Huang Yihao, Mo Shangkun, Jin Yi, Zheng Zhuoning, Wang Hanyi, Wu Shaojuan, Ren Zhuqing, Wu Jian
College of Animal Sciences & Technology/College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China.
College of Animal Sciences & Technology/College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China; Hubei Hongshan Laboratory, Wuhan 430070, China.
Ecotoxicol Environ Saf. 2022 Sep 15;243:114006. doi: 10.1016/j.ecoenv.2022.114006. Epub 2022 Aug 26.
Ammonia is one of the most important toxic metabolites in the intestine of animals. It can cause intestinal damage and associated intestinal diseases through different endogenous or exogenous stimuli. However, the definition of harmful ammonia concentration and the molecular mechanism of ammonia - induced intestinal epithelial injury remain unclear. In this study, we found that the viability of porcine IPEC-J2 intestinal epithelial cells significantly decreased with the increase of NHCl dose (20-80 mM). Ammonia (40 mM NHCl) increased the expression level of ammonia transporter RHCG and disrupted the intestinal barrier function of IPEC-J2 cells by reducing the expression levels of the tight junction molecules ZO-1 and Claudin-1. Ammonia caused elevated levels of ROS and apoptosis in IPEC-J2 cells. This was manifested by decreased activity of antioxidant enzymes SOD and GPx, decreased mitochondrial membrane potential, and increased cytoplasmic Ca concentration. In addition, the expression levels of apoptosis-related molecules Caspase-9, Caspase-3, Fas, Caspase-8, p53 and Bax were increased, the expression level of anti-apoptotic molecule Bcl-2 was decreased. Moreover, the antioxidant NAC (N-acetyl-L-cysteamine) effectively alleviated ammonia-induced cytotoxicity, reduced ROS level, Ca concentration, and the apoptosis of IPEC-J2 cells. The results suggest that ammonia-induced excess ROS triggered apoptosis through mitochondrial pathway, death receptor pathway and DNA damage. This study can provide reference and theoretical basis for the definition of harmful ammonia concentration in pig intestine and the effect and mechanism of ammonia on pig intestinal health.
氨是动物肠道中最重要的毒性代谢产物之一。它可通过不同的内源性或外源性刺激导致肠道损伤及相关肠道疾病。然而，有害氨浓度的定义以及氨诱导肠道上皮损伤的分子机制仍不清楚。在本研究中，我们发现随着NHCl剂量（20 - 80 mM）的增加，猪IPEC-J2肠道上皮细胞的活力显著降低。氨（40 mM NHCl）增加了氨转运体RHCG的表达水平，并通过降低紧密连接分子ZO-1和Claudin-1的表达水平破坏了IPEC-J2细胞的肠道屏障功能。氨导致IPEC-J2细胞中活性氧（ROS）水平升高和细胞凋亡。这表现为抗氧化酶超氧化物歧化酶（SOD）和谷胱甘肽过氧化物酶（GPx）的活性降低、线粒体膜电位降低以及细胞质钙离子（Ca）浓度升高。此外，凋亡相关分子半胱天冬酶-9（Caspase-9）、半胱天冬酶-3（Caspase-3）、Fas、半胱天冬酶-8（Caspase-8）、p53和Bax的表达水平升高，抗凋亡分子Bcl-2的表达水平降低。而且，抗氧化剂N-乙酰-L-半胱氨酸（NAC）有效减轻了氨诱导的细胞毒性，降低了ROS水平、钙离子浓度以及IPEC-J2细胞的凋亡。结果表明，氨诱导的过量ROS通过线粒体途径、死亡受体途径和DNA损伤触发细胞凋亡。本研究可为猪肠道中有害氨浓度的定义以及氨对猪肠道健康的影响和机制提供参考和理论依据。
