分子与细胞营养 MOLECULAR AND CELLULAR NUTRITION

灵芝多糖对大肠杆菌感染IPEC-1细胞免疫功能的影响

  • 翟俊磊 ,
  • 林颖 ,
  • 王美艳 ,
  • 高玉云 ,
  • 王长康 ,
  • 金灵
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  • 1. 福建农林大学动物科学学院(蜂学学院), 福州 350002;
    2. 福建农林大学国家菌草工程技术研究中心, 福州 350002
翟俊磊(1995-),男,河南驻马店人,硕士研究生,研究方向为动物营养与饲料科学。E-mail:1146063270@qq.com

收稿日期: 2021-05-06

  网络出版日期: 2021-12-16

基金资助

福建省自然科学基金项目(2018J01599);福建省教育厅科技类项目(JAT170176)

Effects of Ganoderma lucidum Polysaccharides on Immune Function of IPEC-1 Cells Infected with Escherichia coli

  • ZHAI Junlei ,
  • LIN Yin ,
  • WANG Meiyan ,
  • GAO Yuyun ,
  • WANG Changkang ,
  • JIN Ling
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  • 1. College of Animal Sciences(College of Bee Science), Fujian Agriculture and Forest University, Fuzhou 350002, China;
    2. China National Engineering Research Center of Juncao Technology, Fujian Agriculture and Forestry University, Fuzhou 350002, China

Received date: 2021-05-06

  Online published: 2021-12-16

摘要

本试验旨在探究灵芝多糖对IPEC-1细胞增殖、周期和免疫功能的影响,为灵芝多糖在仔猪肠道健康方面的应用提供理论依据。试验分为空白组和不同浓度(5、10、20、50、100、200、400和800 μg/mL)灵芝多糖组,CCK-8法检测IPEC-1细胞活力,利用PI染色和流式细胞仪检测灵芝多糖对IPEC-1细胞周期的影响。利用肠出血性大肠杆菌(EHEC)构建的IPEC-1细胞炎症模型,设空白组、EHEC组以及不同浓度(5、10、20、50和100 μg/mL)灵芝多糖+EHEC组,测定灵芝多糖处理IPEC-1细胞后对EHEC的抑菌活性;通过酶联免疫吸附测定(ELISA)技术检测IPEC-1细胞上清液中白细胞介素-6(IL-6)、白细胞介素-1β(IL-1β)、肿瘤坏死因子-α(TNF-α)、干扰素-γ(IFN-γ)和白细胞介素-10(IL-10)的分泌量;荧光定量PCR技术检测IPEC-1细胞中IL-6、IL-1βTNF-αIL-10 mRNA相对表达量。结果显示:1)与空白组相比,灵芝多糖作用IPEEC-1细胞48 h后细胞活力下降,且24 h时细胞活力优于12 h时,故选择24 h作为最佳作用时间。灵芝多糖作用24 h,浓度为5~100 μg/mL时细胞活力优于浓度为200~800 μg/mL时。2)5、10、20、50和100 μg/mL灵芝多糖可显著抑制EHEC的增殖;50和100 μg/mL的灵芝多糖处理IPEC-1细胞24 h,显著提高细胞抗EHEC活性(P<0.05)。3)与空白组相比,EHEC组的IL-1β、IL-6和IFN-γ分泌量显著上升(P<0.05),IL-10分泌量显著下降(P<0.05);与EHEC组相比,5、10、20、50和100 μg/mL灵芝多糖+EHEC组的IL-6、TNF-α和IFN-γ分泌量显著降低(P<0.05),20、50和100 μg/mL灵芝多糖+EHEC组的IL-1β分泌量显著降低(P<0.05),5和50 μg/mL灵芝多糖+EHEC组IL-10分泌量显著上升(P<0.05)。4)与空白组相比,EHEC组的IL-1βTNF-α mRNA相对表达量显著上升(P<0.05);与EHEC组相比,50和100 μg/mL灵芝多糖+EHEC组IL-1β mRNA相对表达量显著下降(P<0.05)。由上述结果可知,灵芝多糖能抑制大肠杆菌的增殖,缓解IPEC-1细胞的炎症反应,增强细胞的免疫功能。

本文引用格式

翟俊磊 , 林颖 , 王美艳 , 高玉云 , 王长康 , 金灵 . 灵芝多糖对大肠杆菌感染IPEC-1细胞免疫功能的影响[J]. 动物营养学报, 2021 , 33(12) : 7118 -7130 . DOI: 10.3969/j.issn.1006-267x.2021.12.051

Abstract

To explore the effects of Ganoderma lucidum polysaccharides on the proliferation, cycle and immune function of IPEC-1 cells, and to provide theoretical basis for the application of Ganoderma lucidum polysaccharides in intestinal health of piglets. The experiment was divided into blank group and different concentrations (5, 10, 20, 50, 100, 200, 400 and 800 μg/mL) of Ganoderma lucidum polysaccharides groups. The cell viability of IPEC-1 cells was detected by CCK-8 method, and the effects of Ganoderma lucidum polysaccharides on the cell cycle of IPEC-1 cells was detected by PI staining and flow cytometry. IPEC-1 cell inflammation model was constructed by enterohemorrhagic Escherichia coli (EHEC) and was used for subsequent experiments, which were divided into blank group, EHEC group, and different concentrations (5, 10, 20, 50 and 100 μg/mL) of Ganoderma lucidum polysaccharides+EHEC groups. The antimicrobial activity of Ganoderma lucidum polysaccharides treated IPEC-1 cells against EHEC was determined. The secretion amounts of interleukin-6 (IL-6), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ) and interleukin-10 (IL-10) in the supernatant of IPEC-1 cells were determined by enzyme-linked immunosorbent assay (ELISA) method. The mRNA relative expression levels of IL-6, IL-1β, TNF-α and IL-10 in IPEC-1 cells were detected by fluorescence quantitative PCR. The results showed as follows:1) compared with the blank group, the cell viability decreased after 48 h of Ganoderma lucidum polysaccharides treatment, and the cell viability at 24 h was better than that at 12 h, so 24 h was selected as the best action time. Cell viability at 5, 10, 20, 50 and 100 μg/mL was better than 200, 400 and 800 μg/mL. 2) Ganoderma lucidum polysaccharides with the concentrations of 5, 10, 20, 50 and 100 μg/mL could significantly inhibit the proliferation of EHEC; IPEC-1 cells treated with 50 and 100 μg/mL Ganoderma lucidum polysaccharides for 24 h significantly improved the anti-EHEC activity (P<0.05). 3) Compared with the blank group, the secretion amounts of IL-1β, IL-6 and IFN-γ in EHEC group were significantly increased (P<0.05), while the secretion amount of IL-10 was significantly decreased; compared with the EHEC group, the secretion amounts of IL-6, TNF-α and IFN-γ in the 5, 10, 20, 50 and 100 μg/mL Ganoderma lucidum polysaccharides+EHEC groups was significantly decreased (P<0.05), the secretion amounts of IL-1β in the 20, 50 and 100 μg/mL Ganoderma lucidum polysaccharides+EHEC group were significantly decreased (P<0.05), and the secretion amount of IL-10 in 5 and 50 μg/mL Ganoderma lucidum polysaccharides+EHEC groups was significantly increased (P<0.05). 4) Compared with the blank group, the mRNA relative expression levels of IL-1β and TNF-α in EHEC group were significantly increased (P<0.05); compared with EHEC group, the mRNA relative expression levels of IL-1β in 50 and 100 μg/mL Ganoderma lucidum polysaccharides+EHEC groups was significantly decreased (P<0.05). It is concluded that Ganoderma lucidum polysaccharides can inhibit the proliferation of Escherichia coli, alleviate the inflammatory response of IPEC-1 cells and enhance the cellular immune function.

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