【作者】 刘鹏；

【导师】 孟庆义；

【作者基本信息】 中国人民解放军军医进修学院 ， 急诊医学， 2012， 博士

【摘要】 自十九世纪六十年代以来，百草枯已经成为许多国家广泛使用的除草剂。从七十年代初开始，研究发现百草枯（PQ）对人类和动物具有较强的毒性。百草枯的中毒机制主要是在机体内经过氧化还原过程产生大量的活性氧类物质(ROS)，破坏过氧化-抗氧化平衡，引起脂质、蛋白和DNA的氧化损伤，导致细胞、组织功能障碍。虽然在百草枯中毒的患者中广泛应用了甲泼尼龙，但疗效不明确。由于缺乏特效的解毒剂或有效的治疗方法，百草枯中毒患者死亡率高。尽管人们已经意识到氧化应激在百草枯中毒机制中的重要性，但关于百草枯中毒患者体内总抗氧化态的变化及给予大剂量甲泼尼龙治疗的影响等方面的研究非常有限。本研究旨在评估百草枯中毒小鼠的血清总抗氧化态（TAS）变化及大剂量应用甲泼尼龙的作用。实验主要由以下几部分构成：第一部分小鼠腹腔百草枯染毒后血清总抗氧化态变化的研究目的：本部分研究旨在探讨小鼠腹腔注射百草枯对其血清总抗氧化态的影响。方法：小鼠随机分为对照组和百草枯染毒组（PQ组），PQ组小鼠又分别按照50、70、100、150、200和300mg/kg的百草枯染毒剂量分成6组，给予一次性腹腔注射百草枯；PQ组小鼠分别在染毒后1h和3h留取血样，进行血清2,2-联氮-双(3-乙基-苯并噻唑-6-磺酸)二铵盐﹝ABTS﹞自由基清除率的检测，并与对照组比较；每个血样采集点均有5个样本。结果：1、不同染毒剂量组小鼠染毒后1、3h的血清ABTS自由基清除率显著低于对照组小鼠（P＜0.05）；2、但同一染毒剂量染毒后1、3h测定的血清ABTS自由基清除率无显著性差异（P＞0.05）；3、小鼠染毒后1、3h的血清ABTS自由基清除率与染毒剂量间无显著相关性。结论：百草枯染毒后小鼠的血清ABTS自由基清除率下降，血清总抗氧化态降低，且染毒后1、3h无显著性差异。第二部分百草枯染毒小鼠早期应用甲泼尼龙对血清总抗氧化态的影响目的：本部分研究旨在评估百草枯染毒小鼠早期应用大剂量甲泼尼龙（medrol）对小鼠血清总抗氧化态的影响。方法：小鼠被随机分成对照组、PQ组、PQ+medrol组和medrol组。PQ组小鼠按照一次性腹腔注射50、70、100、150、200和300mg/kg的百草枯染毒剂量分成6组；PQ+medrol组小鼠在腹腔分别一次性注射百草枯50、70、100、150、200和300mg/kg后即刻注射medrol200mg/kg；medrol组小鼠仅给予腹腔一次性注射medrol200mg/kg；PQ组、PQ+medrol组和medrol组小鼠在染毒后1h和3h取血样，进行血清ABTS自由基清除率的检测，并与对照组比较；每个血样采集点均有5个样本。结果：1、PQ组与PQ+medrol组小鼠1、3h的血清ABTS自由基清除率均显著低于对照组（P＜0.05）2、同一染毒剂量的PQ组与PQ+medrol组小鼠同一采血时点测定的血清ABTS自由基清除率比较无显著性差异（P＞0.05）；3、medrol组小鼠在1、3h血清ABTS自由基清除率与对照组比较无显著性差异（P＞0.05）；4、PQ+medrol组小鼠1、3h的血清ABTS自由基清除率与染毒剂量间无明显相关性。结论：本实验研究证实了百草枯对机体过氧化-抗氧化平衡的破坏作用，未发现早期大剂量应用甲泼尼龙可以提高染毒小鼠的血清抗氧化态。第三部分甲泼尼龙可能加速急性百草枯中毒小鼠死亡的初步研究目的：本部分研究拟对急性百草枯中毒小鼠进行甲泼尼龙冲击治疗，明确糖皮质激素能否加速急性百草枯中毒小鼠的死亡。方法：小鼠随机分为对照组、染毒组、治疗组和甲泼尼龙对照组。染毒组30只小鼠被随机分为6组，分别一次性腹腔注射50、70、100、150、200和300mg/kg剂量的百草枯；治疗组小鼠30只，在与染毒组相同处理的基础上，百草枯染毒后一次性腹腔注射甲泼尼龙200mg/kg；甲泼尼龙对照组5只小鼠，仅给予一次性腹腔注射甲泼尼龙200mg/kg；观察小鼠的中毒症状，比较染毒组和治疗组小鼠的半数致死量。结果：给予百草枯后的不同时间内，小鼠出现自由活动减少，濒死时呼吸频率增快、张口呼吸、鼻翼扇动等中毒症状。治疗组在染毒后24h、48h和72h的半数致死量均小于染毒组。甲泼尼龙对照组小鼠在观察期内未出现死亡。结论：腹腔注射大剂量甲泼尼龙有可能加速百草枯中毒小鼠的死亡。更多还原

【Abstract】 Paraquat (PQ) has been a widely used herbicide in many countries since the1960s.From70s, its high toxicity has been reported in animals as well as in human.Mthylprednisolone is widely practiced, but evidence for efficacy is very weak.The highmortality rate observed following paraquat exposure has been attributed to the lack ofan antidote or effective treatment to ameliorate the toxic effects of the poison. Paraquatinduced toxicity is a manifestation of its ability to undergo an alternative reduction andreoxidation known as redox cycling and subsequent generation of reactive oxygenspecies(ROS), which disrupt the prooxidative/antioxidative balance in the body，causeoxidative damage to lipids, proteins, and DNA,and lead to cell and tissueDysfunction.Despite recognizing the importance of oxidative stress as a mechanism ofparaquat toxicity,limited studies exist regarding the variation of total antioxidant status（TAS） and the effect of high doses of methylprednisolone administration in patientswith paraquat poisoning.This study was designed to evaluate the serum TAS and theaction of high doses of methylprednisolone in mice with paraquat poisoning.PartⅠThe study on changes in serum total antioxidant status of micepoisoned with paraquat by ip injectionObjective: The purpose of this study was to evaluate the serum TAS of micepoisoned with paraquat by ip injection.Methods: The mice were randomLy divided into control groups and PQgroups.The mice in PQ groups were divided into6subgroups, which received50,70,100,150,200,and300mg/kg, respectively, of paraquat by i.p. injection. The bloodsamples were taken not only from control group but also from PQ group at1h and3h,respectively, after exposure,in order for the comparative study of ABTS radicalscavenging activity. Blood samples of5mice were taken at each blood sampling point.Results:1.The serum ABTS free radical scavenging activities at either1h or3h after PQ administration in PQ subgroups were significantly lower than in controlgroup（P＜0.05）.2. There was no significant difference in the serum ABTS free radicalscavenging activities in blood samples between the two studied sampling times afterexposure to the same dose of PQ（P＞0.05）.3.There was no significant correlationbetween the exposure dose and the serum ABTS free radical scavenging activities ateither1h or3h after PQ administration.Conclusion: The serum ABTS free radical scavenging activities were reduced andthe serum total antioxidant status was lowed for the mice after PQ administration. Thedifference was not significant between1h value and3h value after PQ administration.PartⅡThe effescts of early use of methylprednisolone on the serum totalantioxidant status in the mice with paraquat poisoningObjective: This study was designed to evaluate the effescts of early use ofmethylprednisolone on the serum total antioxidant status in the mice with paraquatpoisoning.Methods: The mice were randomLy divided into control group,PQ group, PQ+medrol group and medrol group. The mice in PQ group were divided into6subgroups,which received50,70,100,150,200,and300mg/kg, respectively, of paraquat by i.p.injection. The mice in PQ+medrol group all received200mg/kg of methylprednisoloneby i.p. injection immediately after receiving50,70,100,150,200,and300mg/kg,respectively, of paraquat by i.p. injection. The mice in medrol group only received200mg/kg of methylprednisolone by i.p. injection. The blood samples were taken notonly from control group but also from PQ group, PQ+medrol group and medrol group at1h and3h,respectively, after exposure for the comparative study of ABTS radicalscavenging activity. Blood samples of5mice were taken at each blood sampling point.Results:1.The serum ABTS free radical scavenging activities at either1h or3hafter PQ administration in PQ group and PQ+medrol group were significantly lowerthan in control group（P＜0.05）.2. There was no significant difference in the serumABTSfree radical scavenging activities in blood samples between1h value and3h value afterexposure to the same dose of PQ for PQ group and PQ+medrol group，respectively（P ＞0.05）.3.There was no significant difference in the serum ABTS free radical scavengingactivities in blood samples between control group and medrol group at1h and3h,respectively after exposure to PQ（P＞0.05）.4.In PQ+medrol group, there was nosignificant correlation between the exposure dose and the serum ABTS free radicalscavenging activities at either1h or3h after PQ administration.Conclusion:The study confirmed paraquat can disrupt the peroxidation/antioxidantbalance in the body, but it failed to prove that the early use of high doses ofmethylprednisolone can improve the serum total antioxidant status of the mice with PQpoisoning.PartⅢThe high-dose methylprednisolone could accelerate mortality in micewith acute paraquat poisoning:Apreliminary StudyObjective: In this study, the effects of methylprednisolone pulse therapy on micewith acute paraquat poisoning were evaluated,in order to determine if glucocorticoidscould accelerate mortality in the mice with acute paraquat poisoning.Methods:30mice in poisoning group were randomLy divided into6groups,which received50,70,100,150,200,and300mg/kg/d, respectively, of paraquat by i.p.injection. After recieving the same treatment as poisoning group,each of30mice intreatment group received200mg/kg of methylprednisolone by i.p. injection.5mice inMedrol control group only received200mg/kg of methylprednisolone by i.p. injection.Symptoms of poisoning in mice were observed and the LD50values between poisoninggroup and treatment group were compared.Results: The times o f movement of mice reduced after paraquat administration.Before death, the mice had toxicosis symptoms of a low respirator y rate，gasping breathand flapping of nose wing, and so on.The LD50values in treatment group were smallerthan that in poisoning group at either24,48or72h after exposure to paraquat.Conclusion: A single intraperitoneal injection of high-dose methylprednisolonecould accelerate mortality in mice with paraquat poisoning.更多还原