动物营养学报 >

2020 , Vol. 32 >Issue 2: 726 - 735

DOI: https://doi.org/10.3969/j.issn.1006-267x.2020.02.028

反刍与草食动物营养与饲料 RUMINANT AND HERBIVORE NUTRITION AND FEED

壳聚糖对泌乳中期奶牛一氧化氮免疫调节途径的影响

  • 李倜宇 ,
  • 石璐璐 ,
  • 闫素梅 ,
  • 史彬林
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  • 内蒙古农业大学动物科学学院, 呼和浩特 010018
李倜宇(1987-),男,内蒙古锡林郭勒人,博士研究生,研究方向为动物环境与营养。E-mail:litiyu2050@163.com

收稿日期: 2019-08-05

  网络出版日期: 2020-02-21

基金资助

国家自然科学基金项目（31672463）

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Effects of Chitosan on Nitric Oxide Immune Regulating Pathway in Mid-Lactating Dairy Cows

  • LI Tiyu ,
  • SHI Lulu ,
  • YAN Sumei ,
  • SHI Binlin
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  • College of Animal Science, Inner Mongolia Agricultural University, Hohhot 010018, China

Received date: 2019-08-05

  Online published: 2020-02-21

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摘要

本试验利用体内试验和体外试验研究壳聚糖（CHI）对泌乳中期奶牛一氧化氮（NO）免疫调节途径的影响。体内试验：按产奶量、泌乳期、胎次和体重相近的原则，将40头泌乳中期荷斯坦奶牛随机分为5个处理，每个处理8个重复，每个重复1头牛。各处理分别在基础饲粮中添加0、500、1 000、1 500和2 000 mg/kg的CHI。试验期60 d，其中1~30 d为前期，31~60 d为后期。试验期间奶牛自由采食和饮水。体外试验：分为2个子试验，每个试验均采用2×2×2三因子设计，即2个脂多糖（LPS）添加水平（0和10 μg/mL）×2个壳寡糖（COS）添加水平（0和160 μg/mL）×2个抑制剂添加水平（添加和不添加），共8个处理，每个处理6个重复。2个子试验的抑制剂分别是诱导型一氧化氮合酶（iNOS）抑制剂1400W（添加水平1 mmol/L）和核转录因子-κB（NF-κB）抑制剂PDTC（添加水平10 mmol/L）。体内试验结果表明：在试验第60天，血清中NO含量随CHI添加水平增加呈极显著的一次线性（P<0.01）或二次曲线（P<0.01）升高效应，iNOS活性呈趋于显著的一次线性（0.05≤P<0.10）或显著的二次曲线（P<0.05）升高效应；外周血单个核细胞（PBMCs）中iNOS和NF-κB基因表达量均呈趋于显著的一次线性（0.05≤P<0.10）或显著的二次曲线（P<0.05）升高效应；上述指标在1 500 mg/kg添加水平表现为最高。体外试验结果表明：1）当1400W作为抑制剂，COS显著增加iNOS基因表达量（P<0.05）；1400W极显著降低NO含量和iNOS活性（P<0.01）。2）当PDTC作为抑制剂，PDTC显著降低NO含量（P<0.05），极显著降低iNOS和NF-κB的基因表达量（P<0.01）。3）在脂多糖（LPS）刺激的条件下，NO含量、iNOS活性和iNOS基因表达量均极显著增加（P<0.01），而对NF-κB基因表达量无显著影响（P>0.05）；而添加COS又能够缓解LPS引起的NO含量、iNOS活性和iNOS基因表达量的升高。上述结果提示，COS可通过增加iNOS和NF-κB的基因表达量，升高iNOS的活性，从而增加机体内NO的产生，对奶牛免疫功能起到调节作用，而且这种作用呈现剂量依赖效应。此外，COS引起的PBMCs中NO含量的升高，是通过增加iNOS和NF-κB的基因表达量，提高iNOS活性来完成的，且COS对奶牛PBMCs中NO途径的相关指标具有双向调节作用。综上所述，CHI可通过NO分子途径发挥免疫调节功能，且添加水平为1 500 mg/kg时效果最佳。

关键词： 壳聚糖; 奶牛; 一氧化氮; 免疫调节

本文引用格式

李倜宇 , 石璐璐 , 闫素梅 , 史彬林 . 壳聚糖对泌乳中期奶牛一氧化氮免疫调节途径的影响[J]. 动物营养学报, 2020 , 32(2) : 726 -735 . DOI: 10.3969/j.issn.1006-267x.2020.02.028

Abstract

In vivo and in vitro methods were performed in this experiment to study the effects of chitosan (CHI) on nitric oxide (NO) immune regulating pathway in mid-lactating dairy cows. Method in vivo:a total of forty heathy mid-lactating Holstein cows with similar milk yield, days in lactation, parturition date and body weight were randomly divided into five treatments, eight replicates per treatment. The five experimental diets contained, respectively, 0, 500, 1 000, 1 500 or 2 000 mg/kg CHI. Feeding trial lasted for 60 days, and was divided equally into two phases, namely 1 to 30 days (earlier period) and 31 to 60 days (later period). Cows had free access to diets and water during the trial. Method in vitro:experiment consisted of two sub-experiments and adopted 2×2×2 factor design, with 2 lipopolysaccharide (LPS) treatments (0 and 10 μg/mL), 2 chitosan oligosaccharide (COS) treatments (0 and 160 μg/mL) and 2 inhibitors (adding inhibitor and without inhibitor). There were 8 treatments in total, with 6 replicates per treatment. Two sub-experiments used respectively inducible nitric oxide synthase (iNOS) inhibitor 1400W (1 mmol/L) and nuclear transcription factor-κB (NF-κB) inhibitor PDTC (10 mmol/L). The in vivo method results showed as follows:with the increasing of the CHI addition level, NO content showed significant linear (P<0.01) or quadratic (P<0.01) increasing effects, and iNOS activity tended to be linear (0.05 ≤ P<0.10) or significant quadratic (P<0.05) increasing effects on day 60; the gene expressions of iNOS and NF-κB showed linear increasing trends (0.05 ≤ P<0.10) or significant quadratic increasing effects (P<0.05), with 1500 mg/kg being the optimum dosage in vivo. The in vitro method results showed as follows:1) when 1400W as an inhibitor, COS could significantly increase iNOS gene expression in PBMCs (P<0.05); 1400W could significantly decrease NO content and iNOS activity (P<0.01). 2) When PDTC as an inhibitor, PDTC could significantly decrease NO content (P<0.05) and gene expressions of iNOS and NF-κB (P<0.01); there were also interactions between COS and 1400W, PDTC in terms of above indicators. 3) Moreover, in LPS-stimulated conditions, LPS enhanced NO content, iNOS activity and iNOS gene expression significantly (P<0.01), and had no significant effect on NF-κB gene expression (P>0.05). Besides, adding COS could decrease NO content, iNOS activity and iNOS gene expression in comparison with in the absence of LPS stimulation. The results suggested that CHI can dose-dependently affect immune function of dairy cows by increasing gene expression level of iNOS and NF-κB, improving the activity of iNOS, stimulating the releasing of NO from immunological cells. Furthermore, COS-induced improvement of NO contents in PBMCs is a result of up-regulation of iNOS and NF-κB gene expression, and improvement of iNOS activity. COS can dual-directionally regulate key factors of NO pathway in PBMCs. In conclusion, CHI can exert immune regulation function via NO molecular pathway. The addition level of 1 500 mg/kg is considered as the optimum level of CHI.

Key words： chitosan; dairy cow; nitric oxide; immune regulation

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