基于椎间盘退变Wnt-lncRNA-miRNA-mRNA通路探讨补肾活血方疗效机制研究

Traditional Chinese medicine has great advantages in the treatment of intervertebral disc degeneration (IDD). Clinical studies have shown that BushenHuoxue Recipe can effectively relieve symptoms and improve waist function. Previous studies have found that the interaction of Wnt pathway with non-coding RNA plays an integral role in the therapeutic effect of BushenHuoxue Recipe.New research shows that longnon-coding RNA can compete with miRNA for the pathogenesis of IDD, and the function of lncRNA is achieved through Wnt pathway, but whether lncRNA participates in the therapeutic mechanism of BushenHuoxueRecipe has yet to be studied. Therefore, based on the previous research results, and through analysis of chip data, binding site prediction, and cell experiments, the applicant proposed that Bushen Huoxue Recipe can down-regulate the expression of lncRNA TUBAP by inhibiting Wnt pathway, and then mediate miR-483/CTNNB1 and miR-140/CREB1 pathways,promote the proliferation of nucleus pulposus cells, inhibit the degradation of ECM, improve the degeneration microenvironment, and delay IDD.This project intends to explain the pathogenesis of IDD and the therapeutic mechanism of BushenHuoxue Recipe from the perspective of lncRNA, and to verify the molecular, cellular, animal and tissue levels, and to clarify the key mechanism of action of this pathway in the treatment of IDD patients. To provide a solid theoretical foundation for the development of new multi-target drugs in the future, and further promote the modernization of Chinese medicine.

中医药延缓椎间盘退变（IDD）具有显著优势，临床研究显示补肾活血方可有效缓解腰痛症状、改善腰部功能。课题组前期研究证实Wnt通路与非编码RNA的相互作用很可能是补肾活血方发挥疗效的核心机制。新近研究发现，长链非编码RNA（lncRNA）可竞争性结合miRNA参与椎间盘退变，且其功能是通过Wnt通路实现的。那么，lncRNA是否就是补肾活血方延缓椎间盘退变的关键环节？我们通过lncRNA芯片数据分析、结合位点预测、细胞实验验证，提出补肾活血方可能通过抑制Wnt通路下调lncRNA TUBAP表达，进而介导miR-483/CTNNB1和miR-140/CREB1通路促进髓核细胞增殖、抑制ECM降解，从而改善退变椎间盘微环境，最终延缓IDD。本研究从分子、细胞、组织、动物四个层次进行验证，试图阐明补肾活血方延缓IDD的核心药理机制，为今后新型多靶点药物的开发提供实验基础，进一步推动中医药现代化。

椎间盘退变（IDD）是构成众多脊柱退行性疾病的病理基础，是我国45岁以下人群丧失劳动能力的最主要原因。髓核细胞（NPCs）数量减少、基质合成与降解失衡是IDD的始动因素，目前仍缺乏有效针对此类疾病病因的治疗手段。临床研究显示补肾活血方可有效缓解腰痛症状、改善腰部功能，课题组前期研究证实Wnt通路与非编码RNA的相互作用很可能是补肾活血方发挥疗效的核心机制。通过临床组织的qPCR检测发现lncRNA TUBAP在IDD中显著上调；敲低lncRNA TUBAP干预NPCs可促进miR-483、miR-140表达而抑制CTNNB1和CREB1表达。LUC和RIP实验证明lncRNA TUBAP与miR-483、miR-140靶向结合且与TCF4存在结合位点。过表达TCF4和敲低lncRNA TUBAP处理NPCs显示过表达TCF4可对NPCs造成损害作用，而敲低TUBAP则可部分恢复TCF4的该作用。之后利用补肾活血方处理NPCs和IDD小鼠模型，发现该药方可抑制Wnt活性进而抑制TCF4和lncRNA TUBAP表达，诱导miR-140、miR-483表达而抑制CTNNB1、CREB1表达。使用Wnt通路激活剂LiCl和（或）敲低lncRNA TUBAP处理退行性NPCs后，发现LiCl可对NPCs造成损害作用，而敲低TUBAP则可部分恢复LiCl的该作用。本研究通过临床组织检测、体内和体外实验证实敲低TUBAP可通过结合Wnt信号通路关键转录因子TCF4促进miR-140和miR-483表达而抑制CREB1和CTNNB1的表达，从而促进补肾活血方对IDD的改善作用。在课题研究期间，共发表中文核心论文5篇，撰写SCI论文2篇，协助培养博士研究生1名；本研究从lncRNA的角度阐释补肾活血方的作用机制，为推进以补肾活血方为代表的中医药的现代化应用提供依据。