洋金花叶治疗银屑病药效物质基础及作用机制研究

Based on the determination of the treatment for the psoriasis of Datura metel leaves and referring to the strategy of gene reporter in molecular biology, the reporter gene plasmid contained STAT3 response elements were constructed targeting the luciferase STAT3 pathways. Oriented by the expression of the reporter gene, the systematic separation has been developed to split the parts, fractions and constituents, and the chemical structures of the isolated compounds were determined by spectroscopy technique. And, the effective parts and fractions of Datura metel leaves were evaluated by the pharmacodynamics experiments in vitro and vivo. Meanwhile, the influence of the related cytokines and proteins expression around the STAT3 pathway of the pharmacodynamics material basis of the Datura metel leaves were investigated in virtue of the mices psoriasis like animal models induced by imiquimod and the HaCaT cells models induced by IL-22. And, this was necessary to explore the relationships between the effective three levels material basis and cell proliferation, inflammatory reaction, pathways regulating, and to illustrate the pharmacodynamics material basis and mechanisms for the psoriasis treatment of the Datura metel leaves. This research provided the scientific basis for making full use of the rich resources of the Datura metel leaves and developing new drugs to treat the psoriasis, and also provided a new method and a novel pattern to investigate the mechanisms of other drugs for the treatment of psoriasis.

在确定洋金花叶治疗银屑病生物活性的基础上，借鉴“报告基因”策略，构建含有STAT3反应元件的基因质粒，建立靶向STAT3通路的报告基因体系。以报告基因的表达为导向，开展拆分部位、组分和成分三个化学层次的分离及活性评价，结合体内外药效学试验确定有效部位、组分和成分。应用波谱学测试技术确定有效成分的化学结构，结合液质联用技术获取有效部位、组分的化学特征信息。同时采用咪喹莫特诱导的银屑病样小鼠模型和IL-22诱导的HaCaT体外细胞模型，通过Western Blot等方法，围绕银屑病病理过程中与STAT3通路相关细胞因子的水平及表达，探讨三个化学层次的药效物质基础治疗银屑病的作用与细胞增殖、炎症反应和通路调节的相关性，阐明洋金花叶治疗银屑病的作用机制。本研究为充分利用洋金花叶丰富的药用植物资源、研制出疗效好的治疗银屑病的新药提供科学依据，为其他银屑病治疗药物评价和作用机制研究提供新思路、新方法。

本项目阐明了洋金花叶治疗银屑病有效组分和有效成分层面上的药效物质基础，并结合体内、体外药效学研究深入探讨了洋金花叶治疗银屑病的作用机制。.构建了基于报告基因导向的洋金花叶治疗银屑病有效组分和有效成分的药效物质基础体系，拆分得到黄酮和醉茄内酯两个有效组分，且验证二者互不交叉性良好；在阐明洋金花叶治疗银屑病有效组分的化学特征的基础上，分离并鉴定出153个化合物，包括47种醉茄内酯和20种黄酮类化合物(新化合物61个)，并探讨其构效关系，部分醉茄内酯和黄酮类化合物可抑制LPS诱导的RAW264.7炎症细胞的NO释放作用，其中醉茄内酯类化合物的活性优于黄酮类化合物；明确了洋金花叶治疗银屑病的各层次药效物质基础及其作用机制，采用咪喹莫特诱导的银屑病样皮损小鼠模型和IL-22诱导的永生化人角质形成细胞(HaCaT)模型，结合HE染色、ELISA、免疫组化、CCK-8和Western Blot等方法，开展体内、体外药效学和作用机制研究。研究结果表明洋金花叶治疗银屑病有效组分，即醉茄内酯组分可抑制IMQ诱导的小鼠脾脏和胸腺肿大，抑制小鼠皮肤PCNA、involucrin、K16和K17的表达；可显著抑制IL-22诱导HaCaT细胞的增殖及PCNA、involucrin、K16和K17的表达。.此外，为了完善项目研究的完整性、延伸项目研究深度，开展了基于网络药理学、蛋白质组学和代谢组学的洋金花叶治疗银屑病的作用机制研究。.本项目研究深化了对洋金花叶非药用部位的科学认识，为研制出疗效好、具有自主知识产权的治疗银屑病的新药，充分利用洋金花叶丰富的天然植物资源提供重要的科学依据。此外，将报告基因策略用于中药药效物质基础和作用机制的研究，可为其他银屑病治疗药物作用机理的研究提供新思路、新模式。