a Division of Pulmonary and Critical Care Medicine , Pennsylvania State University College of Medicine , Hershey , PA , USA.
b Department of Physiology , Pennsylvania State University College of Medicine , Hershey , PA , USA.
Clin Toxicol (Phila). 2018 Sep;56(9):828-840. doi: 10.1080/15563650.2018.1429615. Epub 2018 Feb 16.
Although methylene blue (MB) had long been proposed to counteract the effects of cyanide (CN) intoxication, research on its mechanisms of action and efficacy has been abandoned for decades. Recent studies on the benefits of MB in post-anoxic injuries have prompted us to reexamine the relevance of this historical observation.
Our study was performed in adult male Sprague-Dawley rats and on HEK293T epithelial cells. First, the effects and toxicity of MB (0-80 mg/kg) on circulation and metabolism were established in four urethane-anesthetized rats. Then nine rats received a lethal infusion of a solution of KCN (0.75 mg/kg/min) and were treated by either saline or MB, at 20 mg/kg, a dose that we found to be innocuous in rat and to correspond to a dose of about 4 mg/kg in humans. MB was also administered 5 min after the end of a sub-lethal exposure to CN in a separate group of 10 rats. In addition, ATP/ADP ratio, ROS production, mitochondrial membrane potential (Δψm) and cellular O consumption rate (OCR) were determined in HEK293T cells exposed to toxic levels of CN (200 µM for 10 min) before and after applying a solution containing MB (1-100 µM for 10 min).
Methylene blue was found to be innocuous up to 50 mg/kg. KCN infusion (0.75 mg/kg/min) killed all animals within 7-8 min. MB (20 mg/kg) administered at the same time restored blood pressure, cardiac contractility and limited O deficit, allowing all the animals to survive, without any significant methemoglobinemia. When administered 5 min after a non-lethal CN intoxication, MB sped up the recovery of lactate and O deficit. Finally, MB was able to decrease the production of ROS and restore the ATP/ADP ratio, Δψm as well as OCR of epithelial cells intoxicated by CN.
The present observations should make us consider the potential interest of MB in the treatment of CN intoxication. The mechanisms of the antidotal properties of MB cannot be accounted for by the creation of a cyanomethemoglobinemia, rather its protective effects appears to be related to the unique properties of this redox dye, which, depending on the dose, could directly oppose some of the consequences of the metabolic depression produced by CN at the cellular level.
尽管亚甲蓝（MB）长期以来被提议用于对抗氰化物（CN）中毒的影响，但对其作用机制和疗效的研究已被搁置了几十年。最近关于 MB 在缺氧后损伤中的益处的研究促使我们重新审视这一历史观察结果。
我们的研究在成年雄性 Sprague-Dawley 大鼠和 HEK293T 上皮细胞上进行。首先，在 4 只乌拉坦麻醉大鼠中确定了 MB（0-80mg/kg）对循环和代谢的作用和毒性。然后，9 只大鼠接受了致命剂量的 KCN（0.75mg/kg/min）输注，并接受了生理盐水或 20mg/kg 的 MB 治疗，我们发现该剂量对大鼠无害，相当于人类约 4mg/kg 的剂量。在另一组 10 只大鼠中，在亚致死暴露于 CN 结束后 5 分钟给予 MB。此外，在暴露于有毒水平的 CN（200µM 10 分钟）之前和之后，用含有 MB（1-100µM 10 分钟）的溶液处理 HEK293T 细胞，测定细胞内 ATP/ADP 比、ROS 产生、线粒体膜电位（Δψm）和细胞 O 消耗率（OCR）。
MB 高达 50mg/kg 时被发现是无害的。KCN 输注（0.75mg/kg/min）在 7-8 分钟内杀死了所有动物。同时给予 20mg/kg 的 MB 恢复了血压、心肌收缩力并限制了 O 不足，使所有动物存活下来，没有明显的高铁血红蛋白血症。当在非致死性 CN 中毒后 5 分钟给予 MB 时，MB 加速了乳酸和 O 不足的恢复。最后，MB 能够降低 ROS 的产生并恢复 CN 中毒上皮细胞的 ATP/ADP 比、Δψm 和 OCR。
目前的观察结果应该使我们考虑 MB 在治疗 CN 中毒中的潜在益处。MB 的解毒特性的机制不能用氰化高铁血红蛋白血症来解释，而是其保护作用似乎与这种氧化还原染料的独特特性有关，根据剂量的不同，它可以直接对抗 CN 在细胞水平上产生的代谢抑制的某些后果。
