中国农业科学 ›› 2021, Vol. 54 ›› Issue (12): 2675-2688.doi: 10.3864/j.issn.0578-1752.2021.12.017
宋美洁1(),欧爱群1,2,薛晓锋1,吴黎明1,寿旗扬3,王凯1(
)
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:基金资助:
SONG MeiJie1(),OU AiQun1,2,XUE XiaoFeng1,WU LiMing1,SHOU QiYang3,WANG Kai1(
)
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(Occludin、Cluadin-1、ZO-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。
宋美洁,欧爱群,薛晓锋,吴黎明,寿旗扬,王凯. 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[J]. 中国农业科学, 2021, 54(12): 2675-2688.
SONG MeiJie,OU AiQun,XUE XiaoFeng,WU LiMing,SHOU QiYang,WANG Kai. Protective Effects of Chinese Propolis Extract Against Lipopolysaccharide- Induced Acute Mastitis and Mammary Barrier Functions in Mice[J]. Scientia Agricultura Sinica, 2021, 54(12): 2675-2688.
表2
实时荧光定量PCR相关引物序列"
基因 Genes | 引物序列 Primer sequence (5′-3′) | 产物长度 Product length (bp) |
---|---|---|
Occludin | F: TCTGCTTCATCGCTTCCTTAG R: GTCGGGTTCACTCCCATTA | 160 |
ZO-1 | F: ACTCCCACTTCCCCAAAAAC R: CCACAGCTGAAGGACTCACA | 166 |
Cluadin-1 | F: AGACCTGGATTTGCATCTTGGTG R: TGCAACATAGGCAGGACAAGAGTTA | 126 |
GAPDH | F: GAGAAACCTGCCAAGTATGATGAC R: TAGCCGTATTCATTGTCATACCAG | 212 |
表4
中国蜂胶乙醇提取物中代表性多酚类化合物的回收率和精密度"
多酚类化合物 Polyphenolic compound | 平均回收率(RSD,n=5) Recovery rate (RSD, n-5) (%) |
---|---|
咖啡酸Caffeic Acid | 100.6-105.6(1.35-3.87) |
p-香豆酸 p-Coumaric acid | 84.6-96.3(0.70-2.10) |
阿魏酸 Ferulic Acid | 92.7-100.4(1.42-3.90) |
咖啡酸苯乙酯 Caffeic acid phenethyl ester | 83.2-101.7(0.96-2.89) |
芹菜素 Apigenin | 86.8-93.4(0.62-3.13) |
柯因Chrysin | 90.3-97.8(0.93-1.91) |
槲皮素 Quercetin | 95.9-98.1(1.01-3.15) |
高良姜素 Galangin | 98.2-117.4(1.23-1.49) |
桑色素 Morin | 70.2-91.7(2.02-6.30) |
松属素 Pinocembrin | 104.5-120.1(0.61-1.73) |
短叶松素 Pinobanksin | 99.1-100.4(0.45-5.11) |
芦丁Rutin | 78.3-85.7(2.03-4.12) |
短叶松素三乙酸酯 Pinobanksin-3-acetate | 96.6-111.2(3.13-6.12) |
杨梅素 Myricetin | 74.6-80.8(2.87-4.40) |
香草酸 Vanillic acid | 81.5-94.2(0.61-3.16) |
柚皮素 Naringenin | 105.2-113.0(0.71-2.72) |
3,4-二甲氧基肉桂酸 3,4-Dimethoxycinnamic acid | 99.4-102.6(0.99-4.67) |
表5
中国蜂胶乙醇提取物中主要的多酚类物质成分"
多酚类化合物 Polyphenolic compound | 分子式 Molecular formula | 保留时间 Rt (min) | 母离子 Parention | 含量 Content (μg·mg-1) |
---|---|---|---|---|
咖啡酸Caffeic Acid | C9H8O4 | 5.487 | 179.035 | 3.23±0.03 |
p-香豆酸 p-Coumaric acid | C9H8O3 | 6.109 | 163.0401 | - |
阿魏酸 Ferulic Acid | C10H10O4 | 6.304 | 193.0506 | - |
咖啡酸苯乙酯 Caffeic acid phenethyl ester | C17H16O4 | 9.503 | 283.0976 | 2.49±0.06 |
芹菜素 Apigenin | C15H10O5 | 7.718 | 269.0455 | 1.68±0.08 |
柯因Chrysin | C15H10O4 | 9.363 | 253.0506 | 7.71±1.01 |
槲皮素 Quercetin | C15H10O7 | 7.225 | 301.0354 | 1.51±0.05 |
高良姜素 Galangin | C15H10O5 | 9.547 | 269.0455 | 12.88±0.57 |
桑色素 Morin | C15H10O7 | 6.919 | 301.0354 | - |
松属素 Pinocembrin | C15H12O4 | 9.461 | 255.0663 | 8.56±0.27 |
短叶松素 Pinobanksin | C15H12O5 | 8.000 | 271.0612 | 8.52±0.25 |
芦丁Rutin | C27H30O16 | 5.599 | 609.1461 | - |
短叶松素三乙酸酯 Pinobanksin-3-acetate | C17H14O6 | 9.609 | 313.0718 | 12.93±0.59 |
杨梅素 Myricetin | C15H10O8 | 6.580 | 317.0303 | 0.81±0.06 |
香草酸 Vanillic acid | C8H8O4 | 5.597 | 167.035 | 0.72±0.01 |
柚皮素 Naringenin | C15H12O5 | 7.767 | 271.0612 | - |
3,4-二甲氧基肉桂酸 3,4-Dimethoxycinnamic acid | C11H12O4 | 7.120 | 207.0663 | 3.49±1.23 |
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