蜂胶对细菌脂多糖刺激下奶牛乳腺上皮细胞炎症相关基因mRNA转录水平和紧密连接蛋白的影响

doi:10.11843/j.issn.0366-6964.2020.05.026

摘要/Abstract

摘要： 本试验旨在研究中国蜂胶乙醇提取物（ethanol extract of Chinese propolis，EECP）对细菌脂多糖（lipopolysaccharide，LPS）刺激下体外培养奶牛乳腺上皮细胞炎症相关基因mRNA转录水平和紧密连接渗透性的影响。EECP中总酚酸和总黄酮含量测定采用福林酚法和硝酸铝法，并建立LPS诱导奶牛乳腺上皮细胞（bovine mammary epithelial cells，MAC-T）炎症模型，采用CCK-8法测定EECP对MAC-T相对增殖率的影响，利用实时荧光定量PCR（RT-qPCR）评估EECP对LPS诱导的MAC-T细胞炎症相关因子（IL-6、IL-8、TNF-α和IL-1β）相对mRNA转录水平；以及对紧密连接蛋白（occludin、ZO-1）相对mRNA转录水平进行检测，并进一步利用免疫荧光技术对紧密连接膜蛋白进行定位，确定EECP对LPS诱导MAC-T细胞炎症紧密连接渗透性的影响。结果显示：EECP中总酚酸含量为106.35 mg没食子酸当量（GAE）·g^-1、总黄酮含量为320.85 mg芦丁当量（RE）·g^-1；CCK-8结果显示EECP的安全浓度为0~15 μg·mL^-1，并可有效提高LPS刺激下MAC-T的活力；LPS刺激显著增加了细胞炎症相关因子IL-6、IL-8、TNF-α和IL-1β mRNA的转录量（P<0.001）；但2.5~15.0 μg·mL^-1 EECP预处理显著降低了IL-6、IL-8、TNF-α和IL-1β mRNA的转录量；与此类似，LPS刺激显著抑制了紧密连接蛋白基因（occludin、ZO-1）mRNA的转录量（P<0.01），而EECP预处理后紧密连接蛋白基因（occludin和ZO-1）mRNA的转录量显著增加（P<0.05）；免疫荧光染色试验也证实EECP能通过上调紧密连接蛋白（occludin、ZO-1）的表达，缓解LPS诱导的乳腺上皮细胞屏障功能紊乱。该结果证实，EECP对细菌脂多糖诱导奶牛乳腺上皮细胞炎症具有良好的保护作用，这为利用中国蜂胶预防奶牛乳腺炎提供了试验基础。

关键词: 中国蜂胶, 抗炎, 紧密连接, 奶牛乳腺上皮细胞

Abstract: This study aimed to investigate the effects of ethanol extract of Chinese propolis (EECP) on transcript levels of inflammation-related genes and tight junctional permeability in bovine mammary epithelial cells stimulated by bacterial endotoxin (lipopolysaccharide,LPS). The total content of phenolic acids and flavonoids in the EECP was determined by Folin-phenol method and ALNO₃ colorimetry, respectively. Moreover, the inflammatory model of bovine mammary epithelial cells (MAC-T) was induced by bacterial lipopolysaccharide and the effect of EECP on the relative cell proliferation rate was detected via CCK-8 method. Real-time quantitative PCR (RT-qPCR) was used to evaluate LPS-induced bovine mammary epithelial cells inflammatory factors (IL-6, IL-8, TNF-α and IL-1β) relative mRNA transcription levels. To determine the effect of EECP on LPS-induced MAC-T cell, inflammation tight junction permeability. We used RT-qPCR to detect the relative mRNA transcription levels of tight junction proteins (occludin, ZO-1), and further used immunofluorescence to localize tight junction membrane proteins. The results showed that the content of total phenolic acids and total flavonoids in EECP were 106.35 mg GAE·g^-1 and 320.85 mg RE·g^-1, respectively. CCK-8 results showed that the safe concentration of EECP was 0-15 μg·mL ^-1, and can effectively increase the viability of MAC-T cells under LPS stimulation. LPS stimulation significantly increased the mRNA transcription levels of cellular inflammation-related factors IL-6, IL-8, TNF-α and IL-1β (P<0.001). However, the mRNA transcription of IL-6, IL-8, TNF-α and IL-1β were significantly reduced under pretreatment with 2.5-15.0 μg·mL^-1 EECP. Similarly, the LPS model group significantly inhibited (P<0.01) the mRNA transcription of tight junction proteins (ZO-1 and occludin), while the mRNA transcription of tight junction proteins was increased by EECP pretreatment (P<0.05). Immunofluorescence staining experiments also confirmed that EECP could alleviate LPS-induced mammary epithelial barrier dysfunction by up-regulating the expression of tight junction proteins occludin and ZO-1. The results confirmed that EECP has a good protective effect on bacterial lipopolysaccharide-induced inflammation of bovine mammary epithelial cells, which provides a basis for the use of Chinese propolis to prevent mastitis in dairy cows.

Key words: Chinese propolis, anti-inflammatory, tight junction, bovine mammary epithelial cells

中图分类号:

欧爱群, 王凯, 吴黎明, 李江红, 彭文君. 蜂胶对细菌脂多糖刺激下奶牛乳腺上皮细胞炎症相关基因mRNA转录水平和紧密连接蛋白的影响[J]. 畜牧兽医学报, 2020, 51(5): 1149-1157.

OU Aiqun, WANG Kai, WU Liming, LI Jianghong, PENG Wenjun. Effects of Propolis on Transcript Levels of Inflammation-related Genes and Tight Junction Proteins of Bovine Mammary Epithelial Cells Stimulated by Bacterial Lipopolysaccharide[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(5): 1149-1157.

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