基于TXNIP介导的线粒体应激探讨活血化瘀法干预糖皮质激素性骨质疏松的机制

Glucocorticoid-induced osteoporosis (GIO) is one of disabling bone metabolic diseases clinically, and the pathogenesis has not been elucidated. Currently, oxidative stress is known as involvement in the pathogenesis of GIO. Previously evidence in our team revealed that skeletal microcirculatory abnormalities associated with mitochondrial stress mediated by TXNIP in GIO rat. We also found that tanshinol considered as active component of water-soluble fraction isolated from Danshen is the material base for the treatment of GIO. Recent publishes revealed that microvascular angiogenesis in skeletal tissue regulates bone formation. Considering the pathogenesis of traditional Chinese Medicine (TCM) associating with in blood stasis of GIO, we hypothesis that mitochondrial stress mediated by TXNIP might down-regulate bony microvascular angiogenesis, contributing to skeletal microcirculatory abnormalities of blood stasis in TCM. This may be potential pathogenesis of GIO. This research aims to observe the changes in mitochondrial stress, microvascular angiogenesis and bone formation, and to confirm inhibitory effect of mitochondrial stress mediated by TXNIP on endothelial progenitor cells(EPCs) in GIO model of rats. The research also aims to elucidate the influence of VEGF/Wnt pathway regulated by TXNIP on angiogenesis and bone formation in vitro using overexpression and siRNA of TXNIP technology. Then the research plans to reveal the inhibitory effect of mitochondrial stress on skeletal microcirculatory abnormalities of blood stasis in the pathology of GIO and the underlying mechanism of tanshinol. The study may be expanded to provide a new theory of agents for prmoting blood circulation and removing blood stasis to treat GIO.

糖皮质激素性骨质疏松（GIO）是临床致残性骨代谢疾病，发生机制尚未阐明。目前，氧化应激被认为参与GIO发病。我们发现，GIO大鼠骨组织微循环障碍，与TXNIP介导的线粒体应激有关，同时丹参的水溶性成分丹参素是其防治GIO的物质基础。新近报道，骨微血管生成调控骨形成。结合GIO血瘀证的中医病机，我们推测：TXNIP介导的线粒体应激负性调控骨微血管生成，造成血瘀证微循环障碍，可能是GIO发病的新机制。本项目拟复制GIO大鼠模型，动态观察线粒体应激和微血管形成与骨形成的变化规律，分析TXNIP介导的线粒体应激对血管内皮祖细胞的影响，在体外应用TXNIP过表达和RNA干扰技术，阐明TXNIP调控VEGF/Wnt通路对血管形成和骨形成的作用，从整体动物、细胞和线粒体水平揭示线粒体应激引起血瘀证微循环障碍在GIO发病中的作用及丹参素的干预机制，为活血化瘀中药防治GIO提供新的理论依据。

糖皮质激素性骨质疏松（GIO）是临床上常见的并发症，长期大剂量应用糖皮质激素 (GC) 易引起类库欣综合征，约30% ~ 50%患者并发GIO。骨形成减少是 GIO 患者的显著特征，机制尚未完全阐明。GIO属“骨痿”范畴。传统中医认为，血脉瘀滞，骨失所养，必致“骨痿”。因此，本项目主要建立了GIO大鼠模型和细胞模型，用丹参素干预，并以经典的活络效灵丹作为对照，结合TXNIP基因敲除小鼠GIP模型中骨组织的差异蛋白表达情况，从体内体外分析TXNIP在GIO发病过程中的作用，并阐明丹参素对改善血瘀证微循环障碍和骨形成低下的作用，进而揭示丹参素对GIO的干预机制。结果发现：（1）丹参素可抑制TXNIP的表达，通过上调VEGF-Wnt信号通路（VEGF，VEGFR2，β-catenin）促进骨微血管生成，改善骨微循环，保护骨形成功能，对抗GC引起的骨量丢失、骨微结构破坏和骨生物力学性能下降的作用，从而发挥抗骨质疏松症的作用；（2）丹参素抑制GIO大鼠骨形成减少的作用， 其机制可能与其通过抑制TXNIP表达，继而下调线粒体氧化磷酸化（Ndufs3，SDHD，Cyt b，COX Ⅳ，ATPB）通路，从而减轻线粒体功能损伤有关。本项目从整体动物、细胞和线粒体水平阐明丹参素对GIO的干预机制，为活血化瘀法防治GIO提供新思路，具有重要意义。