基于代谢组学与琥珀酸/HIF-1α/VEGF炎症信号通路的雷公藤煨制“除剧毒、强疗效”科学性研究

Rheumatoid arthritis (RA), as a multiple factors induced autoimmune disease, was classified as one of the refractory and serious diseases by WHO and China. The latest research indicated that metabolism disorder, accumulated succinate, angiogenesis were involved in many inflammatory diseases. We think that succinate/HIF-1α/VEGF signaling pathway maybe played a key role in joint synovium fibrosis and which possibly was a novel therapeutic target for RA based on previous study. Tripterygium wilfordii was an excellent Chinese medicine with confirmed efficacy for the treatment of RA, but its clinical applications were confined by its severe side effects. According to native usage of Tripterygium wilfordii, our previous investigation indicated that roasting processing can not only significantly reduce the toxicity but also enhance the therapeutic efficacy of Tripterygium Wilfordii. In order to disclose the rationality of roasting processing, obtain excellent leader compounds from Tripterygium wilfordii and find new therapeutic target for RA, this project will focus on: ① To disclose the action material basis involving reducing toxicity and enhancing efficacy of roasted Tripterygium wilfordii by metabonomics and thermal analysis technologies. ②It will be investigated that intracellular succinate is dependent on HIF-1α to regulate angiogenesis, while extracellular succinate regulates angiogenesis is dependent on GPR91, thus exacerbating the inflammation of synovial tissue in RA. ③To disclose the mechanism involving reducing toxicity and enhancing efficacy of roasted Tripterygium wilfordii by metabonomics and succinate/HIF-1α/VEGF signaling pathway.

类风湿性关节炎（RA）是多因素诱导自身免疫疾病，被WHO及我国列为难治性重大疾病。最新研究显示代谢组紊乱、琥珀酸堆积、血管新生、炎症反应在关节功能紊乱中同步存在，结合前期我们提出琥珀酸/HIF-1α/VEGF通路可能是RA治疗新靶点。雷公藤在治疗RA方面具有公认优势，但毒副作用限制了临床应用。受民间习用启发，申请人首次发现雷公藤经烘箱煨制后有显著减毒增效作用，优选温度200℃。为阐明雷公藤煨制科学性，提高临床应用安全与有效性，发现高治疗指数抗RA先导物及进一步新药开发，为RA防治提供新思路。本项目拟开展工作：①采用代谢组学结合热分析技术阐明雷公藤煨制抗RA减毒增效物质基础；② 验证细胞内琥珀酸依赖HIF-1α调节血管新生，同时细胞外琥珀酸通过GPR91调节血管新生，加剧RA滑膜组织炎症新机制；③采用代谢组学结合琥珀酸/HIF-1α/VEGF血管新生通路阐明雷公藤煨制抗RA减毒增效作用机制。

为阐明雷公藤煨制的科学性，项目完成了以下研究。结果为提高雷公藤临床应用安全与有效性及开拓RA防治新思路奠定了坚实基础。.1、采用代谢组学结合热分析技术阐明了雷公藤煨制抗RA减毒增效物质基础。发现煨制后雷公藤中81种成分变化显著，主要为生物碱与三萜。煨制后生物碱类毒性成分大幅减少，高毒性成分雷公藤红素消失，部分转化为低毒性芴酮。表明生物碱和雷公藤红素的变化是雷公藤煨制减毒增效的物质基础。完成了雷公藤红素煨制转化物1-羟基-2，5，8-三甲基-9-芴酮的合成、活性及体内过程研究，为其进一步药用开发奠定了基础。发现一个新奇的无螯合辅助钯催化sp3 C-H/sp2 C-H 分子内氧化偶联反应：一锅法合成新的5-苯基芴酮。.2、采用代谢组学方法阐明了雷公藤煨制抗RA减毒作用机制。发现鞘氨醇、5-L-谷氨酰-牛磺酸、3-甲基二氧吲哚、吲哚-3-甲醛、4,6-二羟基喹啉和棕榈酸，这6种内源性代谢物为总提取物、总生物碱和雷公藤红素组共同的解毒生物标志物。首次发现肝肾毒性标志物6个，分别为5-胆甾烯、半乳糖脑苷脂、6-O-橄榄油酰蔗糖、3-羟基十二酰肉碱、色诺酮B、可妥龙-3-葡萄糖醛酸。生物途径分析表明，毒性和解毒机制可能与鞘磷脂代谢、牛磺酸和次牛磺酸代谢、色氨酸代谢、甘油磷脂代谢、脂肪酸生物合成、戊糖和葡萄糖醛酸相互转化以及类固醇激素生物合成有关。.3、采用代谢组学结合琥珀酸/HIF-1α/VEGF血管新生通路阐明了雷公藤煨制抗RA增效作用机制。给药后，生品总提物和煨制品总提物组大鼠关节炎指数和足肿胀度显著降低，血清中白介素-1β水平显著降低，滑膜组织病理改变明显减轻，琥珀酸堆积大幅下降。与生品总提取物相比，煨制品总提取物组各项指标及滑膜病理变化更趋近正常组，并有显著性差异。经雷公藤煨制品总提取物干预后，有16种差异代谢物明显回调，其中(S)-2-乙酰-2-羟基丁酸、5-羟基吲哚乙酸、N-乙酰色氨酸、肌酸、磷脂酸(8:0/10:0)、白三烯B4与生品总提取物组相比更趋近正常组，并具有显著性差异，为可能增效生物标志物。表明煨制可显著提高雷公藤对CIA模型大鼠治疗效果，其增效机制可能与缬氨酸、亮氨酸和异亮氨酸生物合成、色氨酸代谢、能量代谢、甘油磷脂代谢、花生四烯酸代谢、DNA甲基化和去甲基化等途径有关。