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Punicalagin protects bovine endometrial epithelial cells against lipopolysaccharide-induced inflammatory injury

安石榴苷对脂多糖诱导奶牛子宫内膜上皮细胞炎症损伤的保护作用

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Abstract

Objective

Bovine endometritis is one of the most common reproductive disorders in cattle. The aim of this study was to investigate the anti-inflammation potential of punicalagin in lipopolysaccharide (LPS)-induced bovine endometrial epithelial cells (bEECs) and to uncover the underlying mechanisms.

Methods

bEECs were stimulated with different concentrations (1, 10, 30, 50, and 100 μg/ml) of LPS for 3, 6, 9, 12, and 18 h. MTT assay was used to assess cell viability and to identify the conditions for inflammatory injury and effective concentrations of punicalagin. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to assess gene expression of pro-inflammatory cytokines. Western blotting was used to assess levels of inflammation-related proteins.

Results

Treatment of bEECs with 30 μg/ml LPS for 12 h induced cell injury and reduced cell viability. Punicalagin (5, 10, or 20 μg/ml) pretreatment significantly decreased LPS-induced productions of interleukin (IL)-1β, IL-6, IL-8, and tumor necrosis factor-α (TNF-α) in bEECs. Molecular research showed that punicalagin inhibited the activation of the upstream mediator nuclear factor-κB (NF-κB) by suppressing the production of inhibitor κBα (IκBα) and phosphorylation of p65. Results also indicated that punicalagin can suppress the phosphorylation of mitogen-activated protein kinases (MAPKs) including p38, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK).

Conclusions

Punicalagin may attenuate LPS-induced inflammatory injury and provide a potential option for the treatment of dairy cows with Escherichia coli endometritis.

摘要

目的

评估安石榴苷对脂多糖诱导奶牛子宫内膜上皮细 胞炎症损伤的保护作用,并初步探讨其作用机 制。

创新点

首次证明安石榴苷对脂多糖诱导奶牛子宫内膜上 皮细胞炎症损伤具有保护作用,且此作用与核转 录因子κB(NF-κB)和丝裂原活化蛋白激酶 (MAPK)信号通路的抑制相关。

方法

用不同浓度的脂多糖(1、10、30、50 和100 μg/ml) 刺激奶牛子宫内膜上皮细胞3、6、9、12 和18 h, 筛选出建立炎症损伤的最佳作用浓度和时间。 安石榴苷预处理细胞2 h 后用脂多糖刺激12 h, 用逆转录聚合酶链式反应(RT-PCR)检测炎症 因子白细胞介素1β(IL-1β)、白细胞介素6(IL-6)、 白细胞介素8(IL-8)及肿瘤坏死因子α(TNF-α) 的表达。用蛋白质免疫印迹试验(Western blotting) 的方法检测核因子κB 抑制蛋白α(IκBα)、 磷酸化的p65、p38、c-Jun 氨基末端激酶(JNK) 和细胞外调节蛋白激酶(ERK)的表达水平。

结论

MTT 结果显示,30 μg/ml 脂多糖刺激奶牛子宫内 膜上皮细胞12 h能够造成细胞活力下降和形态改 变(图2 和3)。RT-PCR 结果显示,安石榴苷预 处理后炎症因子显著降低(图4)。Western blotting 结果显示,安石榴苷预处理能显著抑制IκBα 降 解以及p65、p38、JNK 和ERK 的磷酸化表达水 平(图5 和6)。综上所述,安石榴苷对脂多糖 诱导奶牛子宫内膜上皮细胞炎症损伤具有保护 作用,在治疗奶牛子宫内膜炎中具有重要价值。

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Acknowledgments

We are thankful for help from the members of China Agricultural University-Beijing University of Agriculture Traditional Chinese Veterinary Medicine (CAU-BUA TCVM) teaching and research team.

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Authors and Affiliations

  1. Beijing Key Laboratory for Dairy Cow Nutrition, College of Animal Science and Technology, Beijing University of Agriculture, Beijing, 102206, China

    An Lyu, Jia-jia Chen, Hui-chuan Wang, Xiao-hong Yu, Zhi-cong Zhang, Ping Gong, Lin-shu Jiang & Feng-hua Liu

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Correspondence to Lin-shu Jiang or Feng-hua Liu.

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Project supported by the National Key Technology R & D Program of China (No. 2013BAD10B04) and the Importation and Development of High-Caliber Talents Project of Beijing Municipal Institutions (No. CIT&TCD20130324), China

ORCID: An LYU, http://orcid.org/0000-0002-3268-5193

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Lyu, A., Chen, Jj., Wang, Hc. et al. Punicalagin protects bovine endometrial epithelial cells against lipopolysaccharide-induced inflammatory injury. J. Zhejiang Univ. Sci. B 18, 481–491 (2017). https://doi.org/10.1631/jzus.B1600224

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