基于Hippo和TGFβ通路交叉对话对淫羊藿苷干预激素性股骨头坏死微血管内皮细胞损伤的研究

Osteonecrosis of the femoral head (ONFH) is a pathological process that occurs at the femoral head as a result of interruption of blood supply following ischemic insult. One of the most common risk factor for ONFH is use of glucocorticosteroids. Injury of human bone microvascular endothelial cells which result in disordered circulation is the key etiological factors of glucocorticosteroids induced ONFH. However, the pathophysiology of ONFH has not been completely elucidated. In the previous study, we found that TGFβ/Smad signal transduction pathway in bone microvascular endothelial cells was significantly influenced by glucocorticosteroids. Icariin, a flavonoid isolated from the traditional Chinese herbal medicine epimedium brevicornum maxim, has been shown to protect the human bone microvascular endothelial cells from glucocorticosterold induced cell injury by TGFβ/Smad signal transduction pathway. Recent studies have revealed crosstalk between the Hippo/YAP and TGFβ/Smad pathways, thereby providing crucial links between cell apoptosis and cell growth. As for the pathogenesis of glucocorticosteroids induced injury to endothelial cells, the crosstalk between the Hippo /YAP pathway and TGFβ/Smad pathway needs to be further researched to characterize the relation between signal transduction pathway of endothelial cells and glucocorticosteroids .In this study, we will use BMECs model to qualify the effect of crosstalk between Hippo /YAP pathway and TGFβ/Smad pathway on endothelial cell injury induced by glucocorticosteroids. Accompanying with vivo study in animal model and regulation of signal pathway in BMECs model, we will qualify the effect of icariin upon the crosstalk between Hippo /YAP pathway and TGFβ/Smad pathway, which protects the human bone microvascular endothelial cells from glucocorticosterold induced cell injury. Therefore, the mechanism of icariin protecting the bone microvascular endothelial cells from injury induced by glucocorticosteroids is revealed.

骨微血管内皮细胞（BMECs）损伤及其导致的血液循环障碍是激素性股骨头坏死的病理基础，其具体分子机制尚不清楚。申请者前期研究发现激素性BMECs损伤存在着TGFβ通路的下调，而淫羊藿苷在改善BMECs损伤的同时，激活了TGFβ通路。最新研究发现Hippo通路与TGFβ通路通过交叉对话而对BMECs发挥着调控作用。基于此，提出淫羊藿苷通过Hippo与TGFβ通路交叉对话保护激素性BMECs损伤的假说。本项目拟利用激素性BMECs损伤细胞模型，采用过表达及敲降技术，分别激活/抑制Hippo与TGFβ通路，揭示Hippo与TGFβ通路交叉对话调控激素性BMECs损伤的分子机制。通过淫羊藿苷干预Hippo与TGFβ通路的体内、体外研究，结合BMECs细胞功能检测，揭示淫羊藿苷通过Hippo与TGFβ通路交叉对话保护BMECs损伤的机制及靶点，为股骨头坏死防治提供科学依据。

糖皮质激素是导致股骨头缺血坏死的最常见原因之一，糖皮质激素导致的血液循环障碍及骨微血管内皮细胞（BMECs）损伤是激素性股骨头坏死的病理基础，其具体分子机制尚不清楚。我们基于Hippo通路与TGFβ通路通过交叉对话研究糖皮质激素对BMECs损伤及淫羊藿苷的保护作用。本项目中，我们利用BMECs细胞株，应用糖皮质激素干预，研究发现相较于对照组，BMEC中Hippo信号通路的YAP存在不同程度的高表达，而TGFβ信号通路受到抑制，TGF-β1及Smad3表达水平降低。为了研究Hippo与TGFβ信号通路交叉对话介导激素性BMECs损伤的分子机制。我们采用慢病毒转染分别激活/抑制Hippo与TGFβ信号通路，实验结果显示，沉默YAP，随着YAP表达量的下调，TGFβ信号通路的激活有所上调。而过表达YAP，TGFβ信号通路的活化受到显著抑制。该部分实验初步证明在BMEC细胞中TGFβ信号通路受到了YAP的负调控。为了研究YAP调控TGFβ信号通路的分子机制，我们进行转染及免疫共沉淀实验，结果表明YAP与Smad3存在较强的结合，主要共定位于细胞核中。我们建立大鼠激素性股骨头坏死动物模型和激素性BMECs损伤细胞模型，通过淫羊藿苷干预Hippo信号通路与TGFβ信号通路的体内、体外研究，结合BMECs细胞功能检测，初步表明淫羊藿苷通过影响Hippo信号通路YAP表达及TGFβ信号通路Smad3的表达而保护激素性BMECs损伤。通过本研究进一步发展了激素性股骨头坏死的发病机制，进一步明确了淫羊藿苷治疗股骨头坏死的调控靶点与环节，为诊治股骨头坏死疾病提供新认知和新手段。