基于网络药理学研究中药肾康丸治疗糖尿病肾病的分子机制

Diabetic nephropathy （DN）could result in end-stage renal disease and significant mortality in patients with diabetes. Although the mechanism of DN is complex, renal fibrosis play a most critical role during this progress. The progression of fibrosis can be activatied and amplified by endothelial cell (EC) injury and fibrosis which involves series of genes and signal pathways. As an effective traditional Chinese Medicine prescription, Shenkang Wan has been was patented and approved to clinical therapies. Previous study found that Shenkang Wan can protect kidney by blocking fibrosis during DN progression. However, it is difficult to explore the network of mechanism through traditional drug-target methods. In this study, we based on the principle of “multi-components” corresponding to “multi-targets”, which originated in network pharmacology. Firstly, we would set up a network-database of EC fibrosis by iTRAQ methods. Next, we would identify the chemical constituents and predict the targets by UPLC-MS and Surflex-Dock. Third, we would merge the network of multi-components corresponding to multi-targets, screen the active components and selected the optimal combination of ingredients by R-Bioconductor and Cytoscape. Finally, we will validate our results by experiments in vivo. Through high-throughput selection, computer simulation and molecular biological information technology, we trend to explore the mechanism of DN and screen the efficacy components of Shenkang pill based on the "biomolecular network". We believe that our study can provide a new research strategy for the mechanism elucidation and components optimization of Chinese herbal medicine.

糖尿病肾病（DN）已成为终末期肾病和糖尿病患者死亡的主要原因。DN发生机制复杂，肾脏纤维化是其发生发展的最终共同途径，而血管内皮细胞（EC）损伤是启动和促进因素，过程涉及复杂的生物网络信号。中药肾康丸是治疗DN的临床验方，前期研究表明具有保护DN肾脏，延缓DN进程的作用，但单靶点的研究模式难以全面阐明其作用机制。本研究从整体出发，基于网络药理学研究思路，采用iTRAQ技术建立DN进程EC损伤纤维化分子网络；采用UPLC-MS技术结合模拟分子对接方法确定药物有效成分；应用Bioconductor/Cytoscape整合药靶生物网络，运用网络计算筛选活性成分，进行组方优化；再在DN模型上进行表型和靶点验证。以期借助高通量筛选、计算机分子模拟及生物信息技术整合等现代研究手段，从“生物分子网络”这一整体来探寻DN发生的内在机理与肾康丸的药效物质，为中药复方作用机制的阐明和优化提供新的研究策略。

糖尿病肾病（DN）已成为终末期肾病和糖尿病患者死亡的主要原因。DN发生机制涉及多条生物网络信号，目前临床上缺乏有效治疗药物。中药肾康丸是治疗DN的临床验方，前期研究表明具有保护肾功能，延缓DN进程的作用，但单靶点的研究模式难以全面阐明其作用机制。因此本研究从整体出发，基于网络药理学研究思路，采用RNA-seq技术建立DN进程分子网络；UPLC-Q-TOF-MS技术检测肾康丸成分及肾康丸血清药物化学成分检测，结合RNA-seq结果，应用Bioconductor/Cytoscape整合药靶生物网络，结合模拟分子对接方法确定药物有效成分，并在DN动物模型上进行表型和靶点验证。首先建立db/db小鼠DN模型，成模后分为对照组、模型组、肾康丸组和卡托普利组。观察肾康丸对DN小鼠的治疗和肾脏保护作用。结果显示肾康丸能降低db/db小鼠空腹血糖水平，减少24 h尿蛋白，同时改善肾小球硬化。其机制与减轻足细胞突触融合，改善肾小管坏死和恢复足细胞标志性蛋白nephrin和podocin相关。转录组测序结果显示肾康丸干预组和模型组共有718个差异基因，包括430个上调基因和288个下调基因。肾康丸改善足细胞损伤可能与下调AURKB/racgap1/RhoA通路的表达有关。此外，通过UPLC检测肾康丸及肾康丸给药db/db小鼠1 h及3 h血清成分，肾康丸鉴定出125种中药相关化合物，结合血浆肾康丸入血成分，发现共有29个化合物以原型入血。结合差异基因表达分析结果，根据KEGG和GO数据库建立肾康丸作用靶点网络。最终发现肾康丸作用的四个核心靶点：Shcbp1、Psrc1、Dscaml1、Racgap1，并且筛选出两个最佳有效成分：刺芒柄花素和穿心莲内酯。通过动物实验验证该有效成分成分组合，结果显示刺芒柄花素组、刺芒柄花素+穿心莲内酯组和刺芒柄花素2倍剂量+穿心莲内酯组表现出明显的降低空腹血糖水平和改善肾小球硬化的作用。本研究通过建立DN药物靶点信号网络和体内验证该药靶网络，证明肾康丸具有治疗DN和保护肾脏的作用，提示“高通量组学-网络药理学-生物信息技术整合”的模式筛选活性群的方法能有效揭示肾康丸的关键作用成分，并证明肾康丸可通过多靶点、多途径协同改善DN肾脏损伤，有望成为治疗DN的临床新药。这一模式还为揭示其他复方中药方剂药效物质与作用机理提供了强有力的方法与技术支撑。