整合动态生物网络与细胞代谢组学新策略研究大黄酸和姜黄素联用治疗肾纤维化的作用机制

Renal fibrosis is a progressive pathological process which is controlled by multiple signaling pathways. The treatment needs dynamic regulation in multiple pathways. Our previous studies revealed that the rhein and curcumin were the main active components from clinic prescription of Bushen Huoxue Decoction in treating renal fibrosis, but their accurate regulation theory to disease network and the best concentration compatibility remain to be elucidated. On the basis of our preliminary work, the core pathway network in renal fibrosis will be constructed through literature survey and further investigation in this project. Meanwhile, biomarkers are screened and identified by cell metabolomics. Then a (metabolic pathway) protein - metabolite network will be established with the obtained biomarkers. And then it will be integrated with the core pathway network to establish a rhein/curcumin - (signaling pathway) protein - (metabolic pathway) protein - metabolite network. Furthermore, ordinary differential equations are used to simulate the dynamic response of the core biochemical network in renal fibrosis before and after a two-component disturbance. Finally, molecular pharmacology and targeted metabolomics will be used to detect the concentration of proteins and metabolites in the key pathway in NRK-49F cells to validate the results of network simulations. This project aims at elucidating the molecular mechanism of two chemical components by the way of integrating signal transduction pathway with the metabolic pathway, determining the two-component optimal concentration compatibility, developing new methods and strategies for the multi-component drug discovery based on disease networks, and providing a scientific basis for the secondary development of Bushen Huoxue prescription.

肾纤维化是多个信号通路控制的进行性病理学过程，其治疗需针对多个环节进行动态调节。课题组前期研究揭示大黄酸和姜黄素是治疗肾纤维化临床验方—补肾活血方的主要药效成分，然而两者联用对疾病网络的精准调节机制及最佳配伍仍待阐明。本项目在前期工作基础上，拟通过文献挖掘构建肾纤维化核心通路网络，并应用细胞代谢组学方法筛选生物标志物，构建(代谢通路)蛋白-代谢物网络；进而整合两网络构建大黄酸/姜黄素-(信号通路)蛋白-(代谢通路)蛋白-代谢物网络，应用常微分方程组模拟该网络及两组分扰动下的网络动力学行为。最后通过分子药理学及靶向代谢组学技术定量检测NRK-49F细胞中关键通路蛋白及代谢物以验证上述网络模拟结果。从信号通路—代谢通路整合角度定量阐明两组分协同的分子机制，明确两组分最佳配伍并在动物模型上评价疗效。发展基于疾病网络的多组分(组合)药物协同作用的新方法、新策略，为补肾活血方的二次开发提供科学依据。

慢性肾病(chronic kidney disease，CKD)是全球范围内的公共健康问题之一。肾间质纤维化是各种肾慢性疾病发展到终末期的共同生理过程，因此防治肾间质纤维化是治疗慢性肾病的重要治疗策略。本项目基于代谢组学寻找早期CKD的诊断标志物，同时通过代谢网络和分子通路网络揭示大黄酸和姜黄素联用延缓肾间质纤维化的协同作用机制，为大黄酸和姜黄素新药开发提供理论基础。. 首先使用ELISA检测NRK-49F细胞外纤连蛋白的含量，确定了体外大黄酸和姜黄素以及联用时最优给药浓度。然后基于UPLC-QTOF-MS代谢组学对细胞样品进行分析，筛选出3个肾间质纤维化标志物，分别为MG(0:0/20:3(11Z,14Z,17Z))、LysoPE(0:0/18:4(6Z,9Z,12Z,15Z))和PC(18:0/20:3(5Z,8Z,11Z))。其次，基于UPLC-QTOF-MS尿液代谢组学，筛选并鉴定出5个体内肾间质纤维化标志物：L-谷氨酸5-磷酸、α-亚麻酸、二十二碳六烯酸、美立比醇和脱氧肌苷。富集分析显示大黄酸和姜黄素联用可通过参与精氨酸和脯氨酸代谢、不饱和脂肪酸的生物合成、α-亚麻酸代谢、半乳糖代谢以及嘌呤代谢通路延缓肾间质纤维化。. 最后，构建肾间质纤维化分子网络模型，模拟大黄酸和姜黄素联用的协同延缓肾间质纤维化作用机制。结果显示，在5ng/mL大黄酸干预下，TGF-β1、ECM、MCP-1、Cyclin、ɑ-SMA 和 IL-6浓度明显降低；5μM姜黄素干预时，TGF-β1、ECM、MCP-1、Cyclin、ɑ-SMA 和 IL-6浓度有所下降；当大黄酸和姜黄素联合干预时，各物质浓度较大黄酸或姜黄素单用时更为减少，与WB实验结果一致。大黄酸和姜黄素联用时可能通过直接作用于MAPK、NF-κB和JAK/STAT3信号通路从而间接刺激TGF-/Smad 信号通路对肾间质纤维化产生协同保护作用。