动物营养学报 >

2023 , Vol. 35 >Issue 3: 1948 - 1956

DOI: https://doi.org/10.12418/CJAN2023.182

研究论文

复合酸化剂对伊拉兔空肠消化酶活性、免疫功能和紧密连接蛋白表达的影响

  • 杨国峰 , 1 ,
  • 贺佳仪 1 ,
  • 狄斌 1 ,
  • 谢秋萍 2 ,
  • 钟辛悦 1 ,
  • 王长康 1 ,
  • 高玉云 , 1, *
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  • 1 福建农林大学动物科学学院(蜂学学院),福州 350002
  • 2 福建省绿色食品发展中心,福州 350002
*高玉云,副教授,硕士生导师,E-mail:

杨国峰(1997—),男,河南焦作人,硕士研究生,从事动物营养与饲料、动物营养与免疫方向的研究。E-mail:

收稿日期: 2022-08-22

  网络出版日期: 2023-03-16

基金资助

国家自然科学基金项目(31802079)

福建省家禽产业体系岗位专家(KKE19013A)

福建省家禽产业体系岗位专家(2019—2022)

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Effects of Compound Acidifier on Digestive Enzyme Activity, Immune Function and Tight Junction Protein Expression in Jejunum of Ira Rabbits

  • YANG Guofeng , 1 ,
  • HE Jiayi 1 ,
  • DI Bin 1 ,
  • XIE Qiuping 2 ,
  • ZHONG Xinyue 1 ,
  • WANG Changkang 1 ,
  • GAO Yuyun , 1, *
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  • 1 College of Animal Science (College of Bee Science), Fujian Agriculture and Forestry University, Fuzhou 350002, China
  • 2 Fujian Provincial Green Food Development Center, Fuzhou 350002, China
*associate professor, E-mail:

Received date: 2022-08-22

  Online published: 2023-03-16

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摘要

本试验旨在研究复合酸化剂对伊拉兔空肠消化酶活性、免疫功能和紧密连接蛋白表达的影响。选取192只体重相近、健康的(35±2)日龄断奶商品代伊拉兔公兔,随机分为4组,每组6个重复,每个重复8只。4组分别为空白组(基础饲粮)、抗生素组(基础饲粮+60 mg/kg盐霉素)、试验Ⅰ组(基础饲粮+0.1%复合酸化剂)和试验Ⅱ组(基础饲粮+0.2%复合酸化剂),复合酸化剂的有效成分为甲酸和丙酸。试验期28 d。结果表明:1)与空白组相比,试验Ⅰ组空肠黏膜胰蛋白酶活性显著提高(P<0.05);各组间空肠黏膜脂肪酶和α-淀粉酶活性无显著差异(P>0.05)。2)与空白组相比,抗生素组、试验Ⅰ组和试验Ⅱ组空肠黏膜白细胞介素-6(IL-6)和白细胞介素-1β(IL-1β)含量显著降低(P<0.05),空肠黏膜白细胞介素-10(IL-10)含量有提高趋势(P=0.086);各组间空肠黏膜分泌型免疫球蛋白A(sIgA)含量无显著差异(P>0.05)。3)与空白组相比,抗生素组、试验Ⅰ组和试验Ⅱ组空肠黏膜IL-1β mRNA相对表达量显著降低(P<0.05);各组间空肠黏膜IL-6和IL-10 mRNA相对表达量无显著差异(P>0.05)。4)与空白组相比,抗生素组和试验Ⅰ组空肠黏膜闭合小环蛋白-1(ZO-1)和闭锁蛋白(occludin)的mRNA相对表达量显著提高(P<0.05),抗生素组和试验Ⅱ组空肠黏膜闭合蛋白-1(claudin-1)的mRNA相对表达量显著降低(P<0.05);各组间相对表达量闭合蛋白-3(claudin-3)的mRNA相对表达量无显著差异(P>0.05)。综上所述,饲粮添加0.1%复合酸化剂可提高伊拉兔空肠黏膜胰蛋白酶活性,增强免疫能力,促进紧密连接蛋白的表达,其效果与抗生素相似,具有良好的替抗潜力。

关键词： 伊拉兔; 复合酸化剂; 空肠; 消化酶; 免疫; 紧密连接蛋白

本文引用格式

杨国峰 , 贺佳仪 , 狄斌 , 谢秋萍 , 钟辛悦 , 王长康 , 高玉云 . 复合酸化剂对伊拉兔空肠消化酶活性、免疫功能和紧密连接蛋白表达的影响[J]. 动物营养学报, 2023 , 35(3) : 1948 -1956 . DOI: 10.12418/CJAN2023.182

Abstract

This experiment was conducted to investigate the effects of compound acidifier on digestive enzyme activity, immune function and tight junction protein expression in jejunum of Ila rabbits. A total of 192 healthy (35±2)-day-old weaned male commercial Ira rabbits with similar body weight were randomly divided into 4 groups with 6 replicates per group and 8 rabbits per replicate. The four groups were the blank group (basal diet), the antibiotic group (basal diet+60 mg/kg salinomycin), experimental group Ⅰ (basal diet+0.1% compound acidifier) and experimental group Ⅱ (basal diet+0.2% compound acidifier). The effective components in compound acidifier were formic acid and propionic acid. The experiment lasted for 28 days. The results showed as follows: 1) compared with the blank group, the trypsin activity in jejunum mucosa in experimental group Ⅰ was significantly increased (P<0.05), and there were no significant differences in the activities of lipase and α-amylase in jejunal mucosa among all groups (P>0.05). 2) Compared with the blank group, the contents of interleukin-6 (IL-6) and interleukin-1β (IL-1β) in jejunal mucosa in the antibiotic group, experimental group Ⅰ and experimental group Ⅱ were significantly decreased (P<0.05), and the interleukin-10 (IL-10) content in jejunal mucosa had a tendency to be increased (P=0.086). There were no significant differences in the secreted immunoglobulin A (sIgA) content in jejunal mucosa among all groups (P>0.05). 3) Compared with the blank group, the IL-1β mRNA relative expression level in jejunal mucosa in the antibiotic group, experimental group Ⅰ and experimental group Ⅱ was significantly decreased (P<0.05), and there were no significant differences in the mRNA relative expression levels of IL-6 and IL-10 in jejunal mucosa among all groups (P>0.05). 4) Compared with the blank group, the mRNA relative expression levels of zona occludens-1 (ZO-1) and occludin in jejunal mucosa in the antibiotic group and experimental group Ⅰ were significantly increased (P<0.05), and the claudin-1 mRNA relative expression level in jejunal mucosa in the antibiotic group and experimental group Ⅱ was significantly decreased (P<0.05). There were no significant differences in the claudin-3 mRNA relative expression level in jejunal mucosa among all groups (P>0.05). In conclusion, dietary 0.1% compound acidifier can increase the trypsin activity, enhance the immune ability and promote the expression of tight-junction protein in jejunal mucosa of Ila rabbits. Its effect is similar to that of antibiotics, and it has a good potential for substitution.

Key words： Ira rabbits; compound acidifier; jejunum; digestive enzyme; immune; tight junction protein

断奶应激是当前养兔业面临的严重问题。幼兔断奶后,失去了母乳带来的免疫球蛋白和营养物质,加上自身免疫机能发育不完善,肠道消化酶分泌不足,肠道微生物区系不稳定,极易引起腹泻甚至死亡[1]。饲粮添加抗生素可预防断奶后所带来的不良反应,但近年来,抗生素的滥用不仅使细菌产生耐药性,降低畜禽的免疫力,还会导致动物产品及环境中的残留[2]。2006年1月,欧盟禁止在饲料中使用抗生素作为生长促进剂。我国农业农村部第194公告,自2020年1月1日起,退出除中药外的所有促生长类药物饲料添加剂品种。所以,寻找合适的替抗产品是当前的研究重点。酸化剂作为一种绿色无残留饲料添加剂,具有增强机体免疫功能、调节胃肠道pH、增强消化酶活性、改善肠道微生物区系等功能[3⇓⇓-6]。酸化剂种类繁多,甲酸和丙酸作为小分子有机酸化剂,具有极强的抑菌、调控pH和提供能量的功能[7⇓-9]。国内研究报道,饲粮添加0.5%甲酸或二甲酸钾可显著提高肉鸡体增重,降低料重比,提高回肠绒毛高度,改善肉鸡生长性能,促进肠道发育[10]。国外研究报道,饲粮添加甲酸可显著提高断奶仔猪平均日增重,有提高平均日采食和降低料重比的趋势,显著提高空肠微生物区系多样性,改善断奶仔猪生长性能[11]。复合酸化剂在兔生产上的研究较少,且其对肠道免疫和紧密连接蛋白的研究鲜有报道。而本试验前期研究发现,饲粮添加复合酸化剂可以显著提高伊拉兔平均日增重,显著降低腹泻率,并有提高平均日采食量的趋势[12]。因此,本试验采用甲酸和丙酸组成的复合酸化剂来探究其对伊拉兔空肠消化酶活性、免疫功能和紧密连接蛋白表达的影响,以筛选出较为合适的添加剂量,为酸化剂在伊拉兔中的生产应用提供理论依据和实践指导。

1 材料与方法

1.1 试验设计

选取(35±2)日龄的健康断奶商品伊拉兔192只,随机分为4组,每组6个重复,每个重复8只,全为公兔,重复之间体重接近(P>0.05)。4组分别为空白组(基础饲粮)、抗生素组(基础饲粮+60 mg/kg盐霉素)、试验Ⅰ组(基础饲粮+0.1%复合酸化剂)和试验Ⅱ组(基础饲粮+0.2%复合酸化剂)。空白组基础饲粮参照山东省地方标准《肉兔饲养标准》(DB 37/T 1835—2011)营养需要配制,为颗粒料,其组成及营养水平见表1。复合酸化剂由漳州某生物科技有限公司提供(含29%甲酸、6%丙酸、30%木质磺酸盐和35%二氧化硅载体)。试验期28 d,期间幼兔自由采食,充足饮水,按正常免疫程序进行免疫接种。
表1 基础饲粮组成及营养水平(风干基础)

Table 1 Composition and nutrient levels of the basal diet (air-dry basis)%

项目 Items 含量 Content
原料 Ingredients
苜蓿草粉 Alfalfa meal 38.00
玉米 Corn 9.00
麸皮 Wheat bran 16.90
小麦干酒糟及其可溶物 Wheat DDGS 6.00
稻壳粉 Rice husk powder 9.00
豆粕 Soybean meal 5.00
米糠粕 Rice bran meal 10.00
次粉 Wheat middling 3.00
石粉 Limestone 1.20
蛋氨酸 Methionine 0.10
赖氨酸 Lysine 0.20
氯化钠 NaCl 0.60
预混料 Premix1) 1.00
合计 Total 100.00
营养水平 Nutrient levels2)
消化能 DE/(MJ/kg) 9.96
粗蛋白质 CP 15.48
粗纤维 CF 17.35
中性洗涤纤维 NDF 33.56
酸性洗涤纤维 ADF 20.51
酸性洗涤木质素 ADL 5.90
钙 Ca 0.90
总磷 TP 0.51
赖氨酸 Lysine 0.74
蛋氨酸+半胱氨酸 Methionine+cysteine 0.51

1)预混料为每千克饲粮提供 The premix provided the following per kg of the diet:VA 12 000 IU,VD3 900 IU,VE 50 mg,VK3 1 mg,VB1 1 mg,VB2 3 mg,VB6 1 mg,VB12 0.01 mg,烟酸 niacin 30 mg,泛酸 pantothenic acid 8 mg,叶酸 folic acid 0.2 mg,生物素 biotin 0.08 mg,胆碱 choline 100 mg,Cu (as copper sulfate) 10 mg,Fe (as ferrous sulfate) 50 mg,Mn (as manganese sulfate) 8 mg,Zn (as zinc sulfate) 50 mg,I (as potassium iodide) 1 mg,Se (as sodium selenite) 0.05 mg,Co (as cobalt sulfate) 0.25 mg。

2)营养水平为计算值。Nutrient levels were calculated values.

1.2 样品采集

试验第28天09:00开始空腹称重试验兔,从各组的不同重复中分别选择1只接近平均体重的试验兔屠宰,屠宰前1天禁食12 h不禁水。屠宰后打开腹腔,取3 cm左右的空肠中段,用生理盐水冲洗干净,纵向剖开,用干净的载玻片刮取空肠黏膜,放入冻存管中于-80 ℃中保存。

1.3 检测指标及方法

1.3.1 空肠黏膜消化酶活性测定

将空肠黏膜样品取出,解冻后用于测定空肠黏膜中的脂肪酶、α-淀粉酶和胰蛋白酶活性。测定使用南京建成生物工程研究所检测试剂盒,严格按照操作说明书进行测定。

1.3.2 空肠黏膜细胞因子和分泌型免疫球蛋白A(sIgA)含量测定

将空肠黏膜样品取,解冻后利用酶联免疫吸附试验(ELISA)方法测定空肠细胞因子白细胞介素-6(IL-6)、白细胞介素-10(IL-10)、白细胞介素-1β(IL-1β)和sIgA含量。测定使用南京建成生物工程研究所ELISA试剂盒,严格按照操作说明书进行测定。

1.3.3 空肠黏膜细胞因子和紧密连接蛋白表达测定

空肠黏膜总RNA采用TransZol UP Plus RNA Kit RNA提取试剂盒(北京全式金生物技术股份有限公司)提取,提取方法严格按照说明书操作步骤进行。然后根据Reverse Transcription System Kit反转录试剂盒[普洛麦格(北京)生物技术有限公司]进行反转录。最后根据Go Taq® qPCR Master Mix荧光定量试剂盒[普洛麦格(北京)生物技术有限公司]进行实时荧光定量PCR(real-time fluorescence quantitative PCR,RT-qPCR),检测空肠黏膜IL-6、IL-10、IL-1β、闭锁蛋白(occludin)、闭合蛋白-1(claudin-1)、闭合蛋白-3(claudin-3)和闭合小环蛋白-1(ZO-1)的基因表达水平。体系反应条件为:95 ℃预变性5 min;95 ℃变性15 s,退火温度条件下退火10 s,72 ℃延伸10 s,变性至延伸步骤重复进行45个循环。试验所需全部基因引物均由福州尚亚生物技术有限公司设计合成,引物序列见表2。以3-磷酸甘油醛脱氢酶(GADPH)为内参基因,采用2-△△Ct分析方法,计算目的基因的mRNA相对表达量。
表2 引物序列

Table 2 Primer sequences

基因
Genes		 引物序列
Primer sequences (5'—3')		 基因序列号
Gene serial number
3-磷酸甘油醛脱氢酶 GADPH F:GGTAGTGAAGGCTGCTGCTGATG
R:GTCTCGCACTCCAATCTCTGTTCC		 NC_013676.1
白细胞介素-6 IL-6 F:ACGATCCACTTCATCCTGCG
R:GGATGGTGTGTTCTGACCGT		 NC_013678.1
白细胞介素-10 IL-10 F:TCACCGATTTTCTCCCCTGTG
R:ATGTCAAACTCATGGCTT		 NC_013684.1
白细胞介素-1β IL-1β F:TCTGCAACACCTGGGATGAC
R:TCAGCTCATACGTGCCAGAC		 NC_013670.1
闭锁蛋白 Occludin F:CCGTATCCAGAGAGTCCTACAAGT
R:GTCCGTCTCGTAGTGGTCTT		 NC_013679.1
闭合蛋白-1 Claudin-1 F:ACAGCATGGTATGGCAACAG
R:CGAGGACAAGAACAGCAAAGT		 NC_013682.1
闭合小环蛋白-1 ZO-1 F:CCGCTCATACCTTCCTCTCA
R:GTCATTCACCTCCTTCTTGTTCTC		 NC_013685.1
闭合蛋白-3 Claudin-3 F:CCATCATCCAGGACTTCTACAAC
R:AGTAGGCGATCTTGGTGGTC		 NW_003159391.1

1.4 数据统计分析

试验数据用Excel 2021整理后,采用SPSS 26.0统计软件进行单因素方差分析(one-way ANOVA),并用Duncan氏法进行多重比较,结果以“平均值±标准差”表示。P<0.05表示差异显著,P>0.05表示差异不显著,0.05<P≤0.10表示差异有显著趋势。

2 结果与分析

2.1 复合酸化剂对伊拉兔空肠黏膜消化酶活性的影响

由表3可知,与空白组相比,试验Ⅰ组伊拉兔空肠胰蛋白酶活性显著提高(P<0.05);各组间空肠脂肪酶和α-淀粉酶活性无显著差异(P>0.05)。
表3 复合酸化剂对伊拉兔空肠黏膜消化酶活性的影响

Table 3 Effects of compound acidifier on digestive enzyme activity in jejunal mucosa of Ira rabbits

项目
Items		 空白组
Blank
group		 抗生素组
Antibiotic
group		 试验Ⅰ组
Experimental
group Ⅰ		 试验Ⅱ组
Experimental
group Ⅱ		 P值
P-value
脂肪酶 Lipase/(U/g) 73.01±14.51 76.71±80.61 121.06±44.15 125.75±76.95 0.403
α-淀粉酶 α-amylase/(U/mg) 11.06±3.36 10.09±3.18 11.39±3.44 10.76±2.51 0.923
胰蛋白酶 Trypsin/(U/mg) 2 629.62±367.12b 3 032.62±569.09ab 3 630.90±429.15a 3 083.10±552.33ab 0.017

2.2 复合酸化剂对伊拉兔空肠黏膜细胞因子和sIgA含量的影响

由表4可知,与空白组相比,抗生素组、试验Ⅰ组和试验Ⅱ组伊拉兔空肠黏膜IL-6和IL-1β含量显著降低(P<0.05);抗生素组和试验Ⅰ组空肠黏膜IL-10含量有提高趋势(P=0.086);各组间空肠黏膜sIgA含量无显著差异(P>0.05)。
表4 复合酸化剂对伊拉兔空肠黏膜细胞因子和sIgA含量的影响

Table 4 Effects of compound acidifier on contents of cytokines and sIgA in jejunal mucosa of Ila rabbitspg/mL

项目
Items		 空白组
Blank
group		 抗生素组
Antibiotic
group		 试验Ⅰ组
Experimental
group Ⅰ		 试验Ⅱ组
Experimental
group Ⅱ		 P值
P-value
白细胞介素-6 IL-6 115.60±3.16a 105.60±5.22b 102.50±5.83b 99.30±8.76b 0.010
白细胞介素-10 IL-10 609.70±60.55 733.75±87.57 740.00±133.91 715.00±76.32 0.086
白细胞介素-1β IL-1β 71.61±4.09a 63.27±4.87b 62.97±7.64b 57.21±6.84b 0.005
分泌型免疫球蛋白A sIgA 64.23±2.75 66.18±3.28 65.53±1.16 64.34±2.91 0.529

2.3 复合酸化剂对伊拉兔空肠黏膜细胞因子mRNA相对表达量的影响

由表5可知,与空白组相比,抗生素组、试验Ⅰ组和试验Ⅱ组伊拉兔空肠黏膜IL-1β mRNA相对表达量显著降低(P<0.05);各组间空肠黏膜IL-6和IL-10 mRNA相对表达量无显著差异(P>0.05)。
表5 复合酸化剂对伊拉兔空肠黏膜细胞因子mRNA相对表达量的影响

Table 5 Effects of compound acidifier on cytokine mRNA relative expression levels in jejunal mucosa of Ila rabbits

项目
Items		 空白组
Blank
group		 抗生素组
Antibiotic
group		 试验Ⅰ组
Experimental
group Ⅰ		 试验Ⅱ组
Experimental
group Ⅱ		 P值
P-value
白细胞介素-6 IL-6 1.00±0.15 0.94±0.41 0.60±0.01 0.65±0.15 0.155
白细胞介素-10 IL-10 1.00±0.38 1.88±0.32 1.77±0.56 1.28±0.50 0.134
白细胞介素-1β IL-1β 1.00±0.25a 0.48±0.15b 0.39±0.23b 0.49±0.12b 0.019

2.4 复合酸化剂对伊拉兔空肠黏膜紧密连接蛋白mRNA相对表达量的影响

由表6可知,与空白组相比,抗生素组和试验Ⅰ组伊拉兔空肠黏膜ZO-1和occludin的mRNA相对表达量显著提高(P<0.05);抗生素组和试验Ⅱ组空肠黏膜claudin-1的mRNA相对表达量显著降低(P<0.05);各组间空肠黏膜claudin-3的mRNA相对表达量无显著差异(P>0.05)。
表6 复合酸化剂对伊拉兔空肠黏膜紧密连接蛋白mRNA相对表达量的影响

Table 6 Effects of compound acidifier on tight junction protein mRNA relative expression levels in jejunum mucosa of Ira rabbits

项目
Items		 空白组
Blank
group		 抗生素组
Antibiotic
group		 试验Ⅰ组
Experimental
group Ⅰ		 试验Ⅱ组
Experimental
group Ⅱ		 P值
P-value
闭合小环蛋白-1 ZO-1 1.00±0.31b 1.80±0.26a 1.87±0.46a 1.49±0.27ab 0.047
闭锁蛋白 Occludin 1.00±0.16b 2.55±0.71a 2.64±1.20a 1.37±0.12ab 0.047
闭合蛋白-1 Claudin-1 1.00±0.29a 0.49±0.05c 0.88±0.19ab 0.50±0.11bc 0.019
闭合蛋白-3 Claudin-3 1.00±0.07 1.33±0.28 1.57±1.47 1.09±0.70 0.836

3 讨论

3.1 复合酸化剂对伊拉兔空肠黏膜消化酶活性的影响

肠道消化酶是衡量肠道消化能力的重要指标,脂肪酶、α-淀粉酶和胰蛋白酶作为消化脂肪、淀粉和蛋白质的主要酶,其活性大小可间接反映出机体对营养物质的消化吸收程度。这3种酶在肠道中发挥着不同功能,如肠道脂肪酶主要是胰腺分泌的胰脂肪酶,其可将甘油三酯分解为甘油和脂肪酸,为机体提供能量[13];α-淀粉酶可将淀粉分解为葡萄糖和麦芽糖,为机体提供能量,促进机体生长[14];胰蛋白酶可将蛋白质分解为氨基酸和多肽等,再被肠道吸收到机体各个组织中,其在消化吸收中起着不可或缺的作用[15]。总得来说,其都是将体内营养物质分解,从而为机体供能,维持机体生长所需能量。刁慧[16]研究发现,饲粮中添加0.5%苯甲酸可显著提高空肠中胰蛋白酶、淀粉酶和脂肪酶活性,提高断奶仔猪对营养物质的消化率。Ma等[17]研究发现,饲粮添加复合有机酸(甲酸、甲酸铵和丙酸盐等)可显著提高胰腺淀粉酶活性。孙建华等[18]研究发现,饲粮添加复合酸化剂(甲酸、甲酸铵和乳酸)可极显著提高肉鸡空肠胰蛋白酶活性,改善肉鸡消化吸收能力。本试验结果发现,饲粮添加0.1%复合酸化剂(甲酸和丙酸)可显著提高伊拉兔空肠黏膜胰蛋白酶活性,这与前人研究基本相同。其原因可能是饲粮添加酸化剂可降低食糜pH,从而降低胃肠道pH,激活胃蛋白酶,促进蛋白质的分解,进而促进小肠和胰腺中酶的产生和活性提高[19⇓-21]。

3.2 复合酸化剂对伊拉兔空肠黏膜细胞因子含量和mRNA表达及sIgA含量的影响

肠道黏膜免疫系统是机体抵御病原微生物入侵的首道防线,当受外界因素干扰时,肠黏膜易受病原菌的侵入,使机体免疫能力下降。肠黏膜免疫系统主要通过肠道内淋巴细胞发挥作用,如上皮淋巴细胞可分泌白细胞介素-4(IL-4)、IL-10和IL-1β等细胞因子,在维持肠道黏膜完整及调节肠道免疫上发挥着重要作用,细胞因子含量和基因表达量的变化可间接反映机体的健康状况[22]。Tong等[23]研究表明,丙酸可显著抑制小鼠结肠IL-6、IL-1β和肿瘤坏死因子-α(TNF-α)的表达,减缓小鼠肠道炎症。陈慧子等[24]研究发现,生长猪盲肠灌注丙酸可显著提高occludin-1、生长抑素(SST)和G蛋白偶联受体5A(GPRC5A)等免疫相关基因的表达,调节宿主肠道免疫功能,维持机体健康。sIgA是黏膜免疫的主要效应因子,其可通过免疫排斥作用阻止病原微生物黏附于黏膜上皮细胞表面,保护机体健康[25]。何荣香[26]研究发现,饲粮添加复合有机酸(甲酸、甲酸铵和乙酸)可显著提高空肠黏膜中sIgA的含量,增强空肠黏膜免疫功能。本试验结果发现,饲粮添加复合酸化剂可显著降低伊拉兔空肠黏膜促炎因子IL-6和IL-1β的含量,有提高抗炎因子IL-10含量的趋势,并可显著降低IL-1β的mRNA相对表达量,从而增强断奶伊拉兔空肠免疫能力,改善断奶伊拉兔健康状况。其增强肠道免疫能力的原因可能是:1)酸化剂可提高肠道消化酶活性,促进机体消化吸收营养物质,增强机体免疫能力[27];2)酸化剂可改善肠道黏膜形态,增强肠道功能,提高肠道免疫能力[28];3)酸化剂可调节肠道pH,促进肠道有益菌增殖,抑制有害菌增殖,维持肠道健康[29]。

3.3 复合酸化剂对伊拉兔空肠黏膜紧密连接蛋白mRNA表达的影响

紧密连接是相邻上皮细胞之间的细胞间隙,在肠道屏障功能中起重要作用,其结构主要由跨膜蛋白[如闭合蛋白(claudin)和occludin]、连接黏附分子(JAM)和胞质闭合小环蛋白(ZO)组成,这些结构可调节相邻细胞中水、离子和大分子的胞外通透性,加强细胞间连接,避免细胞损伤[30]。而claudin、occludin和ZO-1是紧密连接中极其重要的蛋白分子,其中claudin可维持细胞内环境稳定性,参与分子细胞间传递;occludin具有维持紧密连接稳定性以及小肠通透性的作用;ZO-1可维持上皮细胞极性和肠道屏障通透性,调节基因表达和细胞增殖。这几种蛋白可作为观察各种组织紧密连接屏障功能和通透性功能的指标[31⇓⇓-34]。Tong等[24]研究发现,丙酸可显著提高紧密连接蛋白ZO-1、occludin和黏结蛋白的表达,改善肠道屏障功能,缓解小鼠肠道炎症。Diao等[35]研究发现,胃内灌注短链脂肪酸(乙酸、丙酸和丁酸)可显著提高十二指肠和回肠中occludin和claudin-1的mRNA相对表达量,维持肠道屏障功能,促进肠道发育。陈佳力等[36]研究发现,饲粮添加苯甲酸显著提高空肠黏膜ZO-1和occludin的mRNA相对表达量。本试验结果发现,饲粮添加0.1%复合酸化剂可显著提高伊拉兔空肠黏膜ZO-1和occludin的mRNA相对表达量;饲粮添加0.2%复合酸化剂可显著降低空肠黏膜claudin-1的mRNA相对表达量,从而改善空肠机械屏障功能,增强肠道抵御病原微生物的能力,保障机体内环境稳态,这与前人研究结果基本相似。酸化剂调节肠道机械屏障的原因可能是酸化剂可调节肠道pH,抑制有害菌增殖,减少致病菌黏附和侵袭的可能性,促进肠道发育[37]。

4 结论

饲料添加复合酸化剂可以提高伊拉兔空肠黏膜胰蛋白酶活性,降低空肠黏膜促炎因子IL-6和IL-1β含量和IL-1β mRNA相对表达量,增强空肠免疫能力;同时,促进空肠黏膜紧密连接蛋白ZO-1和occludin的mRNA表达,改善肠道发育,促进伊拉兔健康生长。其中,以添加0.1%复合酸化剂效果较佳。

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