Animal Nutrition Institute, Sichuan Agricultural University, Key Laboratory for Animal Disease-Resistance Nutrition of China, Ministry of Education, Chengdu 611130, China.
Metallomics. 2017 Nov 15;9(11):1562-1575. doi: 10.1039/c7mt00191f.
Vanadium is a metal of high physiological, environmental and industrial importance. However, vanadium-induced oxidative stress can reduce the egg quality of poultry, and be potentially harmful to humans, and the underlying mechanism is not clear. In this study, we investigated the underlying relationship between the oxidant-sensitive mitogen-activated protein kinase (MAPK) signaling pathway and vanadium-induced oxidative stress in oviduct magnum epithelial (OME) cells. Cultured OME cells were treated with 100 μmol L vanadium and/or MAPK inhibitors [P38 MAPK inhibitor, SB203580; extracellular regulated protein kinase 1 and 2 (ERK1/2) inhibitor, U0126; c-JUN N-terminal kinases (JNK) inhibitor, SP600125]. Cell viability, apoptosis, and generation of reactive oxygen species (ROS) were assessed using flow cytometry. The expression of oxidative stress-related genes and their proteins was measured by reverse transcription-polymerase chain reaction and western blotting. Vanadium treatment reduced cell viability, whereas pretreated OME cells with SB203580 and U0126 prevented the reducing effect of vanadium on cell viability (P < 0.05). Likewise, MAPK inhibitors effectively suppressed vanadium-induced apoptosis and ROS generation (P < 0.05). In the OME cells treated with vanadium, SB203580 (P < 0.05) and SP600125 (P = 0.08) increased catalase activity by 89.3% and 55.3%; SB203580 and U0126 increased (P < 0.05) glutathione peroxidase activity by 44.9% and 51.1%, respectively. Incubation of OME cells with MAPK inhibitors also prevents malondialdehyde concentration increase and lactic dehydrogenase activity decrease in response to vanadium (P < 0.05). Vanadium downregulated P38, ERK1/2, JNK, Nrf2, sMaf, GCLC, NQO1 and HO-1 mRNA expression (P < 0.05). In contrast, inhibition of JNK with SP600125 upregulated P38, ERK1/2, JNK, Nrf2, GCLC and HO-1 mRNA expression (P < 0.05); inhibition of P38 with SB203580 upregulated JNK, NQO1 and HO-1 mRNA expression (P < 0.05); and inhibition of ERK1/2 with U0126 upregulated ERK1/2, GCLC and HO-1 mRNA expression (P < 0.05). Moreover, phosphorylation of P38, ERK1/2, JNK, and Nrf2 proteins was enhanced by V incubation; however, SP600125 blocked the phosphorylation of these proteins, whereas SB203580 blocked the phosphorylation of P38 and Nrf2. These results indicate that vanadium inducing oxidative stress in OME cells might be, at least, associated with the phosphorylation of the P38MAPK/JNK-Nrf2 pathway, which reduces the expression of phase II detoxifying enzymes.
钒是一种具有重要生理、环境和工业意义的金属。然而，钒诱导的氧化应激会降低家禽的蛋品质，并且对人类可能有害，其潜在机制尚不清楚。在这项研究中，我们研究了氧化剂敏感丝裂原活化蛋白激酶（MAPK）信号通路与输卵管峡部上皮（OME）细胞中钒诱导的氧化应激之间的潜在关系。用 100μmol L 钒和/或 MAPK 抑制剂[P38 MAPK 抑制剂 SB203580；细胞外调节蛋白激酶 1 和 2（ERK1/2）抑制剂 U0126；c-JUN N 末端激酶（JNK）抑制剂 SP600125]处理培养的 OME 细胞。通过流式细胞术评估细胞活力、细胞凋亡和活性氧（ROS）的生成。通过逆转录聚合酶链反应和 Western blot 测量与氧化应激相关的基因及其蛋白的表达。钒处理降低了细胞活力，而用 SB203580 和 U0126 预处理 OME 细胞可防止钒对细胞活力的降低作用（P<0.05）。同样，MAPK 抑制剂有效抑制了钒诱导的细胞凋亡和 ROS 生成（P<0.05）。在接受钒处理的 OME 细胞中，SB203580（P<0.05）和 SP600125（P=0.08）分别将过氧化氢酶活性提高了 89.3%和 55.3%；SB203580 和 U0126 分别将谷胱甘肽过氧化物酶活性提高了 44.9%和 51.1%（P<0.05）。孵育 OME 细胞也可防止 MAPK 抑制剂对抗钒诱导的丙二醛浓度增加和乳酸脱氢酶活性降低（P<0.05）。钒下调 P38、ERK1/2、JNK、Nrf2、sMaf、GCLC、NQO1 和 HO-1mRNA 表达（P<0.05）。相比之下，用 SP600125 抑制 JNK 上调 P38、ERK1/2、JNK、Nrf2、GCLC 和 HO-1mRNA 表达（P<0.05）；用 SB203580 抑制 P38 上调 JNK、NQO1 和 HO-1mRNA 表达（P<0.05）；用 U0126 抑制 ERK1/2 上调 ERK1/2、GCLC 和 HO-1mRNA 表达（P<0.05）。此外，P38、ERK1/2、JNK 和 Nrf2 蛋白的磷酸化在 V 孵育时增强；然而，SP600125 阻断了这些蛋白的磷酸化，而 SB203580 阻断了 P38 和 Nrf2 的磷酸化。这些结果表明，钒在 OME 细胞中诱导氧化应激可能至少与 P38MAPK/JNK-Nrf2 途径的磷酸化有关，该途径降低了 II 期解毒酶的表达。
