研究论文 RESEARCHPAPER

基于肿瘤坏死因子信号通路探讨槲皮素对小鼠乳腺炎模型的作用机制

  • 王桃 ,
  • 曹璐 ,
  • 米晓钰 ,
  • 吕琛 ,
  • 赵兴绪 ,
  • 张勇
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  • 1. 甘肃农业大学动物医学院, 兰州 730070;
    2. 甘肃省动物生殖生理及繁殖调控重点实验室, 兰州 730070
王桃(1997—),女,甘肃兰州人,硕士研究生,研究方向为兽医产科学。E-mail:w1745157739@163.com

收稿日期: 2021-11-07

  网络出版日期: 2022-06-14

基金资助

甘肃省引导科技创新专项基金资助项目(GSCXZX-2019-1);甘肃省科技厅重点研发-农业类(18YF1NA074);宁夏自治区重点研发(2019BBF02027)

Mechanism of Quercetin on Mice Mastitis Model Based on Tumor Necrosis Factor Signaling Pathway

  • WANG Tao ,
  • CAO Lu ,
  • MI Xiaoyu ,
  • LYU Chen ,
  • ZHAO Xingxu ,
  • ZHANG Yong
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  • 1. College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, China;
    2. Gansu Provincial Key Laboratory of Animal Reproductive Physiology and Reproductive Regulation, Lanzhou 730070, China

Received date: 2021-11-07

  Online published: 2022-06-14

摘要

本试验旨在探讨槲皮素对金黄色葡萄球菌致小鼠乳腺炎的肿瘤坏死因子(TNF)信号通路机制。将90只母鼠按照解剖时间(第3、5、7天)分为3个大组,每组共30只母鼠,再将各大组母鼠分为空白对照组(注射生理盐水)、模型组(注射菌液)及槲皮素高(200 mg/kg)、中(100 mg/kg)、低(50 mg/kg)剂量组,共5个小组,每小组各6只母鼠,分别在治疗第3、5、7天采血,检测血液生理指标,随后颈椎脱臼处死,取乳腺组织,通过苏木精-伊红(HE)染色观察病理组织的变化,实时荧光定量PCR (qRT-PCR)检测核因子抑制蛋白α(IKBα)、IκB激酶β(IKKβ)及核转录因子-κB (NF-κB) mRNA相对表达量,蛋白免疫印迹检测肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)、IKKβ及NF-κB蛋白表达量。结果表明:1)与空白对照组相比,模型组血液白细胞数量极显著升高(P<0.01),乳腺组织病变严重,乳腺组织中IKBαIKKβNF-κB的mRNA相对表达量显著升高(P<0.05)。2)与空白对照组相比,高剂量组NF-κBIKBα的mRNA相对表达量差异不显著(P>0.05),高剂量组IKKβ的mRNA相对表达量在治疗第3、7天差异不显著(P>0.05)。蛋白免疫印迹结果显示,与空白对照组相比,高剂量组TNF-α蛋白相对表达量差异不显著(P>0.05),高剂量组IL-6、IKKβ、NF-κB蛋白相对表达量在治疗第3天差异不显著(P>0.05),在治疗第5、7天差异显著(P<0.05)。综上所述,槲皮素可以通过抑制TNF信号通路中IKBαIKKβNF-κB mRNA相对表达量及TNF-αIL-6、IKKβ、NF-κB蛋白相对表达量来发挥作用,且随着槲皮素添加剂量的升高,其治疗效果也越好,呈现出明显的剂量依赖性。在整个疾病的发生发展过程中,符合疾病发展的规律,在治疗第7天时,基本进入转归期,所有指标基本恢复正常。

本文引用格式

王桃 , 曹璐 , 米晓钰 , 吕琛 , 赵兴绪 , 张勇 . 基于肿瘤坏死因子信号通路探讨槲皮素对小鼠乳腺炎模型的作用机制[J]. 动物营养学报, 2022 , 34(6) : 3972 -3982 . DOI: 10.3969/j.issn.1006-267x.2022.06.056

Abstract

The aim of this study was to investigate the mechanism of tumor necrosis factor (TNF) signaling pathway in mice mastitis induced by Staphylococcus aureus. Ninety female mice were dissected on the 3rd, 5th and 7th day and divided into 3 groups with 30 female mice in each group. Then, female mice in each group were divided into 5 groups:blank control group (injected with normal saline), model group (injected with bacterial solution) and quercetin high (200 mg/kg), medium (100 mg/kg) and low (50 mg/kg) dose groups. Blood samples of 6 female mice in each group were collected on days 3, 5 and 7 of treatment, respectively, to detect blood physiological indicators. Then cervical vertebra was dislocated and sacrificed. The mammary tissues were taken and pathological changes were observed by hematoxylin-eosin (HE) staining. The mRNA relative expression levels of nuclear factor inhibitory protein α (IKBα), IκB kinase β (IKKβ) and nuclear transcription factor-κB (NF-κB) were detected by real-time fluorescence quantitative PCR (qRT-PCR). The protein relative expression levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), IKKβ and NF-κB were detected by Western Blot. The results showed as follows:1) compared with blank control group, the number of white blood cells in model group was extremely significantly increased (P<0.01), the lesions of mammary tissue were serious, and the mRNA expression levels of IKBα, IKKβ and NF-κB in mammary tissue were significantly increased (P<0.05). 2) Compared with the blank control group, the mRNA relative expression levels of NF-κB and IKBα in the high dose group were not significantly different (P>0.05), and the mRNA relative expression level of IKKβ in the high dose group was not significantly different on days 3 and 7 of treatment (P>0.05). Western Blot results showed that there was no significant difference in the protein relative expression level of TNF-α between the blank control group and the high-dose group (P>0.05). The protein relative expression levels of IL-6, IKKβ and NF-κB were not significantly different on day 3 of treatment (P>0.05), but significantly different on days 5 and 7 of treatment (P<0.05). In conclusion, quercetin can inhibit the mRNA relative expression levels of IKBα, IKKβ, NF-κB and the protein relative expression levels of TNF-α, IL-6, IKKβ and NF-κB in TNF signaling pathway, and the therapeutic effect becomes better with the increase of quercetin dosage, presenting a significant dose-dependent effect. In the whole process of the occurrence and development of the disease, it conforms to the law of development of the disease. On day 7 of treatment, the period of conversion basically enter, and all indicators basically return to normal.

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