中国农业科学 ›› 2021, Vol. 54 ›› Issue (12): 2675-2688.doi: 10.3864/j.issn.0578-1752.2021.12.017

• 畜牧·兽医·资源昆虫 • 上一篇    下一篇

蜂胶提取物对脂多糖诱导小鼠急性乳腺炎及乳腺屏障功能的保护作用

宋美洁1(),欧爱群1,2,薛晓锋1,吴黎明1,寿旗扬3,王凯1()   

  1. 1中国农业科学院蜜蜂研究所,北京 100093
    2福建农林大学动物科学学院(蜂学学院),福州 350000
    3浙江中医药大学第二临床医院,杭州 310027
  • 收稿日期:2020-05-06 接受日期:2020-11-25 出版日期:2021-06-16 发布日期:2021-06-24
  • 通讯作者: 王凯
  • 作者简介:宋美洁,E-mail:17853483688@163.com
  • 基金资助:
    国家自然科学青年科学基金项目(31702287)

Protective Effects of Chinese Propolis Extract Against Lipopolysaccharide- Induced Acute Mastitis and Mammary Barrier Functions in Mice

SONG MeiJie1(),OU AiQun1,2,XUE XiaoFeng1,WU LiMing1,SHOU QiYang3,WANG Kai1()   

  1. 1Institute of Apicultural Research, Chinese Academy of Agricultural Sciences, Beijing 100093
    2College of Animal Sciences (College of Bee Science), Fujian Agriculture and Forestry University, Fuzhou 350000
    3Second Clinical Hospital of Zhejiang Chinese Medical University, Hangzhou 310027
  • Received:2020-05-06 Accepted:2020-11-25 Online:2021-06-16 Published:2021-06-24
  • Contact: Kai WANG

摘要:

【背景】奶牛乳腺炎是一种常见、多发的奶牛疾病,不仅危害奶牛健康,同时也造成重大经济损失。传统治疗奶牛乳腺炎的药物主要是抗生素,极易造成抗生素耐药与滥用。开发替代抗生素的方法用于奶牛乳腺炎的防治具有现实意义。蜂胶是西方蜜蜂采集植物树脂,混合自身上颚腺、蜡腺的分泌物形成的具有良好抗炎、抗菌活性的天然产物,对防治奶牛乳腺炎具有潜在开发利用价值。尽管近年来国内外有一些有关蜂胶防治乳腺炎方面的报道,但蜂胶如何影响乳血屏障功能,目前国内外尚无相关研究报道。【目的】利用细菌脂多糖(lipopolysaccharides, LPS)诱导的小鼠急性乳腺炎模型,评估中国蜂胶乙醇提取物对小鼠急性乳腺炎的保护作用及其对乳腺细胞紧密连接蛋白表达的影响,为后续利用蜂胶防治奶牛乳腺炎的深入研究奠定理论基础。【方法】采用超高效液相色谱串联三重四级杆质谱(UHPLC-QqQ-MS/MS)对中国蜂胶乙醇提取物中主要的多酚类化合物进行种类和含量测定。试验分为空白对照组、LPS模型组、中国蜂胶提取物试验组以及地塞米松阳性对照组。ICR雌性小鼠连续灌胃中国蜂胶提取物或阳性对照药物7d后,模型组、试验组和阳性对照组经第四、五对乳头管注射1 mg·kg-1体重 LPS,建立小鼠急性乳腺炎模型,24 h后处死,收集乳腺组织样本。以苏木精-伊红(H.E)染色法、天狼星红染色法评估小鼠乳腺组织病理变化;利用酶联免疫吸附法(ELISA)测定炎症因子释放量;利用荧光定量PCR测定小鼠乳腺组织中紧密连接蛋白(OccludinCluadin-1ZO-1) mRNA的表达情况;最后利用免疫组化技术研究小鼠乳腺紧密连接蛋白(Occludin和ZO-1)表达和分布情况,利用以上指标综合评判中国蜂胶乙醇提取物的抗乳腺炎活性及其对乳腺紧密连接蛋白的影响。【结果】基于UHPLC-QqQ-MS/MS,建立了中国蜂胶乙醇提取物中主要的17种多酚类化合物的精确定量方法,检测结果表明含量最高的5种化合物及其含量分别为:高良姜素(12.88±0.57 μg·mg-1)、短叶松素3-乙酸酯(12.93±0.59 μg·mg-1)、松属素(8.56±0.27 μg·mg-1)和短叶松素(8.52±0.25 μg·mg-1)。动物试验结果表明,灌胃中国蜂胶提取物能够显著缓解LPS注射导致的乳腺组织中炎性细胞浸润、缓解乳腺腺泡结构损伤;同LPS组相比,中国蜂胶提取物灌胃处理可有效抑制LPS导致的小鼠乳腺组织中炎症因子(IL-1β,IL-6和IL-10)的释放,并可提高小鼠乳腺中紧密连接蛋白的mRNA表达,缓解LPS注射所导致的乳腺上皮细胞紧密连接的破坏。【结论】中国蜂胶提取物对脂多糖诱导小鼠乳腺炎具有良好的预防效果,并可有效促进乳腺紧密连接蛋白的表达,维持乳腺中紧密连接结构完整性,保护血乳屏障,但蜂胶对紧密连接影响调控的分子机理还需进一步深入研究。

关键词: 中国蜂胶, 脂多糖, 乳腺炎, 血乳屏障

Abstract:

【Background】 Dairy cow mastitis is a common and frequent dairy cow disease, which not only threatens the health of dairy cows, but also causes significant economic losses. The traditional medicine for the treatment of dairy cow mastitis is largely dependent on the usage of antibiotics, which easily caused antibiotic resistance/abuse. Therefore, it is with practical significance to develop an alternative approach of antibiotics for the prevention and treatment of mastitis in dairy cows. Propolis is a natural product with good anti-inflammatory and antibacterial activities. Propolis is collected by western honeybees from plant resin and mixing with bees’ maxillary gland and wax gland secretions. Accordingly, propolis has great potential for the prevention and treatment of mastitis in dairy cows. Despite that there have some previous reports on the prevention and treatment of mastitis using propolis, scant information is available on the effects of propolis on the breast blood barrier function. 【Objective】The aim of this study was to evaluate the protective effect of ethanol extract of Chinese propolis, EECP, against bacterial lipopolysaccharide- induced mouse acute mastitis, and we focused on its effect on the expression of tight junction proteins in mammary epithelial cells. This outcome of this study would lay a foundation for the in-depth study for the usage of propolis for prevention and treatment on dairy cow mastitis. 【Method】 The type and content of the main polyphenolic compounds in EECP were determined by ultra-performance liquid chromatography tandem triple quadrupole mass spectrometry (UHPLC-QqQ-MS/MS). Female ICR mice were divided into four groups, including the normal control group, model group (injecting LPS into the mammary gland, 1mg·kg-1 b.w.), EECP group and positive control group (dexamethasone). The female mice were subjected to continuous gavage by EECP or positive control drugs for seven days. The LPS model group, EECP group and positive control group were injected with 1 mg·kg-1 LPS through the fourth and the fifth pair of nipple tubes, to induce acute mastitis in mice. After 24 h, they were killed for collecting the mammary tissue samples. Hematoxylin-Eosin (HE) staining method and Sirius red staining method were used to evaluate pathological changes in mouse mammary tissue. Enzyme-linked immunosorbent assay (ELISA) was used to determine the release of inflammatory factors and quantitative real-time PCR was applied to determine mouse mammary tissue gene expressions of tight junction proteins (Occludin, Cluadin-1, ZO-1). Finally, the immunohistochemical technology was used to study the expression and distribution of mouse mammary tight junction proteins (Occludin and ZO-1). These above indicators were applied to comprehensively evaluate the anti-mastitis activity and its effect on mammary tight junction proteins by EECP. 【Result】 Based on UHPLC-QqQ-MS/MS, an accurate quantitative method was established for the 17 main polyphenolic compounds in EECP. The results showed that the five compounds with the higher content and their contents were: galangin (12.88 ±0.57 μg·mg-1), 3-O-Acetylpinobanksin (12.93±0.59 μg·mg-1), pinocembrin (8.56±0.27 μg·mg-1) and pinobanksin (8.52±0.25 μg·mg-1). Animal study results showed that after a week of regular oral administration of EECP reduced on the infiltrations of inflammatory cells in mammary gland under LPS stimulation, and the structural damages were alleviated by EECP. Moreover, Chinese propolis administration inhibited the overexpression of inflammatory factors (IL-1β, IL-6, IL-10) in mouse mammary gland tissue, and increased the transcription level of tight junction protein genes (Occludin, Claudin-1, ZO-1) mRNA, and enhanced the expression of tight junction protein Occludin, ZO-1. 【Conclusion】EECP showed good preventive effect against lipopolysaccharide-induced mastitis in mice, with potent anti-inflammatory effects. It also increased the expressions of tight junction proteins in the mammary gland, which maintained the integrity of the tight junction structure in the mammary gland and protected the blood-milk barrier. Nevertheless, the molecular mechanism of Chinese propolis on the regulation of tight junctions needed to be clarified in the future research.

Key words: Chinese propolis, lipopolysaccharide, mastitis, blood-milk barrier