Scientia Agricultura Sinica ›› 2021, Vol. 54 ›› Issue (12): 2675-2688.doi: 10.3864/j.issn.0578-1752.2021.12.017

• ANIMAL SCIENCE·VETERINARY SCIENCE·RESOURCE INSECT • Previous Articles     Next Articles

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 E-mail:17853483688@163.com;kaiwang628@gmail.com

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

Fig. 1

Mouse mammary lipopolysaccharide injection"

Table 1

RNA reverse transcription reaction system"

试剂
Re agent
体积
Volume (μL)
PrimeScript RT Enzyme Mix 2
Total RNA 2
RNase Free DH2O 加至10 Add up to 10

Table 2

The primer sequence of related genes in RT-qPCR"

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

Table 3

Real-time fluorescent quantitative PCR reaction system"

成分 Components 体积 Volume (μL)
TB green 5
PCR上游引物 Forward primers 0.2
PCR下游引物 Reverse primers 0.2
cDNA 0.2
ddH2O 4.4
Total 10

Table 4

Recoveries and precisions of the 17 polyphenols in Chinese poplar propolis"

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)

Table 5

Major polyphenolic constituents in EECP"

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

Fig. 2

Histopathological changes of mouse mammary glands induced by LPS based on H&E staining (×100) A: Normal control group; B: LPS injection model group; C: Positive control (Dexamethasone) group; D: Chinese propolis ethanolic extract treatment group. The same as Fig.3, Fig.6 and Fig.7"

Fig. 3

Histopathological changes of mouse mammary glands induced by LPS based on picrosirius red staining (×100)"

Fig. 4

Inflammatory cytokines in the mammary gland with mastitis by ELISA Data in the figure are the mean±SD, One-way ANOVA. * Different letters in the same row means significant difference between the treatments(P<0.05), same letter in the same row means not significant difference between treatments (P>0.05). The same as Fig.5"

Fig. 5

Effect of EECP on mRNA transcription levels of tight junction genes"

Fig. 6

IHC analysis on the Occludin expression of mouse mammary glands induced by LPS (×100)"

Fig. 7

IHC analysis on the ZO-1 expression of mouse mammary glands induced by LPS (×100)"

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