【作者】 李云鹏；

【导师】 董兆君；

【作者基本信息】 第三军医大学 ， 军事预防医学， 2008， 博士

【摘要】 我国高原地区幅员辽阔,低压缺氧是高原地区的主要环境特征,大部队进驻高原容易产生缺氧反应,严重时可导致肺水肿、脑水肿等急性疾病。氢氰酸是速杀性化学战剂,能强烈抑制细胞呼吸链功能,造成组织氧利用障碍和能量产生减少。高原缺氧条件下氰类毒剂中毒时,氰化物毒性增加,死亡率大幅度提高,救治药物的药效也显著下降,其机制尚不清楚。由于缺氧和氰化物的作用靶点都是机体内氧利用和能量产生过程的关键环节,推测可能是缺氧导致动物对氰化物更为敏感,或者是氰化物代谢异常所致。一氧化氮是一种重要的第二信使,其参与的病理生理过程有血管舒张、血小板聚集、凋亡及神经传递。而细胞色素氧化酶（cytochrome C oxidase,COX）是细胞呼吸链上的重要递氢体,氰化物就是主要通过抑制COX的活力而导致中毒。研究发现,一氧化氮通过抑制COX活力调控细胞呼吸。而且,抑制效力取决于氧气与一氧化氮的相对浓度。近期研究发现,大鼠海拔5000m连续减压15d,COX活力持续降低。而我们先期的研究业已证实,高原4000m缺氧和氰化钠中毒较之平原单纯氰化钠中毒,COX活力降低更为明显,其机制不明。因此,我们设想一氧化氮可能在调控COX的活性中起重要作用,本研究旨在为高原缺氧条件下氰化物的中毒救治提供理论依据。因此,本研究采用低压舱模拟高原缺氧环境,研究缺氧和氰化钠中毒对COX的影响机制和干预措施的效果。结果:1.本研究通过测定家兔和大鼠的血氰浓度,对比研究了平原和模拟高原缺氧条件下氰离子的体内过程规律。主要毒代学参数如下:平原条件下,t_1/2为58.174,AUC（0-t）为126.388 mg/（L·min）,AUC_（0-∞）:168.566 mg/（L·min）,CL/F为0.013L/min/kg,V/F为0.945L/kg,MRT值为46.193。模拟高原条件下:t_1/2为61.116,AUC（0-t）为209.554mg/（L·min）,AUC_（0-∞）值为340.074 mg/（L·min）CL/F为0.007L/（min·kg）,V/F为0.58L/kg,MRT（0-t）值为51.142。同时观察了与氰离子代谢相关因素在缺氧条件下的变化特点。结果提示:（1）模拟高原缺氧条件下氰化钠中毒血红蛋白含量升高;（2）高铁血红蛋白含量变化不明显（P＞0.05）;（3）尿中硫氰酸盐排出受抑制;（4）COX、硫氰酸生成酶及3-巯基-丙酮酸硫转移酶活性降低。2.肝、肾病理学研究显示:模拟高原缺氧条件下氰化钠中毒导致的肝、肾损伤重于平原,平原组大鼠氰化钠中毒肝细胞以充血、淤血、颗粒样变为主要病变,在中毒6小时基本恢复。肾脏病变以肾小球充血、水肿,肾小管胞浆嗜酸性变为主要病变,高原重于平原,且病变持续加重,不易恢复;3.Western blot实验分析结果显示,与对照组比较,高原缺氧和氰化钠中毒COX亚基Ⅰ、Ⅳ蛋白表达明显升高,NRF-1蛋白表达降低。4.RT-PCR检测结果显示:（1）高原缺氧和氰化钠中毒及人参皂甙干预组COX I mRNA的转录水平明显上调（P＜0.05）;（2）平原氰化钠中毒组COXⅣmRNA表达增高（P＜0.01）;（3）氰化钠中毒大鼠NRF-1 mRNA均降低,高原氰化钠中毒牛磺酸干预组降低明显（P＜0.05）。5.平原及高原氰化钠中毒大鼠肝脏总NOS活性降低;iNOS活性升高,平原iNOS活性升高更明显。平原NO含量升高并在中毒1h达到峰值,高原NO含量先升高后降低。6.与平原组比较,氯霉素、L-NAME干预氰化钠中毒大鼠肝脏COX活性均出现了降低,氯霉素干预COX活性下降更为明显。7.牛磺酸、人参皂甙单独及复方使用均可拮抗氰化钠中毒所致COX活性降低。结论1.模拟高原缺氧条件下氰化钠在体内的代谢动力学呈一室模型。缺氧明显影响氰化钠在体内的代谢过程,氰化物代谢异常可能是高原氰化钠毒性增加的主要原因。2.光镜及超微病理均提示高原组病理损伤持续加重,不易恢复。3.高原缺氧和氰化钠中毒可抑制肝脏COX活性,减少肝脏NO的产生。4.高原缺氧和氰化钠中毒导致COXⅠ和COXⅣ蛋白水平上调,NRF-1蛋白表达下调。表明高原缺氧和氰化钠中毒可促进COX亚基Ⅰ、Ⅳ蛋白的合成。5.L-NAME干预对COX活性有保护作用。6.牛磺酸、人参皂甙单独及联合使用均可减弱氰化钠中毒所致COX活性降低,有一定的保护作用。更多还原

【Abstract】 China is a vast plateau region. Hypobaric hypoxia at high altitude is the main feature of the environment. Hypoxia response can lead to severe pulmonary edema, cerebral edema and other acute diseases when the troops enter high altitude area. Hydrocyanic acid is a fast-killing chemical warfare agent, which can strongly inhibit cell respiratory chain function, causing obstacles to the use of oxygen and generate energy reduction. Cyanide toxicity, mortality has been greatly improved induced by cyanide administration under the condition of plateau hypoxia. Meanwhile, treatment efficacy of the drug decreased significantly but the poisonous mechanism remains unclear. To understand the reason, we proposed that hypoxia and cyanide intoxication may lead to hypoxia animals are more sensitive to the abnormal metabolism of cyanide ion.Nitric Oxide（NO） participates in the pathological and physiological processes of vasodilatation, platelet aggregation, apoptosis and neural transmission as an important second messenger. Cytochrome oxidase is an essential hydrogen carrier in cell respiration chain. Cyanide is mainly through inhibiting the activity of cytochrome oxidase resulting in poisoning. It has been found that NO regulates the cell respiration by inhibiting the activity of cytochrome oxidase. Moreover, the effect of inhibition depends on the relative concentration between oxygen and NO. Researchers found that the cytochrome oxidase activity continued reducing in rats under the condition of decompression continuously 15days on 5000m above sea level. Our previous research has confirmed that the activity of cytochrome oxidase reduced more pronounced at 4000m high altitude hypoxia combined sodium cyanide poisoning compared with the plain poisoning, with the mechanism unknown. Therefore we suppose the NO may play an important role in regulating the activity of cytochrome oxidase. The purpose of this study is to provide a theoretical evidence for the treatment of cyanide poisoning under plateau hypoxia. In view of this, the low-pressure oxygen cabin simulated plateau environment was used to study the effects of plateau hypoxia combined NaCN intoxicatin on COX and the role of preconditioning.Results: 1.The aim of this study was to measure the blood cyanide ion concentration of rabbits and rats under the condition of plateau hypoxia for understanding its metabolism mechanism. The main toxicokinetics parameters for plain toxicant group were as follows: t_1/2 （58.174min）, AUC_0～t（ 126.388 mg/（L·min））, AUC（0～∞）168.566 mg/（L·min）, CL/F（0.013L/min/kg）, V/F（0.945L/kg） and MRT（0～t） value （46.193）. For plateau group were: t_1/2 （61.116）, AUC_0～t（209.554mg/L·min）, AUC_0～∞ （340.074 mg/（L·min））,CL/F（0.007L/min/kg）, V/F（0.58L/kg） and MRT（0～t） value（51.142）. Correlative factors of metabolism were also investigated under hypobaric hypoxia. Compared with plain NaCN intoxication group, we found that （1） hypoxia combined with NaCN intoxication induced hemoglobin concentration enhancement; （2） MHb content did not change significantly （P>0.05）; （3） Urinary thiocyanate discharge was inhibited;（4） the activity of cytochrome oxidase, rhodanese and 3-MST decreased. 2. Pathologic diagnosis showed that liver and kidney injury induced by NaCN intoxication at high altitude was more serious than in plain intoxicant. 3. Compared with control group, Western blot experimental results suggested that the expression of COX I and COX IV protein markedly elevated. Meanwhile, NRF-1 protein expression decreased. 4. RT-PCR results revealed that: （1）The transcriptional level of COX I mRNA for plateau hypoxia combined NaCN intoxicant and Saponius of Panax Ginseng preconditioning group was up-regulation （P<0.05）; （2） The transcriptional level of COX IV mRNA for NaCN plain intoxication group enhanced （P<0.01）; （3） NRF-1 mRNA expression level decreased for all group.Furthermore,plateau hypoxia combined NaCN intoxication treated with taurine preconditioning decreased significantly（P<0.05）. 5. Both plain and plateau NaCN administration produced the decrease of total NOS activity and the increase of iNOS activity of rat liver. The activity of iNOS at 308m increased more significantly than at 4000m. NO concentration of plain group reached a peak value at 1h. However, the NO concentration of plateau group increased at the time point of 30min, and then decreased. 6. Compared with plain group, chloramphenicol and L-NAME preconditioning produced the decrease of COX activity, and the former declined more significantly. 7. Taurine and Saponius of Panax Ginseng used alone or compound can be antagonistic to the decrease of COX acitivity induced by NaCN intoxication.Conclusion 1.Under the condition of plain and plateau environment, the pharmacokinetics of rabbits induced by NaCN injection was characterized by one-compartment model. Hypoxia could markedly disturb the metabolism process of NaCN in vivo. Cyanide metabolic abnormalities may be mainly account for the increase of cyanide toxicity at high altitude. 2.Light microscope and ultrastructural pathological study results suggested that pathology damage sustained increase, not easy to restore. 3. Plateau hypoxia combined NaCN intoxication can affect the COX activity, and inhibit NO production of the liver. 4. Plateau hypoxia combined NaCN intoxication resulted in up-regulation of COX I and COX IV protein expression level, and the down regulation of NRF-1 protein expression. It showed that plateau hypoxia combined NaCN intoxication promote the synthesis of COX I and COX IV protein. 5. L-NAME played a protective role on the COX activity.6. Taurine and Saponius of Panax Ginseng used alone or compound had a protective effect on the decrease of COX induced by NaCN intoxication.更多还原