亚急性暴露下喹烯酮对SD大鼠免疫毒性及其作用机制研究

发布时间：2018-07-25 18:59

【摘要】：喹烯酮(Quinocetone，简称QCT)是由中国农业科学院兰州畜牧与兽药研究所研究合成的一类喹恶啉类药物，具有喹恶啉-l,4-二氧化物（quinoxaline-1,4-dioxides,QdNOs）的基本结构，可抑制肠道内多种细菌的生长和繁殖，包括金葡菌、沙门氏菌及大肠杆菌等，主要用于牛、猪及家禽以发挥其促生长、改善肠道菌群、促进蛋白质吸收及合成作用。近年来，有研究发现喹恶啉类衍生物卡巴氧和喹乙醇，具有潜在的诱突变性和致癌性。由于喹烯酮与该两种药物具有相同的基本结构，喹烯酮药物的使用安全性也逐渐受到人们的关注。相关资料显示，QCT可能导致哺乳动物细胞株的强致突变作用，对HepG2细胞株引起DNA损伤。但目前有关QCT引起的免疫毒性研究有限，因此选用Sparague-Dawley大鼠（简称SD大鼠）进行喹烯酮亚慢性暴露，以评价喹烯酮对于哺乳动物的免疫毒性作用，并通过氧化应激状态对其相关机制进行探讨。目的：探讨亚急性QCT暴露对SD大鼠的免疫毒性作用，并初步探讨QCT致免疫毒性与氧化应激的相互关系。方法：健康雄性SD大鼠32只，随机分为以下四组：正常对照组(control):0mg/kg/day；喹烯酮低剂量组(QCT-H):50mg/kg/day；喹烯酮中剂量组(QCT-M):800mg/kg/day；喹烯酮高剂量组(QCT-H):2400mg/kg/day。每日记录体重及进食量，喂养至28天采用颈椎脱臼处死，记录脾脏、胸腺等主要脏器重量，留置一半脾脏作组织病理检查，剩余部分制备单细胞悬液，以丝裂原刺激法测量T、B淋巴细胞增殖能力、以YAC-1细胞作靶细胞测定NK细胞杀伤活性。彗星实验评价脾分离细胞的DNA损伤程度。制作脾组织冰冻切片，进行DHE染色，观察荧光强度，并计算其平均荧光密度。制备脾线粒体匀浆，检测GSH水平及GPx，SOD和CAT活力。结果：（1）高剂量QCT组SD大鼠脾脏组织表现出脾窦淤血改变；T、B淋巴细胞，NK细胞活性显著降低，差异有显著性(P 0.01)。（2）彗星实验结果显示喹烯酮暴露组OTM值与Tail DNA值较对照组均可见剂量依赖性增加，中、高剂量组与对照组检测结果存在显著性差异(P 0.01)。（3）脾细胞ROS水平呈剂量依赖性升高，喹烯酮高剂量组GSH、CAT、GPx与对照组相比均出现显著降低(P 0.01)。结论：高剂量喹烯酮亚急性暴露能够引起SD大鼠免疫靶器官组织损伤，且能导致脾组织DNA损伤，提示其对哺乳动物存在潜在的免疫毒性。喹烯酮亚急性暴露引起的免疫毒性跟其代谢中产生过量的ROS以及对抗氧化系统的抑制有直接关系。创新点：对喹烯酮亚急性暴露引起的SD大鼠免疫毒性进行评价，并从DNA损伤、氧化应激方面探讨其产生免疫毒性作用的机制。
[Abstract]:Quinocetone (QCT) is a class of quinoxaline drugs studied and synthesized by Lanzhou Institute of Animal Husbandry and Veterinary Medicine, Chinese Academy of Agricultural Sciences. It has the basic structure of quinoxaline-14-dioxides (QdNOs) and inhibits the growth and reproduction of many bacteria in the intestine. Including Staphylococcus aureus, Salmonella and Escherichia coli, mainly used in cattle, pigs and poultry to promote growth, improve intestinal flora, promote protein absorption and synthesis. In recent years, it has been found that quinoxaline derivatives carbamoxy and quindoquindox have potential mutagenicity and carcinogenicity. Because quinolone and these two drugs have the same basic structure, the safety of quinolone has been paid more and more attention. The relevant data showed that QCT might lead to strong mutagenicity in mammalian cell lines and DNA damage to HepG2 cell lines. However, the current studies on immunotoxicity induced by QCT are limited. Therefore, Sparague-Dawley rats (SD rats) were selected for subchronic exposure to quinolones in order to evaluate the immunotoxicity of quinolones to mammals. The mechanism of oxidative stress was discussed. Aim: to investigate the immunotoxicity of subacute QCT exposure to SD rats and the relationship between QCT induced immunotoxicity and oxidative stress. Methods: 32 healthy male Sprague-Dawley rats were randomly divided into four groups: normal control group (control): 0 mg / kg / day); low dose quinolone group (QCT-H): 50 mg / kg / day; QCT-M: 800 mg / kg / r / day; QCT-H: 2400mg / kg / day. Body weight and food intake were recorded daily. After 28 days of feeding, cervical vertebrae dislocated and killed, spleen, thymus and other main organs weight were recorded, and half of the spleen was kept for histopathological examination, and the remaining part was used to prepare single cell suspension. Mitogen stimulation was used to measure the proliferation of T _ (B) B lymphocytes, and YAC-1 cells were used as target cells to determine the cytotoxicity of NK cells. Comet assay was used to evaluate the degree of DNA damage in splenic isolating cells. The frozen sections of spleen tissue were made and DHE staining was performed to observe the fluorescence intensity and calculate the average fluorescence density. Spleen mitochondria homogenate was prepared to detect the level of GSH and the activity of SOD and CAT. Results: (1) the splenic tissue of SD rats in high dose QCT group showed a marked decrease in NK cell activity in splenic sinus congestion. The difference was significant (P0.01). (2). The results of comet assay showed that the OTM value and Tail DNA value in the quinolone exposed group were increased in a dose-dependent manner as compared with those in the control group. There was a significant difference between the high dose group and the control group (P0. 01). (3). The level of ROS in splenocytes was increased in a dose-dependent manner, and the GSH-CATGPx in the high dose group of quinolone was significantly lower than that in the control group (P0. 01). Conclusion: high dose quinolone subacute exposure can damage the immune target organs of SD rats and lead to DNA damage in spleen tissue, suggesting that it has potential immune toxicity to mammals. The immunotoxicity induced by subacute exposure to quinolone is directly related to the production of excessive ROS in its metabolism and the inhibition of the antioxidant system. Innovation: the immunotoxicity of SD rats induced by subacute exposure to quinolone was evaluated, and the mechanism of its immune toxicity was discussed from the aspects of DNA damage and oxidative stress.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R114

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