College of Animal Science, Inner Mongolia Agricultural University, Hohhot, P.R. China.
J Anim Sci. 2018 Apr 14;96(4):1305-1316. doi: 10.1093/jas/sky037.
It is known that physiological overproduction of nitric oxide (NO) contributes to oxidative stress and inflammation. Our published studies indicated that vitamin A (VA) reduces NO-induced oxidative stress in bovine mammary epithelial cells (BMECs) by increasing antioxidant enzyme activities. However, the precise mechanism is unclear. The present study was conducted to examine the protective effects of VA on NO-induced damage to BMECs in vitro using diethylenetriamine nitric oxide (DETA-NO) as the NO donor and to explore the intracellular signaling mechanisms of VA that involve nuclear factor erythroid 2-related factor (Nrf2) and nuclear factor kappa-B (NF-κB). Subconfluent BMECs were divided into 10 treatment groups with 6 replicates per treatment and were cultured with dimethyl sulfoxide (DMSO, vehicle negative control) or 0, 0.05, 0.1, 0.2, 0.5, 1, 2, 3, or 4 μg/mL of VA for 24 h and then incubated in the absence or presence of DETA-NO (1,000 μmol/liter) and VA for an additional 6 h. The results showed that exposure to DETA alone decreased cell proliferation compared with the negative control. Pretreatment with VA promoted the proliferation of BMECs, increased the activities of antioxidative enzymes including selenoprotein glutathione peroxidase (GPx) and thioredoxin reductase (TrxR) and their gene and protein expression but decreased NO and interleukin 1 (IL-1) contents in a quadratic manner (P < 0.05). In addition, the expression of mRNA and protein of factors that are related to NF-κB or Nrf2 signaling pathways in BMECs were regulated by VA in a quadratic dose-dependent manner; VA at a concentration of 1 μg/mL exhibited the strongest effect. Together, these results suggest that VA promotes antioxidant functions of BMECs by regulating the synthesis of selenoproteins including GPx and TrxR and by reducing concentrations of IL-1 and NO in vitro by modulating Nrf2 and NF-κB signaling pathways.
已知一氧化氮（NO）的生理过度产生会导致氧化应激和炎症。我们已发表的研究表明，维生素 A（VA）通过增加抗氧化酶的活性来减少牛乳腺上皮细胞（BMEC）中由 NO 诱导的氧化应激。然而，确切的机制尚不清楚。本研究旨在使用二乙三胺五乙酸一氧化氮（DETA-NO）作为 NO 供体，在体外研究 VA 对 BMEC 中由 NO 诱导的损伤的保护作用，并探讨 VA 涉及核因子红细胞 2 相关因子（Nrf2）和核因子 kappa-B（NF-κB）的细胞内信号转导机制。亚汇合 BMEC 分为 10 个处理组，每个处理组有 6 个重复，并分别用二甲基亚砜（DMSO，阴性对照）或 0、0.05、0.1、0.2、0.5、1、2、3 或 4μg/mL 的 VA 培养 24 小时，然后在无或有 DETA-NO（1000μmol/L）和 VA 的情况下再培养 6 小时。结果表明，单独接触 DETA 会降低细胞增殖，与阴性对照相比。VA 的预处理促进了 BMEC 的增殖，增加了抗氧化酶的活性，包括硒蛋白谷胱甘肽过氧化物酶（GPx）和硫氧还蛋白还原酶（TrxR）及其基因和蛋白表达，但以二次方式降低了 NO 和白细胞介素 1（IL-1）的含量（P<0.05）。此外，VA 以二次剂量依赖性方式调节 BMEC 中与 NF-κB 或 Nrf2 信号通路相关的因子的 mRNA 和蛋白表达；VA 在 1μg/mL 的浓度下表现出最强的效果。综上所述，这些结果表明，VA 通过调节包括 GPx 和 TrxR 在内的硒蛋白的合成，并通过调节 Nrf2 和 NF-κB 信号通路来降低 IL-1 和 NO 的浓度，从而促进 BMEC 的抗氧化功能。
