摘要
随着生活水平的逐步提高,人们对动物源性食品的需求量越来越大,兽药残留作为食品安全的重要组成部分已成为人们普遍关注的一个社会热点问题。兽药残留超标不仅直接对人体产生急慢性毒性作用,引起细菌耐药性增强,还通过环境和食物链的作用间接对人体健康造成潜在危害,直接影响着我国养殖业的发展及国际化进程。另一方面,许多药物在动物体内残留的产生机制还不清楚,药物在动物体内的代谢过程还不明确。因此,加强对兽药残留机制的研究,减少和控制兽药残留的发生显得尤为重要,这对制定动物源性食品中兽药残留的监控措施也具有理论指导意义。
细胞色素P450酶系在药物代谢中起着重要的作用,也是药物产生相互作用的重要酶学基础。在动物养殖过程中,联合用药所产生的药物之间的相互作用是造成兽药残留的众多影响因素之一。恩诺沙星属于氟喹诺酮类抗菌药物,在动物养殖过程中具有广泛的应用。近年来,有关氟喹诺酮类药物对细胞色素P450酶活性影响的研究屡见报道。随着现代分子生物学技术与基因工程技术的发展与应用,药物作用机制和病理机制的研究已经深入到分子水平和基因水平,然而在恩诺沙星的作用下,鸡细胞色素P450各亚型在分子水平上的变化以及相应酶活性产生变化的分子机制还不清楚。本课题拟以食用动物鸡为研究对象,从体内、体外两个方面,通过观察恩诺沙星给药后鸡细胞色素P450基因转录、蛋白表达和酶活性的变化,探讨恩诺沙星对鸡细胞色素P450影响的分子机制,以期为揭示兽药残留的影响机制及制定动物源性食品中兽药残留的监控措施提供理论依据。本实验首先建立鸡原代肝细胞培养体系,利用Real-Time PCR方法检测鸡原代肝细胞细胞色素P450基因表达量的变化。结果表明,与对照组比较,各剂量组CYP1A5、CYP2H、CYP3A mRNA表达量均降低,而CYP2E mRNA表达量则呈现出低剂量组降低,高剂量组升高的趋势。
在建立鸡肝微粒体体外孵育体系的基础上,采用紫外分光光度法测定细胞色素P450酶系的活性。结果显示,与对照组比较,各剂量组AND、ERND活性均显著降低(P<0.05),NCCR活性仅在高剂量组显著降低(P<0.05)。同时,利用Western-Blot方法和生物发光法分别对CYP1A、CYP3A的蛋白表达量及酶活性进行检测,数据表明,恩诺沙星对鸡肝微粒体体外孵育体系中CYP1A、CYP3A蛋白表达量和酶活性均呈现出抑制性作用,且随着反应时间的增加,抑制程度也逐渐加深。
体外实验结果提示,恩诺沙星对鸡肝细胞细胞色素P450具有选择性抑制作用,恩诺沙星在体内是否会产生同样作用,影响程度是否与体外实验相同,本实验进一步从体内观察恩诺沙星对鸡细胞色素P450的影响。首先采用蔗糖梯度离心法制备鸡肝微粒体蛋白,采用BCA法检测鸡肝微粒体总蛋白质含量,CO差示光谱法检测鸡肝微粒体总CYP450含量。结果表明,与对照组比较,中、高剂量组肝微粒体总蛋白质含量和总CYP450含量均显著降低(P<0.05)。利用体外实验建立的方法分别对鸡体内肝细胞色素P450基因水平、蛋白水平和酶活性进行检测,结果表明:(1)与对照组比较,CYP1A4 mRNA表达量仅在中剂量组显著降低(P<0.05);CYP1A5和CYP2H mRNA表达量在中、高剂量组均显著降低(P<0.05);CYP3A mRNA表达量在各剂量组均显著降低(P<0.05);CYP2C、CYP2E mRNA表达量在各剂量组的差异与空白对照组相比均无统计学意义;(2)中剂量恩诺沙星对AND、ERND的活性产生抑制性作用,高剂量恩诺沙星对NCCR、AND、ERND活性产生抑制作用;(3)恩诺沙星对鸡肝细胞色素CYP1A、CYP3A的蛋白表达和酶活性均呈现出抑制性作用,并且随着剂量的增加,抑制程度也逐渐加深。低剂量时对CYP3A蛋白表达的抑制作用强于CYP1A,中、高剂量时对CYP1A蛋白表达有较强抑制作用;而相同给药剂量下对CYP3A酶活性的抑制作用比CYP1A强。
综上所述,本研究主要取得了如下研究结果:(1)建立鸡原代肝细胞分离培养体系,分离的肝细胞存活率达90%以上,更换无血清培养液后能够继续存活2-5天,基本满足体外药物代谢及毒理学评价的需要;(2)建立鸡肝微粒体体外孵育体系,其细胞色素P450酶系活性保留较好,可用于药物与细胞色素P450相互作用及体外代谢清除等方面的研究,为兽药代谢的体外评价奠定了基础;(3)改进蔗糖密度梯度离心法制备鸡肝滑面微粒体,经透射电镜检测污染细胞器成分较少见,Western-Blot未检查到易污染细胞器,为鸡亚细胞组分的进一步研究奠定了基础;(4)成功建立鸡细胞色素P450酶系活性的检测方法,为研究药物代谢在鸡体内的变化规律提供了酶学方法;(5)体内、体外实验结果均提示EF对鸡细胞色素P450具有选择性抑制作用,不同浓度EF对鸡肝细胞色素P450的抑制程度不同,呈现剂量-效应趋势;(6)中、高剂量EF可以在基因转录水平和蛋白水平降低CYP1A和CYP3A的表达,从而导致相应酶活性的降低,而低剂量EF对CYP1A5和CYP3A酶活性具有抑制作用;但在蛋白水平上均未观察到相应的变化,仅CYP3A在基因转录水平有降低。这种mRNA变化与对应蛋白含量及酶活性变化的不一致性,可能反映了翻译后水平的蛋白稳定性调节,即分子水平的酶诱导性蛋白调节;(7)低剂量EF对CYP1A5和CYP3A可以产生抑制作用,提示兽医临床上,EF与经CYP1A5和CYP3A代谢的药物联合使用时,应注意这种抑制作用所产生的影响,在食用动物的养殖中应适当延长休药期以减少兽药残留的发生。
With the gradual improvement of people’s living, the demand of animal food is increasing greater. Veterinary drug residues as an important social issue of food safety have already drawn common concern of the whole society. Veterinary drug residues not only do harm to people’s health directly or indirectly, but also impact the development of the national economy. And the excess of the veterinary drug residues level could influence the export of meat products. On the other hand, for many drugs, both the production mechanism of residues and metabolic process has not been clear in edible animals. So it is essential to reinforce the study on the mechanisms of veterinary drugs in the body of animals and to reduce veterinary residues.
Cytochrome P450s play an important role in drug metabolism. They are the enzymatic foundation of drug-drug interaction. Drug-drug interaction caused by combined medication is one of the influence factors on veterinary drug residues in domestic animal breeding. Enrofloxacin (EF), a fluorinated quinolone carboxylic acid derivative, is a chemotherapic agent with extensive use in veterinary medicine in view of its broad spectrum of activity. Recently, reports about the effects of fluorinated quinolone on cytochrome P450 have continuously increased. But the changes of chicken hepatic cytochrome P450 happened in molecular level and the mechanism of enzyme activity changes remains unclear. Taking edible animal--chicken as the research object, this study was designed to investigate the effects of EF on chicken hepatic cytochrome P450 in genetic transcription, protein and enzyme activity levels from both in vitro and in vivo. And this dissertation is to discuss the molecular mechanism of the effect so as to reveal the generation mechanism of veterinary drug residues, and to provide a theoretical basis for the safety of clinical combined medication.
The chicken primary hepatocytes were cultured successfully in serum-free medium. The expression of chicken primary hepatocytes cytochrome P450 mRNA was determined by Real-Time PCR. The results showed that the expression level of CYP1A5, CYP2H, and CYP3A mRNA decreased in both experimental groups compared with the control, while the expression level of CYP2E mRNA decreased in low dose and increased in high dose.
The incubation system of chicken hepatic microsome was successfully established. The activity of cytochrome P450 was determined by ultraviolet spectrophotometry. Compared with the control groups, the activity of AND and ERND in both experimental groups obviously reduced (P<0.05). The activity of NCCR obviously decreased (P<0.05) only in high dose group. The protein expression and enzyme activity of CYP1A and CYP3A were respectively determined by Western-Blot and bioluminescence. The results showed that EF presented rejection capability on protein expression and enzyme activity of CYP1A and CYP3A and deepened with reaction time increasing.
In vitro experiment, EF had the function of selective inhibition on chicken hepatic cytochrome P450. To investigate whether there are the same effect and degree in vivo, further experiment was carried out in vivo. Sucrose gradient centrifugation (SDGC) method was employed to extract chicken hepatic microsome. The concentration of chicken hepatic microsome protein was determined by BCA and the total CYP450 was determined by CO difference spectrum. The results showed that both the concentration of chicken hepatic microsome protein and the total CYP450 in middle and high dose groups obviously reduced (P<0.05).
By using the methods established in vitro, the results in vivo showed that: (1) compared with the control group, the expression level of CYP1A4 mRNA obviously decreased (P<0.05) only in middle dose group; the expression level of CYP1A5 and CYP2H mRNA obviously decreased (P<0.05) both in middle and high dose groups; the expression level of CYP3A mRNA obviously decreased (P<0.05) in all experimental groups; but there was no significant difference of the expression level of CYP2C and CYP2E mRNA in all experimental groups. (2) EF in middle dose inhibited the activity of AND and ERND, and in high dose inhibited the activity of NCCR, AND and ERND. (3) EF presented rejection capability on protein expression and enzyme activity of CYP1A and CYP3A and deepened with reaction time increasing. EF in low dose inhibited the expression of CYP1A protein stronger than CYP3A, while in middle and high dose it had the contrary effect. Moreover, with the same dosage, EF inhibited the activity of CYP3A stronger than CYP1A.
Based on the results of this study, conclusions can be drawn as follows: (1) the chicken primary hepatocytes were isolated and cultured successfully. The survival of separated cells was more than 90% and could survive two to five days while medium was replaced with serum-free, which basically meet with the requirement of evaluation on toxicology and drug metabolism in vitro. (2) chicken liver microsome incubation system was established and the activity of cytochrome P450 was almost remained. Thus this method can be used for the study of interaction between drug and cytochrome P450 and vitro metabolism removing, and lay a foundation for evaluating veterinary metabolism in vitro; (3) the chicken hepatic smooth microsomes were prepared by the improved SDGC method, and this would establish a foundation for further study on subcellular fractions; (4) the method for determining contents and activities of hepatic microsomal cytochrome P450s in chickens was established, and this would provide an enzymatic analysis of drug metabolism in chickens; (5) the study results both in vitro and in vivo showed that EF could inhibit the chicken hepatic cytochrome P450 in genetic transcription and protein level, but different concentration of EF caused different influence which hinted dose-response relationship; (6) for CYP1A and CYP3A, EF in middle and high dose may inhibit the expression both in genetic transcription and protein levels, and corresponding enzymatic activities were down-regulated at the meanwhile. However, EF in low dose only down-regulated the enzymatic activities and mRNA expression, and there was no significant difference for protein between low dose group and the control. The unconsistency between mRNA, protein and enzymatic activities might relate with the protein stable regulation in post-translation level i.e. enzyme induction protein regulation in molecular level; (7) as EF in low dose can inhibit CYP1A and CYP3A, we should pay more attention to the effect caused by inhibition while EF combined administration with drugs metabolizing by CYP1A and CYP3A. To reduce the veterinary drug residues, withdrawal time should be lengthened in edible animals’cultivation.
引文
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