畜牧兽医学报 ›› 2023, Vol. 54 ›› Issue (11): 4805-4816.doi: 10.11843/j.issn.0366-6964.2023.11.034

• 临床兽医 • 上一篇    下一篇

桔青霉素诱导小鼠肠道屏障功能障碍的机制研究

杨梦然1,2, 杨成林1,2, 吴悠1,2, 王思琪1,2, 孔祥祎1,2, 宁灿1,2, 肖文广1,2, 范慧1,2, 邬静1,2*, 袁志航1,2*   

  1. 1. 湖南农业大学动物医学院, 长沙 410128;
    2. 湖南农业大学 畜禽保健湖南省工程研究中心, 长沙 410128
  • 收稿日期:2023-01-31 出版日期:2023-11-23 发布日期:2023-11-26
  • 通讯作者: 袁志航,主要从事兽医药理与毒理学研究,E-mail:zhyuan2016@hunau.edu.cn;邬静,主要从事动物中毒病与营养保健研究,E-mail:wujing@hunau.edu.cn
  • 作者简介:杨梦然(1998-),女,湖南邵东人,硕士,主要从事兽医药理与毒理学研究,E-mail:ymr980218@stu.hunau.edu.cn
  • 基金资助:
    国家自然科学基金(31802238;31572563);湖南省自然科学基金(2021JJ30343)

Study on the Mechanism of Citrinin Induced Intestinal Barrier Dysfunction in Mice

YANG Mengran1,2, YANG Chenglin1,2, WU You1,2, WANG Siqi1,2, KONG Xiangyi1,2, NING Can1,2, XIAO Wenguang1,2, FAN Hui1,2, WU Jing1,2*, YUAN Zhihang1,2*   

  1. 1. College of Veterinary Medicine, Hunan Agricultural University, Changsha 410128, China;
    2. Hunan Engineering Research Center of Livestock and Poultry Health Care, Hunan Agricultural University, Changsha 410128, China
  • Received:2023-01-31 Online:2023-11-23 Published:2023-11-26

摘要: 旨在探讨内质网应激在桔青霉素诱导肠道屏障功能障碍中的作用。试验选用24只初始体重为(27.8±1.5)g的7周龄昆明小鼠,随机分为4组,分别为对照组(5%乙醇)和桔青霉素低(1.25 mg·kg-1)、中(5 mg·kg-1)、高(20 mg·kg-1)剂量组。适应性饲养1周后,对照组按0.1 mL·10g-1体重灌胃5%乙醇,毒素处理组分别灌胃不同剂量桔青霉素,持续14 d。试验结束后,采血并分离血清,测量小肠长度并收集小鼠空肠样本。HE染色观察肠道病理变化;ELISA检测血清中二胺氧化酶(diamine oxidase,DAO)、D-乳酸(D-lactic acid,D-LA)、内毒素(endotoxin,ET)的含量;检测空肠组织抗氧化酶的活性及活性氧(reactive oxygen species, ROS)的释放。为进一步探究内质网应激在桔青霉素诱导肠道损伤中的作用,选用32只昆明小鼠随机分为4组,分别为对照组(5%乙醇)、桔青霉素中剂量(5 mg·kg-1)组、内质网应激抑制剂4-苯基丁酸苯基丁酸(4-Phenylbutyric acid,4-PBA)(240 mg·kg-1)组和桔青霉素(5 mg·kg-1)+4-PBA(240 mg·kg-1)组。4-PBA经腹腔注射预处理小鼠1 h后,用桔青霉素对小鼠进行灌胃处理。14 d后,检测空肠组织病理损伤,氧化损伤,细胞凋亡情况及内质网应激相关蛋白表达,并用Spearman相关性分析确定内质网应激信号通路与肠道氧化应激、肠道屏障功能障碍之间的相关性。结果显示,不同剂量的桔青霉素会造成肠道组织损伤、氧化应激及屏障功能障碍。与桔青霉素中剂量组相比,4-PBA预处理能提高小鼠空肠总超氧化物歧化酶(total superoxide dismutase, T-SOD)、过氧化氢酶(catalase, CAT)的活性和总抗氧化能力(total antioxidant capacity, T-AOC),降低ROS和丙二醛(malondialdehyde, MDA)含量,抑制细胞凋亡及内质网应激,降低血清中DAO、D-LA和ET的含量,升高空肠组织中紧密连接蛋白ZO-1、Occludin和 Claudin-1 mRNA 表达,缓解桔青霉素诱导的空肠组织损伤,且内质网应激信号通路与肠道氧化应激及肠道屏障功能障碍之间存在显著相关性。综上表明,内质网应激在桔青霉素诱导的空肠屏障功能障碍中具有重要调控作用。

关键词: 桔青霉素, 内质网应激, 氧化应激, 细胞凋亡, 肠道屏障功能障碍

Abstract: The objective of this study was to investigate the role of endoplasmic reticulum stress (ERS) on citrinin-induced intestinal barrier dysfunction. Twenty-four 7-week-old Kunming mice weighed (27.8±1.5) g were divided into 4 groups randomly: control group(5% ethanol), citrinin of low-(1.25 mg·kg-1), medium-(5 mg·kg-1) and high-(20 mg·kg-1) dose groups. After conventionally raised for one week, the mice in control group were administrated orally by 5% ethanol according to 0.1 mL·10 g-1 body weight and the mice in toxin groups were exposed to different dosage of citrinin for 14 days. At the end of the experiment, the blood was collected, the serum was separated, the length of small intestine was measured and the jejunum samples were collected. The pathological changes of intestine were observed by HE staining, while the content of diamine oxidase (DAO), D-lactic acid(D-LA) and endotoxin (ET) were detected by ELISA. Besides, the activities of antioxidative enzymes and the release of reactive oxygen species (ROS) of jejunum were examined. In order to explore the role of ERS on citrinin-induced intestinal injury, 32 Kunming mice were randomly divided into 4 groups: control group(5% ethanol), medium-dose citrinin group (5 mg·kg-1), 4-PBA group (240 mg·kg-1) and citrinin + 4-PBA group. The mice were exposed to citrinin after intraperitoneal injection of 4-PBA for 1 h. The pathologic injury, oxidative damage, apoptosis and the expression of ER stress related proteins in jejunum were tested after 14 days. Besides, Spearman correlation analysis was used to determine the correlation between ERS signaling pathway and intestinal oxidative stress as well as intestinal barrier dysfunction. The results showed that different dosage of citrinin could trigger intestinal damage, oxidative stress and intestinal barrier dysfunction. Pretreated with 4-PBA up-regulated total antioxidant capacity (T-AOC) and the activities of catalase (CAT), total superoxide dismutase (T-SOD), decreased the production of ROS and malondialdehyde (MDA), inhibited cell apoptosis and ERS, down-regulated the content of DAO, D-LA and ET, increased the mRNA expression of ZO-1, Occludin and Claudin-1, finally relieved the damage of jejunum caused by citrinin. In addition, the endoplasmic reticulum stress signaling pathway was significantly correlated with intestinal oxidative stress and intestinal barrier dysfunction. In summary, the study indicated that ERS plays an important regulatory role in citrinin-caused barrier dysfunction of jejunum.

Key words: citrinin, endoplasmic reticulum stress, oxidative stress, apoptosis, intestinal barrier dysfunction

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