动物营养学报 >

2018 , Vol. 30 >Issue 5: 1816 - 1826

DOI: https://doi.org/10.3969/j.issn.1006-267x.2018.05.024

分子营养 Molecular Nutrition

体外法研究pH与脂多糖或组胺的交互作用对奶山羊瘤胃上皮细胞紧密连接蛋白mRNA表达量的影响

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  • 1. 内蒙古农业大学动物科学学院, 呼和浩特 010018;
    2. 内蒙古农牧业科学院动物营养与 饲料研究所, 呼和浩特 010031
孙燕勇(1988-),女,河北张家口人,硕士研究生,从事反刍动物营养研究。E-mail:840353512@qq.com

收稿日期: 2017-11-07

  网络出版日期: 2018-05-06

基金资助

国家自然科学基金项目（31472124，31101739）；内蒙古自然科学基金项目（2015MS0380）；杨胜先生门生社群项目（B2016011）；现代农业（奶牛）产业技术体系建设专项资金（CARS-36）；2016年度内蒙古自治区青年创新人才项目

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Interaction Effects of pH and Lipopolysaccharide or Histamine on mRNA Expression Levels of Tight Junction Proteins of Rumen Epithelium of Dairy Goats in Vitro

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  • 1. College of Animal Science and Technology, Inner Mongolia Agricultural University, Hohhot 010018, China;
    2. Research Institute of Animal Nutrition, Inner Mongolia Academy of Animal Sciences, Hohhot 010031, China

Received date: 2017-11-07

  Online published: 2018-05-06

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摘要

本试验旨在采用体外法研究pH与脂多糖（LPS）或组胺（HIS）的交互作用对奶山羊瘤胃上皮细胞紧密连接蛋白mRNA表达量的影响。选用8只体况良好，体重、泌乳量相近的萨能奶山羊作为瘤胃上皮供体。采用3×3双因素试验设计，山羊屠宰后采集瘤胃上皮，插入到尤斯灌流仪器（Ussing chamber）半室中央，在浆膜侧半室加入5 mL缓冲液，黏膜侧加入配制好的不同处理的培养液5 mL，每个处理3个重复。试验1：因素1为pH，分别为7.4、5.5、5.2；因素2为LPS浓度，分别为0、30、60 kEU/mL。试验2：因素1为pH，分别为7.4、5.5、5.2；因素2为HIS浓度，分别为0、0.5、10.0 ng/mL。采集培养80 min后的瘤胃上皮，测定其紧密连接蛋白Claudin-1、Claudin-4、Claudin-7、Occludin、zonula occludens-1（ZO-1）mRNA表达量。结果显示：1）pH与LPS的交互作用对Claudin-1、Claudin-7、ZO-1 mRNA表达量影响显著（P<0.05）。与pH 7.4×0 kEU/mL LPS组相比，降低pH或添加LPS均显著降低了Claudin-1、Claudin-7 mRNA表达量（P<0.05），ZO-1 mRNA表达量有整体降低的趋势，在pH 5.2×60 kEU/mL LPS组最高。2）pH与HIS的交互作用对Claudin-1、Claudin-7、ZO-1 mRNA表达量影响显著（P<0.05）。pH 5.5×0.5 ng/mL HIS组Claudin-1 mRNA表达量最低，但与pH 5.2×0.5 ng/mL HIS组差异不显著（P>0.05）。pH 7.4×10.0 ng/mL HIS组Claudin-7 mRNA表达量显著低于pH 7.4×0 ng/mL HIS组（P<0.05），但与pH 5.5×10.0 ng/mL HIS组差异不显著（P>0.05）。与pH 7.4×0 ng/mL HIS组相比，降低pH或添加LPS有提高ZO-1 mRNA表达量的趋势，且pH 5.2×10.0 ng/mL HIS组显著提高（P<0.05）。结果提示，亚急性瘤胃酸中毒（SARA）发生后，pH与LPS或HIS交互作用于瘤胃上皮，降低瘤胃上皮紧密连接蛋白mRNA表达量，进而增大瘤胃上皮黏膜通透性。

关键词： 亚急性瘤胃酸中毒; 奶山羊; 瘤胃上皮; 组胺; 脂多糖; 尤斯灌流仪器; 紧密连接蛋白

本文引用格式

孙燕勇, 高民, 徐明, 宋利文, 李洋, 李超, 陈利青, 胡红莲 . 体外法研究pH与脂多糖或组胺的交互作用对奶山羊瘤胃上皮细胞紧密连接蛋白mRNA表达量的影响[J]. 动物营养学报, 2018 , 30(5) : 1816 -1826 . DOI: 10.3969/j.issn.1006-267x.2018.05.024

Abstract

The objective of this study was to investigate the interaction effects of pH and lipopolysaccharide (LPS) or histamine (HIS) on mRNA expression levels of tight junction proteins of rumen epithelium of dairy goats in vitro. Eight healthy Saanen dairy goats with similar body weight and milk yield were used as the donors of rumen epithelium. A 3×3 two-factor experiment design was adopted. After slaughtering, rumen epithelium was collected and inserted in the middle of half chamber in a Ussing chamber. Buffer at 5 mL was added to the serosal side, and different treatments of culture mediums at 5 mL were added to the mucosal side, each treatment had 3 replicates. Experiment 1, factor 1 was pH, and was 7.4, 5.5, 5.2, respectively; factor 2 was LPS concentration, and was 0, 30 and 60 kEU/mL, respectively. In experiment 2, factor 1 was pH, and was 7.4, 5.5, 5.2, respectively; factor 2 was HIS concentration, and was 0, 0.5 and 10.0 ng/mL, respectively. The rumen epithelium was collected after cultured for 80 min, and the expression levels of Claudin-1, Claudin-4, Claudin-7, Occluding and zonula occludens-1 (ZO-1) mRNAs were measured. The results showed as follows:1) the interaction between pH and LPS had significant effects on expression levels of Claudin-1, Claudin-7 and ZO-1 mRNAs (P<0.05). Compared with pH 7.4×0 kEU/mL LPS group, the decrease of pH or the supplementation of LPS significantly decreased expression levels of Claudin-1 and Claudin-7 mRNAs (P<0.05), and tended to rise expression level of ZO-1 mRNA, while it was the highest in pH 5.2×60 kEU/mL LPS group. 2) The interaction between pH and HIS had significant effects on expression levels of Claudin-1, Claudin-7 and ZO-1 mRNAs (P<0.05). pH 5.5×0.5 ng/mL HIS group had the lowest expression level of Claudin-1 mRNA, which was not significantly different from that in pH 5.2×0.5 ng/mL HIS group (P>0.05). Compared with pH 7.4×0 ng/mL HIS group, pH 7.4×10.0 ng/mL HIS group had significantly lower expression level of Claudin-7 mRNA, which was not significantly different from that in pH 5.5×10.0 ng/mL HISgroup (P>0.05). Compared with pH 7.4×0 kEU/mL LPS group, the decrease of pH or the supplementation of HIS tended to rise expression level of ZO-1 mRNA, and that in pH 5.2×10.0 ng/mL HIS group was significantly increased (P<0.05). The results suggest that after subacute rumen acidosis (SARA) occurs, pH interacts with LPS or HIS to decrease the expression levels of tight junction proteins in the rumen epithelium, which in turn increases the permeability of rumen epithelial mucosa.

Key words： subacute rumen acidosis; dairy goats; rumen epithelium; histamine; lipopolysaccharides; Ussing chamber; tight junction proteins

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