雷公藤制剂对消化系统的毒性机理研究

	雷公藤制剂对消化系统的毒性机理研究
	代曼云
导师	李飞
关键词	代谢组学，雷公藤，药源性肝损伤，消化性溃疡，FXR，ET-1，肠道血管屏障 Metabolomics, Tripterygium wilfordii, Drug-induced liver injury, peptic ulcer disease, FXR, ET-1, intestinal vascular barrier.
摘要	研究背景与目的：“支持中医药振兴发展，推进中医药综合改革”被再次写进今年的政府工作报告中。至此，从2013年以来，“中医药发展”的关键词连续十年被写入政府工作报告。足以见得中医药发展的潜力与国家对中医药发展的支持力度。从中药雷公藤提取制作的雷公藤片和雷公藤多苷片临床上主要用于治疗风湿热瘀、类风湿性关节炎、肾病综合症、自身免疫性肝炎等疾病。研究报道，雷公藤毒性在传统中草药中排第6位，己成为半个世纪以来发生中毒最多的中药之一，毒性反应主要集中在消化系统、生殖系统和血液系统。由于中药成分复杂易引起药物相互作用，因此，探究药物相互作用在雷公藤消化系统损伤中的作用与机制对雷公藤制剂的生产和临床高效安全的用药具有重要理论和实践意义。研究方法：本研究以临床常用雷公藤制剂雷公藤多苷片和雷公藤片为研究对象，利用动物与细胞模型，基于代谢组学、病理学、生物化学等分析技术探究雷公藤制剂对消化系统毒性作用的主要毒性成分和分子机制。利用代谢组学技术和HPLC分析技术对两种药片关键药物成分进行比对与绝对定量；通过分析病理、电镜、二胺氧化酶活力（DAO）、伊文思蓝外渗评价肠道损伤、肠道机械屏障与胃肠道血管屏障（Gastrointestinal vascular barrier，GVB）通透性；通过血浆、肝脏、盲肠内容物代谢组学分析探究雷公藤甲素（TP）/雷公藤红素（Cel）肝、肠损伤中产生贡献的主要代谢通路；利用过氧化物酶体增殖物激活受体α（peroxisome proliferators-activated receptors，PPARα）敲除小鼠和激动剂探究PPARα对雷公藤片肝毒性的预防作用；利用法尼醇受体（Farnesoid X receptor，FXR）敲除小鼠探究FXR在肝、肠损伤中的作用与机制；利用抗生素消灭肠道菌群模拟无菌小鼠肠道FXR抑制、肠道FXR抑制剂Gly-MCA、肠道FXR激动剂Fex探究肠道FXR在TP/Cel的肝肠损伤作用；利用FXR激动剂GW4064在FXR野生型和敲除型小鼠中探究激活状态下FXR在TP/Cel肝、肠损伤中的作用；利用内皮缩血管肽(endothelin，ET)-1抑制剂17β-环戊丙酸酯（Estradiol cypionate，Estr）、受体抑制剂波生坦（Bosentan，Bos）和腺病毒过表达ET-1探究ET-1在TP/Cel肝、肠损伤中的作用与机制；利用 HUVEC细胞体外探究FXR-ET-1-JNK-GSMDE焦亡与GVB的关系；利用HepG2和肝原代细胞探究TP损伤与ET-1-JNK炎症通路的关系。利用FXR-WT/KO小鼠探究FXR-ET-JNK在临床用药雷公藤和阿司匹林肠损伤中的作用。研究结果：雷公藤片在小鼠中肝毒性明显大于雷公藤多苷片，并且Cel的含量是雷公藤多苷片的21倍，Cel可增加TP的肝毒性并且存在剂量依赖关系；代谢组学分析发现TP/Cel肝损伤中肝脏发生明显脂质代谢紊乱和氧化应激，使用PPARα激动剂非诺贝特可有效缓解TP/Cel肝毒性，并且与IL6-STAT3通路有关联。进一步分析发现TP/Cel联合用药后引起的肝、肠损伤（大体观、生化、DAO、损伤长度、电镜分析、病理分析）伴有明显的凝血功能下降和胆汁酸代谢紊乱与FXR通路显著抑制（Shp、Ostb、Osta、Fgf15、I-Babp）；敲除FXR或抑制肠道FXR后可明显增加TP和TP/Cel诱导的肝、肠损伤，激活全身FXR或仅激活肠道FXR可有效阻止TP/Cel诱导的肝、肠损伤；ET-1抑制剂和受体抑制剂能明显缓解TP和TP/Cel诱导的肝、肠损伤且不影响FXR通路，过表达ET-1后明显增加TP诱导的肝、肠损伤。分子生物学技术分析显示ET-1-JNK介导的炎症在TP/Cel诱导的肝、肠损伤中发挥重要作用；HUVEC细胞体外验证显示FXR-ET-1/JNK-Caspase3-GSDME 细胞焦亡参与GVB的损伤。FXR-ET-JNK在临床用药雷公藤和阿司匹林肠损伤中同样发挥重要作用。研究结论与意义：TP与Cel的药物相互作用在雷公藤肝、肠损伤中担任着非常重要的角色，总结为以下几个方面：（1）Cel在雷公藤片中含量明显高于雷公藤多苷片，TP/Cel药物相互作用介导了雷公藤引起的肝、肠损伤，Cel应该被纳入雷公藤制剂生产标准体系；（2）Cel抑制肠道FXR介导了TP/Cel药物相互作用诱导的肝、肠损伤：抑制肠道FXR促使TP诱导激活肠道ET-1-JNK损伤通路，破坏GVB诱发溃疡出血；FXR-ET-1-JNK调节的GVB受损后通过增加菌群的等有害物质移位加重TP/Cel肝损伤；（3）FXR-JNK/ET-1-Caspase3-GSDME介导了GVB损伤；（4）FXR-JNK/ET-1 损伤通路同样介导了雷公藤片和阿司匹林诱导的肠道溃疡损伤。本论文发现并阐述了TP/Cel药物相互作用致肝、肠损伤中的作用与分子机制，肠道FXR和ET-1作为缓解雷公藤消化系统损伤的药物预防与治疗靶点提供理论基础；也为药源性肝损伤和消化性溃疡的预防与治疗提供新机遇；同时也为肠道FXR通过GVB调节其他系统疾病提供新的研究思路。; Research background and purpose: "Supporting the revitalization and development of traditional Chinese medicine and promoting comprehensive reform of traditional Chinese medicine" was mentioned again in this year's government report. Since 2013, the keyword "TCM development" has been written into the government report for ten consecutive years. We can see the potential of TCM development and the state's support for TCM development. Tripterygium wilfordii tablets (TGT) and tripterygium wilfordii polyglycosides tablets (TWT) extracted from Tripterygium wilfordii are mainly used in the treatment of rheumatic heat stasis, rheumatoid arthritis, nephrotic syndrome, autoimmune hepatitis, and other diseases. It was reported that the toxicity of Tripterygium wilfordii ranked the third in traditional Chinese herbal medicine, and has become one of the most toxic traditional Chinese medicines in half a century. The toxic reactions mainly in the digestive system, reproductive system and blood system. Due to the complex composition of Traditional Chinese medicine, it is easy lead to drug interactions. Therefore, it is of great theoretical and practical significance for the production, efficient and safe clinical use of Tripterygium wilfordii preparations to explore the mechanism of drug interaction in digestive system injury from tripterygium wilfordii. Research methods: In this study, TGT and TWT were used as research objects, animal and cell models were used to explore the main toxic components and molecular mechanism of the digestive toxicity pairs of Tripterygium Wilfordii preparations based on metabonomics, pathology, biochemistry and other analysis techniques. The key components of the two tablets were quantified by Metabolomics and HPLC. The permeability of intestinal mechanical barrier and intestinal vascular barrier (GVB) were evaluated by pathology, electron microscopy, diamine oxidase activity (DAO) and Evans blue extravasation. The plasma, liver and cecum metabonomics analysis were used to explore the main metabolic pathways contributing to hepatoenteric injury fromTP/Cel. PPARα-KO mice and the agonists of PPARα to investigate the of PPARα in the liver induced by TWT. FXR-KO mice were used to explore the role and mechanism of FXR in the liver and intestine injury induced byTP/Cel. Intestinal FXR inhibitor Gly-MCA and intestinal FXR agonist Fex, and sterile mice (Clearing the intestinal flora by antibiotics) to explore the role of intestinal FXR in liver and intestine injury induced byTP/Cel. The role of FXR activator GW4064 in liver and intestine injury ofTP/Cel was investigated in FXR wild-type and knockout mice. Endothelin-1 inhibitor Estr, receptor inhibitor Bos and overexpression of ET-1 by adenovirus were used to explore the role and mechanism of ET-1 in liver and intestine injury induced byTP/Cel. HUVEC was used to explore the relationship between FXR-ET-1-JNK-GSMDE pyroptosis and GVB in vitro; HepG2 and primary hepatocytes were used to
语种	中文
	2022-05
学位授予单位	中国科学院大学
文献类型	学位论文
条目标识符	http://ir.kib.ac.cn/handle/151853/75098
专题	昆明植物所硕博研究生毕业学位论文
推荐引用方式 GB/T 7714	代曼云. 雷公藤制剂对消化系统的毒性机理研究[D]. 中国科学院大学,2022.

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