28日龄和第35日龄断奶仔猪体重较对照组降低18.45%和28.39%(P<0.05);以电化学法测得断奶组仔猪血清中NO的浓度分别提高199.42%和34.00%(P<0.05);O_2浓度下降和6.32%和11.81%。;28日龄断奶仔猪SOD活性降低47.81%,NO活性显著上升(P<0.05),MDA含量提高34.74%(P>0.05);断奶组仔猪肠道D-木糖含量,肠蔗糖酶、麦芽糖酶、淀粉酶、脂肪酶、肠胰胰蛋白酶均显著降低(P<0.05);回肠pH值升高7.11%(P<0.05);二胺氧化酶(DAO)活性断奶组和修复组仔猪分别显著下降32.31%和30.00%(P<0.05);断奶仔猪的十二指肠、空肠和回肠的隐窝深度显著下降。与断奶组相比,21日龄、35日龄修复组仔猪血清NO浓度分别显著下降30.86%和22.81%(电化学法),修复组仔猪血清中O_2浓度均上升7.76%(P<0.05);修复组仔猪SOD活性显著提高61.48%(P<0.05)和6.15%,28日龄、35日龄修复组NO活性,MDA含量分别显著降低50.07%,52.46%, 33.45%(P<0.05)和38.15%;修复组仔猪麦芽糖酶活力、肠胰蛋白酶活性、胰胰蛋白酶活性分别显著提30.54%,70.11%和100.35%(P<0.05),回肠pH值显著降低5.36%(P<0.05)。各组仔猪肠道绒毛长度、肠道胰蛋白酶活性与自由基H2O_2检测量(μM)、NO检测量(nM)之间均存在着负相关关系(P<0.05),其相关系数和回归关系显著(P<0.05)。
结论:氧化应激会导致动物体内产生大量自由基,使其抗氧化能力下降,对肠道结构和功能造成损伤。在氧化应激过程中,自由基H2O_2、NO和肠道绒毛与肠胰蛋白酶的活性是肠道损伤与修复的主要生物标志物。使用复合抗氧化剂能够显著提高仔猪抵抗氧化应激的能力,减轻肠道损伤,是有效的干预断奶应激的方法。
Objective:In this experiment we studied the changes between free radicals metaboli and the morphological structure, physiological function of intestinal tract under oxidative stress and discussed the effects of composite antioxidant. To investigate the relationship among free radicals metaboli, composite antioxidant and intestinal tract, we also studied the effects of composite antioxidants on reparation.
Methods: Experiment 1: 36 healthy SD rats as 42 days of age were randomly divided into three groups with twelve rats in each group. The control group and induction group was fed basal diet and the repair group was fed with composite antioxidant including vitamin C, vitamin E, tea polyphenols and microbial antioxidants. The feeding trial lasted for 22 days. On the 5th、9th、13th、17th day, rats in induction and repair group received an injection of 0.8 mg LPS/kg while rats in control group received of same amount of saline. On the 22nd day of experiment, 6 rats in each group were randomly selected, received D-xylose supplementation. The content of serum MDA, activities of liver SOD, GSH-Px, lipase, maltase and the length, width of intestinal villi were determined.
Experiment 2: 12 litters of 14-day-old piglets were randomly divided into three groups with 4 litters each. The control group and weaning group were fed basal diet and the repair group was fed composite antioxidant including vitamin C, vitamin E, tea polyphenols, Alpha Lipoic Acid and microbial antioxidants. The feeding trial lasted for 22 days, the induction and repair group were weaned on 21-day-old. On the 21, 28, 35- day-old, the piglets were took blood and on the 24-day-old, 1 piglets in each group were randomly selected, received D-xylose supplementation. The content of serum/liver MDA, NO, activities of serum/liver SOD, GSH-Px, CAT, digestive enzymes and the structure of intestinal villi were determined.
Results: Experiment 1: The ADG of rats in induction group was lower (P<0.05) than that in control group and the concentration of MDA was higher (P<0.05) and the activity of GSH-Px was lower (P<0.05). The liver coefficient, D-(+)-Xylose concentration in rats’serum, the activities of lipase and maltase in induction were 11.13%, 72.72%, 66% and 65.74% lower (P<0.05) respectively than that in control group. The villus height was lower (P<0.05) and the crypt depth was deeper (P<0.05) than that in control group. While the concentration of MDA was 49.31% lower, the activities of SOD and GSH-Px were 40.96% and 35.04% higher than that in induction group; the liver coefficient, D-(+)-Xylose concentration in rats’serum, the activity of maltase and the villus height were higher; the crypt depth were shallower than that in induction group.
Experiment 2: Compared with the control group, 28, 35-day-old piglets in weaning group noticeable decrease the weight by 18.45% and 28.39%, the concentration of O_2 by 6.32% and 11.81%, the activities of serum SOD by 47.81%(P<0.05). And also the activities of trypsase, lipase, amylase, maltase, invertase and DAO were lower (P<0.05). Weaning group piglets on 21, 35-day-old increased the concentration of NO by 199.42% and 34.00%, the contents of serum MDA on 35-day-old by 78.14%, the pH value of ileum by7.11 and the content of serum NO was also higher (P<0.05). Compared with the weaning group, the repair group remarkable decreased the concentration of NO by 30.86% and 22.81%, the contents of NO on 28, 35-day-old by 50.07% and 52.46%, MDA by 33.45% (P<0.05) and 38.15%, the pH value of ileum by 5.36%. The repair group piglets dramaticly increased the concentration of O_2 on 28, 35-day-old by 7.76%, the activity of SOD by 61.48% and 6.15%, and the activities of maltase, trysase in intestine and pancreas were 30.54%, 70.11% and 100.35% higher (P<0.05). The related coefficient and linear regression among the concentration of H2O_2, NO, the length of intestine villus and the activities of trypsinase were notable (P<0.05).
Conclusion: The oxidative stress induced by LPS or weaning can cause animal to produce a large number of free radicals, the ability of antioxidation was decreased and had damage to the structure and function of intestinal tract. The key points of oxidative stress and repairation in animal body were H2O_2, NO, intestine villus and trypsinase. Adding composite antioxidants to the stress and weaning piglets can significantly increase the ability of antioxidantion, reduces peroxidation and also have an effect on intestine reparation, enhancing the function of digestion. Adding composite antioxidants has a good effect on oxidative stress.
引文
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