基于骨细胞网络Akt信号偶联Connexin43探讨牛膝-杜仲药对干预糖皮质激素性骨质疏松症的作用机制

Glucocorticoids (GCs) are frequently used in clinical medicine to treat non-infectious inflammatory diseases. However, prolonged GC administration is the most common cause of secondary osteoporosis. An estimated 30–50% of patients receiving long-term GC therapy sustain osteoporotic fractures. In our previous study, we found high dose of GCs inhibited bone remodeling, induced bone cells into autophagy or apoptosis. β-Ecdysone (βEcd) which is from Niuxi, could improve bone mineral density of model mice. The aim of this research is to explore the mechanism of GC induced osteoporosis (GIOP) by studying the effects of high dose of GCs on bone osteocytes activity, differentiation, autophagy and apoptosis; to explore the functions of Kidney tonify herbal pair Niuxi- Duzhong, and their active ingredients via observing the interventions in excess GCs. . Methods: After drug efficacy study based on Zebrafish experiment platform, the research will do: ① In vitro study: The MLO-Y4 osteocyte-like cells will be cultured and treated with GC (Dexamethasone). Using GC antagonist RU486 as controls, the treated groups will be received βEcd and Duzhong active ingredients to observe their interventions in high dose of GCs. The cells activities and differentiations will be observed by staining, SiRNA, gene transfection, or molecular biology methods after inhibiting Akt or silencing Connexin 43. Experiments will focuse on the common regulator Akt of Wnt-MEKK2/β-catenin signaling, PI3K/Akt signaling, and FoxO signal transduction pathways, as well as Connexin 43 in osteocyte network. ② In vivo study: The Connexin 43 KO mice will be divided into placebo group, low dose of GC group, high dose of GC group. GC groups will be received Prednisolone. In high dose of GC group, it includes GC with positive medicine controls (Alendronate group, Chinese medicine "Xianlinggubao" group), GC with herbal pair or GC with active ingredients. After 4 weeks, the bone formation will be observed via lumbar cryostat sections under the fluorescence microscope. The bone mineral densities will be measured by Micro CT. The autophagy and apoptosis genes expressions will be detected by PCR Array and Western-blot. . This research will be hoped to support experiments data to explore new Chinese medicine formula based on Niuxi - Duzhong and rich in traditional Chinese medicine theory "Kidney governing bone and generating marrow".

糖皮质激素GC诱导的骨质疏松症（GIOP）是继发性骨质疏松最常见类型，发生率达30～50％。前期研究发现GC抑制骨重塑，诱导骨细胞发生自噬乃至凋亡；牛膝活性成分β蜕皮甾酮可有效干预GIOP模型小鼠骨密度下降。牛膝-杜仲为高频出现的治疗骨质疏松补肾药对。本项目以MLO-Y4骨样细胞、斑马鱼、Connexin43敲除小鼠为研究对象，以GC处理制作病理细胞模型及GIOP动物模型。在斑马鱼实验平台经药效配比筛选后，围绕骨细胞网络Wnt-MEKK2/β-catenin、PI3K/Akt、FoxO途径共有调节蛋白Akt与连接蛋白Connexin43，探析GIOP分子机制；观察牛膝-杜仲干预模型小鼠疗效；牛膝活性成分β蜕皮甾酮联合杜仲活性成分，对模型细胞与动物信号通路异常环节与靶点的干预作用。以期揭示牛膝-杜仲有效物质基础，发现新的药理作用及其靶点，提供创制新药方实验依据，丰富“肾主骨生髓”科学内涵。

项目背景：糖皮质激素GC诱导的骨质疏松症（GIOP）是继发性骨质疏松最常见类型，发生率达30～50％。牛膝-杜仲为中医临床高频出现的治疗骨质疏松补肾药对。.研究内容：本项目以斑马鱼、C57BL/6小鼠、MLO-Y4骨样细胞、骨细胞特异性Connexin43（CX43）敲除小鼠为研究对象，以GC处理制作GIOP动物模型与病理细胞模型，进行牛膝-杜仲药对干预GIOP的配比与机制研究。.实验结果：1.通过GIOP斑马鱼模型、均匀设计实验，发现牛膝杜仲配伍具干预GIOP作用，牛膝和杜仲质量浓度比例为1：1时，干预GIOP效果最佳。此比例与经典组方中配伍比例较为一致。2.以牛膝活性成分β-蜕皮甾酮（βEcd）为加载因素，以杜仲各活性成分为影响因素，正交设计实验筛选出干预GIOP的最佳组合，发现βEcd联合松脂醇二葡萄糖苷（PDG）可较有效抑制高浓度GC引起的碱性磷酸酶降低及骨矿化量、骨密度降低。该组合通过抑制地塞米松诱导的氧化应激，更好的保护细胞，效果优于单独使用βEcd或PDG。3.建立GIOP小鼠模型与病理骨细胞模型，βEcd-PDG组合促进GIOP模型小鼠骨形成，增加骨密度、增强骨质量，并抑制骨细胞的凋亡水平。高浓度GC诱发MLO-Y4骨样细胞产生氧化应激，抑制骨细胞活性。βEcd-PDG组合使用可改善骨细胞氧化应激、抑制凋亡，提高骨细胞的活性，其协同作用优于单独用药。同时，Akt/CX43信号通路是βEcd和PDG可能的作用靶点之一。以骨细胞特异性CX43敲除小鼠模型显示，生物力学指标明显下降，CX43与骨质量密切相关。.科学意义：项目从骨细胞网络角度探析GIOP的分子机制与牛膝-杜仲药对干预模型的疗效与机制，并筛选出活性成分最佳组合。揭示了牛膝-杜仲有效物质基础，发现新的药理作用及其靶点，提供创制新药方的实验依据，丰富了中医“肾主骨生髓”科学内涵。