动物营养学报 >

2022 , Vol. 34 >Issue 11: 7350 - 7360

DOI: https://doi.org/10.3969/j.issn.1006-267x.2022.11.052

研究论文 RESEARCH PAPER

葡萄糖对脂多糖刺激下的奶牛乳腺上皮细胞炎症相关基因表达的影响

  • 张金友 ,
  • 宋金婷 ,
  • 李伟 ,
  • 庄雨龙 ,
  • 李长志 ,
  • 宋伟红
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  • 1. 黑龙江八一农垦大学动物科技学院, 大庆 163319;
    2. 黑龙江省农垦科学院畜牧兽医研究所, 哈尔滨 150038
张金友(1979—),男,河北沧州人,副教授,博士,研究方向为动物生殖生理与乳腺免疫。E-mail:zhangjinyou868@aliyun.com

收稿日期: 2022-05-24

  网络出版日期: 2022-11-14

基金资助

黑龙江省自然科学基金联合引导项目（LH2019C082）；黑龙江省农垦总局重点科研计划项目（HKKYZD190304）；黑龙江八一农垦大学学成、引进人才科研启动计划（XYB202017）

收起

Effects of Glucose on Expression of Inflammation Related Genes in Dairy Cow Mammary Epithelial Cells Stimulated by Lipopolysaccharide

  • ZHANG Jinyou ,
  • SONG Jinting ,
  • LI Wei ,
  • ZHUANG Yulong ,
  • LI Changzhi ,
  • SONG Weihong
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  • 1. College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University, Daqing 163319, China;
    2. Institute of Animal Husbandry and Veterinary of Heilongjiang Academy of Land Reclamation Science, Harbin 150038, China

Received date: 2022-05-24

  Online published: 2022-11-14

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摘要

本试验旨在研究葡萄糖对脂多糖（LPS）刺激下的奶牛乳腺上皮细胞炎症基因表达的影响，为了解不同能量水平对乳腺上皮细胞免疫反应能力的影响提供依据。从健康的奶牛乳腺中分离乳腺上皮细胞，经纯化后将细胞分别在0.18、0.50、0.90和4.50 g/L葡萄糖条件下培养24 h，以及在含有上述4个葡萄糖浓度的培养液中同时添加1 μg/mL LPS继续培养24 h。培养结束后收集细胞提取RNA，通过实时荧光定量PCR检测细胞中白细胞介素-8（IL-8）、诱导型一氧化氮合成酶（iNOS）、乳铁蛋白（LF）、白细胞介素-6（IL-6）、转化生长因子-β1（TGF-β1）和肿瘤坏死因子-α（TNF-α） mRNA相对表达量。结果显示：IL-8、iNOS和LF的mRNA相对表达量在4种葡萄糖浓度之间无显著差异（P>0.05）；IL-6的mRNA相对表达量在葡萄糖浓度为0.18和0.50 g/L时显著高于葡萄糖浓度为0.90和4.50 g/L时（P<0.05）；TGF-β1的mRNA相对表达量在葡萄糖浓度为0.18和0.50 g/L时显著高于葡萄糖浓度为4.50 g/L时（P<0.05）；TNF-α的mRNA相对表达量在葡萄糖浓度为0.18 g/L时比葡萄糖浓度为0.50和4.50 g/L时显著升高（P<0.05）。添加1 μg/mL LPS除了在0.90和4.50 g/L葡萄糖浓度下极显著下调TGF-β1的mRNA相对表达量（P<0.01）之外，对其他5个基因的表达均产生了不同程度的刺激作用。随着葡萄糖浓度的降低，LPS对iNOS、LF和TNF-α基因表达的刺激作用被加强；而随着葡萄糖浓度的降低，LPS对IL-8和IL-6基因表达的刺激作用被减弱。综上可知，虽然低浓度葡萄糖通过促进LF基因的表达提升了乳腺组织消除病原菌的能力，但低浓度的葡萄糖仍然可以通过降低乳腺上皮细胞中IL-8和IL-6基因表达减弱中性粒细胞的募集作用，提高TNF-α、TGF-β1和iNOS基因的表达增加发生免疫抑制及细胞损伤的机会，对奶牛乳腺的免疫功能造成不利影响。因此，低浓度的葡萄糖总体上增加了乳腺炎症发病的可能。

关键词： 奶牛; 乳腺上皮细胞; 脂多糖; 葡萄糖; 炎症

本文引用格式

张金友 , 宋金婷 , 李伟 , 庄雨龙 , 李长志 , 宋伟红 . 葡萄糖对脂多糖刺激下的奶牛乳腺上皮细胞炎症相关基因表达的影响[J]. 动物营养学报, 2022 , 34(11) : 7350 -7360 . DOI: 10.3969/j.issn.1006-267x.2022.11.052

Abstract

The purpose of this experiment was to study the effects of glucose on the expression of inflammation related genes in dairy cow mammary epithelial cells stimulated by lipopolysaccharide (LPS), so as to provide basis for understanding the effects of different energy levels on the immune response of mammary epithelial cells. Mammary epithelial cells were isolated from the mammary glands of healthy dairy cows. After purification, the cells were cultured for 24 h under the conditions of 0.18, 0.50, 0.90 and 4.50 g/L glucose, respectively, and 1 μg/mL LPS was added to the culture medium containing the above four glucose concentrations for 24 hours. After culture, the cells were collected to extract RNA, and real-time fluorescence quantitative PCR was used to detect the mRNA relative expression levels of interleukin-8 (IL-8), induced nitric oxide synthase (iNOS), lactoferrin (LF), interleukin-6 (IL-6), transforming growth factor-β1 (TGF-β1) and tumor necrosis factor-α (TNF-α) of genes in cells. The results showed as follows:there were no significant differences in the mRNA relative expression levels of IL-8, iNOS and LF among the four glucose concentrations (P>0.05); the mRNA relative expression level of IL-6 g in 0.18 and 0.50 g/L glucose was significantly higher than that in 0.90 and 4.50 g/L glucose (P<0.05); the mRNA relative expression level of TGF-β1 in 0.18 and 0.50 g/L glucose was significantly higher than that in 4.50 g/L glucose (P<0.05); the mRNA relative expression level of TNF-α in 0.18 g/L glucose was significantly higher than that in 0.50 and 4.50 g/L glucose (P<0.05). The addition of 1 μg/mL LPS significantly extremely down-regulated the mRNA relative expression level of TGF-β1 in 0.90 and 4.50 g/L glucose (P<0.01), it stimulated the expression of the other five genes in varying degrees. With the decrease of glucose concentration, the stimulating effect of LPS on iNOS, LF and TNF-α gene expression was enhanced (P<0.05); with the decrease of glucose concentration, the stimulating effect of LPS on IL-8 and IL-6 gene expression was weakened (P<0.05). In conclusion, although the low concentration of glucose can enhance the ability of mammary tissue to eliminate pathogenic bacteria by promoting the expression of LF gene. However, low concentration of glucose can still cause more adverse effects on the immune function of mammary epithelial cells in dairy cows by reducing the expression of IL-8 and IL-6 genes to weaken the recruitment of neutrophils, and increasing the expression of TNF-α, TGF-β1 and iNOS to increase the chance of immunosuppression and cell damage in mammary epithelial cells. Therefore, the low concentration of glucose increases the possibility of mastitis in general.

Key words： dairy cows; mammary epithelial cells; lipopolysaccharide; glucose; inflammation

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