基于PI3K/AKT调控体系探索紫草素抗类风湿性关节炎滑膜增生和血管新生的作用及机制

Synovitis hyperplasia and angiogenesis induce articular destruction of rheumatoid arthritis (RA). PI3K/AKT signaling pathway regulates the proliferation, survival, migration, invasion, adhesion and secretion of synovial fibroblasts (FLS) and vascular endothelial cells (EC) and angiogenesis in RA, which has become a new target for the treatment of RA. Radix Arnebiae, a traditional Chinese medicine, has been used effectively in the treatment of RA clinically with the functions of heat clearance and blood activation. Our previous results has shown that shikonin, the main immunoregulatory and antiinflammatory component of Radix Arnebiae, inhibited pannus formation in the joints of collagen induced arthritis(CIA) rats. However, its mechanism of this action remains unclear. The aim of this study is to investigate the effect of shikonin on synovial hyperplasia and angiogenesis of RA targeting PI3/AKT signaling pathway. CIA rats will be used to assess the effect of shikonin on RA. In vitro experiments, RA-HFLS three-dimensional micromass organ culture model will be used to assess synovial hyperplasia, and RA-HFLS and HUVEC co-culture is to assess the tube formation for the first time. MicroCT, double-stranded siRNA gene silencing, immunohistochemitry and western blot and real-time PCR will be also used. The result of this study will help to clarify the mechanism of Radix Arnebiae for RA treatment.

滑膜炎症增生和血管新生介导类风湿性关节炎（RA）的关节破坏，PI3K/AKT通过调控滑膜成纤维细胞（FLS）和血管内皮细胞（EC）功能异常在此过程中扮演重要角色，是RA治疗新靶点。清热活血中药紫草临床治疗痹证（RA）疗效好，我们前期实验也证明其主要免疫抗炎活性成分紫草素具有较好的抑制RA滑膜血管翳增生作用，然相关机制不明。本研究借助与人类RA临床特征最为接近的CIA大鼠模型，并引进国际最先进的拟滑膜增生的RA-HFLS三维micromass培养模型和拟血管生成微环境的RA-HFLS与HUVEC共培养诱导管腔形成体系，结合microCT、siRNA基因沉默、real-time PCR等技术，探索紫草素对RA滑膜增生和血管新生的作用，并通过“PI3K/AKT活化—FLS和EC功能异常”这一调控体系揭示相关机制。这将为临床合理应用紫草和进一步开发为治疗RA的新型“抗风湿改善病情药”提供实验依据。

清热活血中药紫草临床治疗痹证（RA）疗效好，实验研究证明其主要免疫抗炎活性成分紫草素具有抗炎、镇痛作用，有效治疗CIA，然作用机制不明。本研究采用CIA模型和体外FLS、EC培养模型，研究紫草素对RA滑膜增生和血管新生的作用，并从“PI3K/AKT活化—FLS和EC功能异常”切入探索其作用机制。研究发现紫草素能抑制CIA大鼠炎症关节中滑膜增生和血管新生，降低大鼠胸主动脉环微血管芽生及鸡胚尿囊膜血管新生能力，抑制FLS和/或HUVEC的增殖、迁移、粘附、侵袭能力以及管腔形成能力，从而具有抗RA滑膜增生和血管新生的作用；进一步，初步阐释了其作用机制与抑制CIA大鼠和体外FLS和/或HUVEC细胞中促滑膜增生和血管新生调控因子（VEGF 、IL-1β、TNF-α、TGF-β、PDGF、VEGFR2）的表达水平及负向调控下游PI3K/AKT和MAPK信号通路有关。相关结果为临床合理应用紫草和进一步开发为治疗RA的新型“抗风湿改善病情药”提供实验依据。