兰州大学机构库 >药学院
高原低氧环境下药物代谢酶CYP3A1和UGT1A1的调控机制研究
Alternative TitleThe regulatory mechanism of CYP3A1 and UGT1A1 in hypobaric hypoxia
敏琼
Thesis Advisor封士兰 ; 王荣
2019-06-05
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name硕士
Degree Discipline药学
Keyword高原低氧 CYP3A1 UGT1A1 细胞因子 调控机制
Abstract高原自然环境的特点是低压、低氧、气候干燥寒冷及紫外线强等,其中低氧是影响人体的重要因素。这种低氧环境会影响药物在体内的转化过程,然而目前高原用药的方式和剂量与平原无异,这会影响高原人群用药的安全性和有效性。药物代谢酶作为药物在体内进行生物转化的重要工具,在药物的解毒和转化过程中起重要作用。目前关于低氧状态下药物代谢酶的调控机制尚不明确,因此探究一些调控因素对酶的调控作用非常必要。CYP3A4(大鼠体内为CYP3A1)和UGT1A1是人体内最重要的药物代谢酶,能够代谢临床50%以上的药物,所以本实验选取CYP3A1和UGT1A1作为I相代谢酶和II相代谢酶的代表,考察急进高原后大鼠肝脏内两个药物代谢酶的变化情况,并结合两个调控因素核受体和细胞因子的变化对机制进行探讨。本研究将大鼠急进至青海玉树(海拔4010m)进行低氧处理,待低氧不同时间(12h、24h、36h、48h)进行实验,对照组大鼠在甘肃兰州(海拔1500m)正常喂养。本课题主要从以下几个部分进行研究:①考察高原低氧环境对大鼠肝脏的影响。分析对照组和低氧不同时间组大鼠的血气、生化指标,摘取肝脏做病理切片观察组织病理改变;②探究高原低氧环境对大鼠肝脏内药物代谢酶CYP3A1和UGT1A1表达的影响,用Real-time PCR检测mRNA表达的变化,用Western-blot检测蛋白表达的变化;③研究高原低氧环境对药物代谢酶调控因素核受体(PXR和CAR)和细胞因子的影响,用Real-time PCR和Western-blot检测核受体PXR和CAR的表达变化,用蛋白芯片技术检测肝组织细胞因子的含量变化,并分析两种调控因素与药物代谢酶的相关性;④在细胞实验中进行机制验证,控制单一变量验证调控因素对药物代谢酶的调控。第一部分结果表明:大鼠急进高原后血液中可利用氧气浓度降低,pH值降低,酶类物质ALT和AST增加,总蛋白降低,提示肝组织发生生理性变化;肝脏病理结果显示,高原组大鼠肝细胞轻度坏死,周围血管轻微充血,中央静脉水肿且伴有炎性浸润,说明肝脏功能可能受损。肝脏作为药物代谢的主要器官,其功能的受损对药物代谢酶的合成、表达等均会产生影响,进而影响药物代谢过程。第二部分结果表明:大鼠急进高原后肝脏内CYP3A1和UGT1A1的mRNA和蛋白表达水平显著降低,说明在高原环境中部分药物在体内的代谢将会减慢,清除速率降低,药物在体内驻留时间延长,进而有可能增加药物毒性。第三部分结果表明: 代谢型核受体PXR和CAR的mRNA表达水平从低氧12h后即显著降低,综合文献可以推测在高原低氧状态下PXR和CAR可能作为这两种酶的上游基因而调控CYP3A1和UGT1A1的转录;蛋白芯片结果表明,大鼠急进高原后肝组织内IFN-γ、IL-4和IL-6的表达量在低氧48h后有显著升高,而IL-10、IL-13和MCP-1在低氧24h后就显著升高。通过相关性分析得出结论:IFN-γ、IL-6与CYP3A1高度负相关,IL-6与UGT1A1高度负相关,在第四部分的细胞实验中验证该相关性,确定IFN-γ和IL-6对CYP3A1和UGT1A1的调控作用。第四部分结果表明: IFN-γ孵育BRL大鼠正常肝细胞12h后CYP3A1、UGT1A1的mRNA表达显著降低,但随着孵育时间的延长,其mRNA表达逐渐升高,有上调趋势,蛋白表达趋于正常。IFN-γ相关转录因子STAT1的mRNA表达显著增加,说明IFN-γ的调控作用可能通过蛋白激酶STAT1的激活来实现的。而IL-6在低氧状态孵育12h、24h后CYP3A1的mRNA表达显著降低,UGT1A1的mRNA表达在孵育12h后显著降低,随着孵育时间的延长,CYP3A1和UGT1A1的mRNA表达水平有上升后又回复正常的过程。IL-6孵育48h后,UGT1A1的蛋白表达显著降低,CYP3A1无显著变化。为了探究IL-6的调控机制,我们研究了IL-6相关转录因子的mRNA表达,发现C/EBPβ的表达在36h时显著升高,说明C/EBPβ参与了IL-6对药物代谢酶的调控。整个实验结果说明在低氧状态下IFN-γ和IL-6参与CYP3A1和UGT1A1的调控,但这种调控与时间关系密切,随着孵育时间的延长,可能调动了细胞的反馈机制促使酶的表达趋于正常。本研究通过动物实验和整体实验研究了高原低氧环境对药物代谢酶CYP3A1和UGT1A1的调控作用,可以得出结论:大鼠急进高原后肝脏产生炎性反应,这是抑制药物代谢酶CYP3A1和UGT1A1表达的主要因素。此外,核受体PXR和CAR也可能参与了CYP3A1和UGT1A1的调控。
Other Abstract

The features of high altitude are hypopiesia, hypoxia, strong ultraviolet rays, cold and dry environment, hypoxia is the important factor influenced organism among these features. But there is no difference in the method and dosage of taking medicine between high altitude and plain, the safety and efficiency of plateau people wil be effected when they take medicine to cure desease. It is well known the drug-metabolizing enzyme is the major tool that take part in the process of drugs’ biotransformation, catalysis and detoxification. Currently, the regulatory mechanism of enzymes in hypoxia is unclear, so it is necessary to investigage the influence of hypoxia to enzyme. This project chose CYP3A1 and UGT1A1 as the symbol, and research the change of CYP3A1 and UGT1A1 after acute exposure to high altitude. because nuclear receptor and cytokines are regulatory factors of enzyme, then we combined the change of nuclear receptor and cytokines to discuss the regulatory mechanism. Healthy adult Wistar rats were randomly divided into Control group(normal feeding in Lanzhou, Gansu), High altitude group(hypoxia 12h, 24h, 36h, 48h). the high altitude group was managed by acute exposure to Yushu, Qinghai. This project was divided into four parts as follows: Part one, to study the effect of hypoxia on high altitude, we investigated the indicator of blood gas and biochemical, and then observed the pathologic change of liver; Part two, we explored the change of drug metabolism enzyme CYP3A1 and UGT1A1 in plateau, the Real-time PCR and Western blot were utilized to detect the gene and protein expression of CYP3A1 and UGT1A1. Part three, to estimate the changes of regulatory factors after exposure to high altitude, we used Real-time PCR and Western blot to detect the expression of PXR and CAR and used protein chip to study the changes of cytokines, and then analysed the correlation of regulatory factors(PXR, CAR and various cytokines) and drug metabolism enzyme(CYP3A1 and UGT1A1); Part four, because of the complexity of organism, we proved the correlation and mechanism of cytokine and enzymes in vitro;

The results of Part one showed available O2, pH and total protein decreased, the ALT and AST increased when the rats were exposed in high altitude, this results remind us that the liver maybe had physiologic changes. Besides, we utilized pathological section find that the morphology and function of liver were negative effected. The pathological result shown that the liver’s cell was slightly damaged, blood vessel appeared congestion and central veins appeared edema and inflammatory infiltration, these appearances showed us the liver’s function may be damaged. Liver is the key organ of the metabolism, whose dysfunction would influence the synthesis and expression of drug-metabolizing enzyme so as to interfere the metabolic process. The results of Part two demonstrated the expression of CYP3A1 and UGT1A1 have significantly decreased after acute exposure to high altitude, we can learn that metabolism of some drugs would be slower than normal situation, and the clear rate maybe decrease so as to the increase of residence time would induce toxity of drug. The results of Part three have shown that the mRNA expression of PXR and CAR was decreased after 12h of acute exposure to high altitude, and so as to we can guess the PXR and CAR regulated the transcription of CYP3A1 and UGT1A1 as the upstream gene according to references; then we can learn that the contents of IFN-γ, IL-4 and IL-6 were increased after 48h of acute exposure to high altitude and the contents of IL-10, IL-13 and MCP-1 were increased after 24h. Finally, we formed the conclusion by analysis of correlation that IFN-γ and IL-6 have strongly negative correlation with CYP3A1, IL-6 have strongly negative correlation with UGT1A1, and eventually we verified the correlation in Part four and comfirmed the regulation of IFN-γ and IL-6. Finally, the results of Part four indicated that IFN-γ would decrease the expression of CYP3A1 and UGT1A1 mRNA after 12h of incubation and the expression would gradually increase as the extension of incubated time. Similarly, IL-6 would decrease the expression of CYP3A1 mRNA after 12h and 24h of incubation and the expression of UGT1A1 mRNA after 12h of incubation. As the extension of time, the expression of CYP3A1 and UGT1A1 mRNA have increased and tended to normal. The whole project showed that IFN-γ and IL-6 play a role in the regulation of CYP3A1 and UGT1A1 transcription, and this process is related to time of incubation, we guess that the feedback system would regulate the expression of enzyme to normal as the extension of incubated time. Our project aimed to investigate the regulatory mechanism of CYP3A1 and UGT1A1 in the high altitude, we can make a major conclusion as followes: the liver’s inflammation reduced the expression of CYP3A1 and UGT1A1 when the rats were exposed in high altitude. Besides, the Nuclear receptor PXR and CAR maybe regulate CYP3A1 and UGT1A1.

Pages68
URL查看原文
Language中文
Document Type学位论文
Identifierhttp://ir.lzu.edu.cn/handle/262010/343904
Collection药学院
Affiliation药学院
First Author AffilicationSchool of Pharmacy
Recommended Citation
GB/T 7714
敏琼. 高原低氧环境下药物代谢酶CYP3A1和UGT1A1的调控机制研究[D]. 兰州. 兰州大学,2019.
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