酰胺类模药APAP在家蚕体内的代谢途径研究

【作者】 徐旭；

【导师】 徐世清； 司马杨虎；

【作者基本信息】 苏州大学 ， 发育生物学， 2013， 硕士

【摘要】 对乙酰氨基酚（APAP）是一种广泛应用于临床的退烧镇痛类药物，利用APAP成分及其衍生物开发新型退烧镇痛类药物的研究还在广泛进行。APAP也是临床和基础研究中肝损伤建模和酰胺类药物毒理研究的模式药物。本实验旨在研究APAP在家蚕体内的代谢及损伤情况，探索利用无脊椎模式动物家蚕在药物开发和毒理学研究领域实验动物替代的可能性。研究结果报告如下：1APAP在家蚕使用的剂量换算医学实验动物替代的基本问题之一是剂量互换。目前家蚕与小鼠和人类的剂量尚无可靠的换算公式，根据体表面积经验计算公式得出的APAP对家蚕经口暴露的LD50为1664.7mg/kg，与实验测得的LD502450.3~3226.85mg/kg，相差50%以上。通过系统调查APAP对5龄2日家蚕皓月品种的时间-剂量影响的死亡效应，获得APAP经口暴露的安全剂量为600mg/kg，损伤剂量为3600mg/kg，损伤临界剂量为1800mg/kg。2APAP在家蚕体内的组织代谢及产物以APAP对家蚕的安全剂量、损伤剂量和损伤临界剂量经口暴露皓月品种5龄2日幼虫。HPLC分析显示，APAP在家蚕体内的主要代谢产物是糖基化产物，其次是硫酸化产物，与小鼠体内高度相似。糖基化主要发生在消化管，而硫酸化代谢产物主要在血淋巴中生成，脂肪体不是APAP累积和正常代谢的场所。家蚕与小鼠对APAP代谢的不同点在于家蚕丝腺的作用。损伤剂量和损伤临界剂量经口暴露时，蚕体能够将APAP原药转运入丝腺储存，从而降低血淋巴和其它组织中的原药含量，并在血淋巴中APAP原药浓度低于0.2mg/L时向血淋巴开始释放。这在研究高浓度模药长时间作用，特别是研究模药对体内生殖腺等具有生理屏障组织的影响时具有特殊意义。3APAP暴露后组织中UGTs酶基因表达特征及酶活变化家蚕5龄2日幼虫的消化管和血淋巴中UGTs酶本底活性略高于脂肪体。安全剂量600mg/kgAPAP经口暴露家蚕皓月品种5龄2日幼虫，消化管和血淋巴中UGTs酶活性分别在给药后24min和16min有微弱上升，而脂肪体中的UGTs酶活性在给药后8min内上调了3倍，并在24min时超过对照6倍，暗示脂肪体的糖基化酶活性对APAP是敏感的。给药后Ugt30，Ugt86和Ugt89这3个基因在消化管和脂肪体中的转录本水平都显著上升，其中Ugt30和Ugt89基因上调表达更快速，在消化管中30min就上升至峰值，脂肪体则没有消化管敏感。4高浓度APAP对家蚕组织的损伤研究经口给药3600mg/kg的APAP后，家蚕幼虫2h后表现出行动迟缓和取食减少，还伴有吐液和身体收缩等中毒症状，蚕体甚至出现黑化现象。组织形态和组织切片观察结果显示，18h内消化管组织出现局部溃烂，消化管内膜结构被严重破坏，细胞膜也出现破损，柱状细胞、微绒毛和杯状细胞等主要结构被破坏分辨不清。表明该剂量APAP能够导致家蚕幼虫消化管的吸收营养物质功能受到严重破坏。APAP暴露后12h，脂肪体细胞缩短成圆形，细胞膜增厚或破损，细胞内空泡增加、细胞核变小细胞排列变得杂乱松散；至给药后18h，组织中空泡增大，出现了细胞核完全破坏的无核细胞。表明脂肪体发生了严重损伤。3600mg/kg的APAP经口暴露后，消化管中ROS含量在给药后8min时出现一时的增加，随后很快恢复至阳性对照水平，至消化管组织形态严重损坏的处理后12-18h，处理组中没有出现大量的ROS，表明APAP对消化管的损伤可能不是其它常见的药物组织氧化损伤。高浓度APAP经口给药后12h，家蚕血淋巴中死亡细胞的比例没有增加，但APAP处理后32min内造血器官中的ROS不断增加，至12h以后才逐渐恢复至对照NS处理组水平。说明APAP对家蚕血淋巴中的血球细胞可能没有损伤，但在一定时间内会导致造血器官中发现ROS增加，可能会影响该组织的造血功能。更多还原

【Abstract】 Acetaminophen (APAP) is an operative medicine widely used for the treatment offever and analgesia, and is a pattern drug of amide class widely used for liver injury model,too. Along with the conversion of medicine research pattern, the alternatives to animaltesting have become an important aspect of toxicology development. For drug toxicity test,the experimental animals used are mostly mammals such as mice. The drug administeredby orally exposure stays in the stomach for a relatively long period, and are easily digestedby gastric fluid, making the observation of the influence of drug on digestive tract difficult.In this study, Bombyx mori as an invertebrate, was used as the experimental animal.1Appropriate dose of APAP in Bombyx larvaeOne of the topic problems for alternatives to animal testing is the conversion of dose.Before, there is no reliable formula for the dose of clinical drugs from mice and human tosilkworm. According to the experiment the LD50of APAP in silkworm is2450.3~3226.85mg/kg, over50%more than1664.7mg/kg which got by the empiricalformula. Our results showed that a safe dose for day2of5th instar larvae of haoyue strainis600mg/kg, while the damage dose is3600mg/kg and the critical dose is1800mg/kg.2The metabolites of APAP in multiple tissues of Bombyx larvaeThe HPLC results showed that the products of glycosylation and sulfation are the twomain metabolites of APAP in multiple tissues of Bombyx larvae after orally explosion on2d of5thinstar. The metabolites are similar with the mice. There are tissue metabolismdifferences of APAP in Bombyx larvae, the glycosylation metabolic target organ might bedigestive tract while sulfation target organ might be haemolymph.The silkgland is a special organ in Bombyx larvae instead of in mice or human. Thesilkworm larvae are able to transfer the original medicine of APAP to the silkgland viahaemolymph from digestive tract, and the physiological effect might be store the redundantAPAP and the critical concentration could be0.2mg/ml at this stage. This phenomenon isuseful for the long-term toxicology studies.3Changes of activities of UGTs and its gene expression levelsThe activity of UGTs in digestive tract is higher than that in fat body on a2d of5thinstar larva of silkworm. However, this activity increased in digestive tract andhaemolymph start at24min and16min, respectively, after oral exposed to600mg/kg APAP.While the active is rapid rised in fat body, showed2times higher than its positive control at8min, and5times at24min, and suggested that the fat body is very sensitive to APAPbut might not been a main metabolic tissue.The results of gene expression patterns showed that Ugt30, Ugt86and Ugt89are allupregulated expression level in digestive tract and fat body. A peek level of Ugt30andUgt89gene mRNA in digestive tract presents to30min later, while they slowly increasedin the fatbody, supported the above results and guess.4The injury in silkmorm tissues caused by APAPAfter oral injected APAP of3600mg/kg, the larvae showed behaviors of sluggish andfeeding disturbance, accompanied by vomiting and body poisoning symptoms such asshrinkage even melanism at2hours later. The results from morphology and sectionsshowed that there were local fester in digestive tract. The structure of tunica intima wasseverely damaged, and the microvilli distinguish and cytomembranes of mast and gobletcells were damaged gravely. Follow the cells of fat body shorten then become a circle at12hours later, while the intracellular cavitations increased and the cytomembrane thickenedor damaged, the even losed nuclei, indicated that the fat body was suffered serious damageby APAP.ROS was temporary increased in digestive tract at8min after oral injected3600mg/kg of APAP, nevertheless the ROS was almost disappeared at the quite late of12h-18h,and even the tissue was serious injured, suggested that the damage of APAP may not been aoxidative damage pathway as a common drug in the digestive tract. The ROS inhematopoietic organs was been increasing in32min and was recovered gradually after12h,meanwhile, the hematocyte has not been cut significantly than control. These resultsindicated that the blood cell injury in vivo induced by APAP is different from that in mice.更多还原