Xu Haibin, Hao Shu, Gan Fang, Wang Hong, Xu Jing, Liu Dandan, Huang Kehe
College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, Jiangsu Province, China; Institute of Nutritional and Metabolic Disorders in Domestic Animals and Fowls, Nanjing Agricultural University, Nanjing 210095, Jiangsu Province, China.
College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, Jiangsu Province, China; Institute of Nutritional and Metabolic Disorders in Domestic Animals and Fowls, Nanjing Agricultural University, Nanjing 210095, Jiangsu Province, China.
Chem Biol Interact. 2017 Jun 25;272:107-116. doi: 10.1016/j.cbi.2017.05.016. Epub 2017 May 18.
Ochratoxin A (OTA) frequently contaminates a wide variety of food and feeds, inducing immune toxicity of animals. However, the underlying mechanism remains unclear. In the present study, porcine alveolar macrophages (PAMs) cell line 3D4/21 were chosen as a model, and treated by various concentrations of OTA. The results showed that OTA induced immune toxicity of PAMs in a dose-dependent manner, as demonstrated by cell viability, LDH release, Annexin V/PI staining, Bcl-2/Bax mRNA ratio, and pro-inflammatory cytokines expression. OTA increased intracellular ROS levels of PAMs by flow cytometry and confocal microscopy. NAC, a commonly used antioxidant, alleviated the OTA-induced increase of ROS production, apoptosis, and LDH release and decrease of cell viability. Furthermore, OTA enhanced the mRNA and protein expression of TLR4 and MyD88 and the phosphorylation of ERK1/2, p38, and NF-κB p65. Knockdown of TLR4 by using a TLR4-specific siRNA alleviated the OTA-induced immune toxicity. These data indicate that OTA induced immune toxicity via ROS-relative TLR4/MyD88 signaling pathway in PAMs.
赭曲霉毒素A（OTA）经常污染各种各样的食品和饲料，诱导动物产生免疫毒性。然而，其潜在机制仍不清楚。在本研究中，选用猪肺泡巨噬细胞（PAMs）细胞系3D4/21作为模型，并用不同浓度的OTA进行处理。结果表明，OTA以剂量依赖的方式诱导PAMs产生免疫毒性，这通过细胞活力、乳酸脱氢酶（LDH）释放、膜联蛋白V/碘化丙啶（Annexin V/PI）染色、Bcl-2/Bax mRNA比值以及促炎细胞因子表达得以证明。通过流式细胞术和共聚焦显微镜检测发现，OTA增加了PAMs细胞内活性氧（ROS）水平。常用抗氧化剂N-乙酰半胱氨酸（NAC）减轻了OTA诱导的ROS产生增加、细胞凋亡、LDH释放以及细胞活力降低。此外，OTA增强了Toll样受体4（TLR4）和髓样分化因子88（MyD88）的mRNA和蛋白表达以及细胞外信号调节激酶1/2（ERK1/2）、p38和核因子κB p65（NF-κB p65）的磷酸化。使用TLR4特异性小干扰RNA（siRNA）敲低TLR4可减轻OTA诱导的免疫毒性。这些数据表明，OTA通过ROS相关的TLR4/MyD88信号通路在PAMs中诱导免疫毒性。
