Luo Yan, Ma Huiming, Niu Shibo, Li Xu, Nie Lihong, Li Guanghua
Key Laboratory of Fertility Preservation and Maintenance of Ministry of Education, Ningxia Medical University, Yinchuan, Ningxia 750004, P.R China.
Institute of Basic Medical Sciences, Ningxia Medical University, Yinchuan, Ningxia 750004, P.R China.
Exp Ther Med. 2021 May;21(5):421. doi: 10.3892/etm.2021.9865. Epub 2021 Feb 25.
Stress induced by changes in the internal or external environment in humans and animals leads to intestinal epithelial damage, in a manner that is associated with impaired intestinal barrier function. However, the role of the stress hormone norepinephrine (NE) in impairments in barrier function remains poorly understood. In the present study, a rat heat-exposed model was used to observe changes in the tight junction proteins Occludin and zonula occludens-1 (ZO-1), in addition to those in protease-activated receptor 2 (PAR-2) and transient receptor potential ankyrin 1 channel (TRPA1) in colon. The levels of plasma NE were detected using an ELISA kit. Different concentrations of NE were used to culture the human colon cell line Caco-2 for 6 and 24 h to investigate the cell viability using Cell Counting Kit-8 assay, whilst the expression levels of Occludin, ZO-1, PAR-2 and TRPA1 were examined using western blotting and immunofluorescence in Caco-2 cells and immunohistrochemistry in rat colon tissues. Although there was no clear histological damage to the rat colonic mucosa, there were decreased expression levels of tight junction proteins Occludin and ZO-1 after heat exposure. In addition, PAR-2 expression was increased by heat exposure. It was found that TRPA1 expression was concentrated to the luminal surface of the colon in the heat exposed group compared with that in the control group. After the administration of increasing concentrations of NE for 6 h, treatment did not affect cell viability. Furthermore, after application of NE for 24 h, cell viability gradually increased as the NE concentration was elevated from 10 to 100 µM. However, no significant increase in viability was observed when the cells were treated with 120 and 160 µM NE. Occludin expression was decreased when 10 µM NE was applied for 6 or 24 h. By contrast, 60 µM NE significantly downregulated Occludin expression in the 6 h group, but caused an insignificant decrease in the 24 h group. It was found that ZO-1 expression was upregulated after treatment with 10 µM NE for 6 h, whilst downregulation was observed after treatment with 10 µM NE for 24 h. PAR-2 protein expression was increased after application of NE for both 6 and 24 h, but not after treatment with 60 µM NE. In addition, TRPA1 expression was not affected by the treatment of NE, but increased positive staining was observed on the luminal side of the mucosa, which appeared to be concentrated in the cells of the luminal side in the rat colon after heat exposure. Collectively, the present results suggested that expression of tight junction proteins Occludin and ZO-1, in addition to that of PAR-2, can be regulated by NE, which may contribute to impairments in barrier function observed during heat stress.
人类和动物体内或外部环境变化所诱导的应激会导致肠道上皮损伤，其方式与肠道屏障功能受损有关。然而，应激激素去甲肾上腺素（NE）在屏障功能损伤中的作用仍知之甚少。在本研究中，使用大鼠热暴露模型观察结肠中紧密连接蛋白闭合蛋白（Occludin）和闭合小带蛋白1（ZO-1）的变化，以及蛋白酶激活受体2（PAR-2）和瞬时受体电位锚蛋白1通道（TRPA1）的变化。使用酶联免疫吸附测定试剂盒检测血浆NE水平。用不同浓度的NE培养人结肠癌细胞系Caco-2 6小时和24小时，使用细胞计数试剂盒-8检测法研究细胞活力，同时使用蛋白质免疫印迹法和免疫荧光法检测Caco-2细胞中Occludin、ZO-1、PAR-2和TRPA1的表达水平，以及大鼠结肠组织中的免疫组织化学。虽然大鼠结肠黏膜没有明显的组织学损伤，但热暴露后紧密连接蛋白Occludin和ZO-1的表达水平降低。此外，热暴露使PAR-2表达增加。结果发现，与对照组相比，热暴露组中TRPA1表达集中在结肠的管腔表面。给予递增浓度的NE处理6小时后，处理不影响细胞活力。此外，给予NE处理24小时后，随着NE浓度从10 μM升高到100 μM，细胞活力逐渐增加。然而，当细胞用120 μM和160 μM NE处理时，未观察到活力显著增加。当应用10 μM NE处理6小时或24小时时Occludin表达降低。相比之下，60 μM NE在6小时组中显著下调Occludin表达，但在24小时组中导致的降低不显著。结果发现，用10 μM NE处理6小时后ZO-1表达上调，而用10 μM NE处理24小时后观察到下调。给予NE处理6小时和24小时后PAR-2蛋白表达均增加，但用60 μM NE处理后未增加。此外，TRPA1表达不受NE处理的影响，但在黏膜管腔侧观察到阳性染色增加，热暴露后大鼠结肠管腔侧细胞中似乎集中出现这种情况。总的来说，目前的结果表明，紧密连接蛋白Occludin和ZO-1以及PAR-2的表达可受NE调节，这可能导致热应激期间观察到的屏障功能损伤。
