淫羊藿苷对原发性骨质疏松骨髓脂肪酸的作用及规律研究

The findings of our previous studies suggested marrow adipogenesis occurs in synchrony with deterioration of trabecular microarchitecture and bone marrow adipocytes steady-state lost is the main pathophysiological basis of the changes of bone microstructure in osteoporosis. The significant increase in adipocyte volume in osteoporosis is not merely a "passive" vacuum filling process to compensate deterioration in bone structure. Mechanism research of Icariin for prevention and treatment of osteoporosis was mostly limited to osteoblast and osteoclast per se. The project was directed by the traditional Chinese medicine theory "kidney-essence-marrow-bone" and based on the hypothesis of inhibiting bone marrow adipocytes generation for prevention and treatment of osteoporosis. Metabolomics method using Magnetic Resonance Spectroscopy and gas chromatograph-mass spectrometry will be applied to quantify the changing regularity of bone marrow fatty acids of primary osteoporosis model in rabbits treated with Icariin. Micro-CT scanning and biomechanical testing in vitro were performed on lumbar vertebrae in rabbits. Marrow peroxisome proliferation-activated receptor r2 mRNA was measured by quantitative Real-Time PCR. Histopathology of lumbar vertebrae was underwent for the mean density and diameter of adipocytes. The purpose of this study is to explore the common metabolic features of bone marrow fatty acids of primary osteoporosis treated with Icariin, which clarify the effect and action mechanism of Icariin on prevention and treatment of osteoporosis. Our second objective was to prove the hypothesis that down-regulating excess fat of bone marrow is a new mechanism for osteoporosis management and prevention, which will generate innovative breakthrough in the mechanism study of osteoporosis and provide new theoretical basis and clinical application for the treatment of osteoporosis from a new perspective.

课题组前期研究表明骨髓脂肪失稳态是骨质疏松(OP)骨超微构筑改变的重要基础，骨髓脂肪增多不是骨小梁衰败的简单代偿性填充。淫羊藿苷(ICA)抗OP机理研究大部分以成骨细胞和破骨细胞两种终末细胞为研究对象，建立在这种"就骨论骨"研究理论基础上的治疗就难免单纯局限于破骨细胞和成骨细胞本身。本项目拟在"肾-精-髓-骨"中医理论指引下，以抗"骨髓脂肪过剩"也是治疗OP的机制为假说，基于磁共振波谱、色谱-质谱联用技术的代谢组学方法对ICA干预去势雌兔OP模型骨髓脂肪酸进行定量分析，并对椎体行显微CT扫描、生物力学试验及骨髓PPARr2 mRNA表达及病理检查，捕捉ICA抗OP骨髓脂肪酸共同的代谢特征，从骨髓代谢组学水平阐明ICA抗OP的作用及其机制；证明抗骨髓脂肪过剩是防治OP的机制，其结果将对OP防治的机理研究有创新性突破，有望从新的视角为OP防治提供新的理论基础及临床应用前景。

原发性骨质疏松(OP)最根本的原因是雌激素缺乏引发骨形成与骨吸收之间平衡失调，导致骨重建紊乱。课题组前期研究表明骨髓脂肪失稳态是OP骨超微构筑改变的重要基础，骨髓脂肪增多并不是骨小梁衰败的简单代偿性填充。以往淫羊藿苷(ICA)抗OP机理研究大部分以成骨细胞和/或破骨细胞两种终末细胞为研究对象，建立在这种"就骨论骨"研究理论基础上的治疗就难免单纯局限于破骨细胞(抑制骨吸收)和/或成骨细胞(促进骨形成)本身。然而，ICA如何影响骨髓脂肪组织仍不清楚。. 本项目以抗"骨髓白色脂肪过剩"也是治疗OP的机制为假说，在"肾-精-髓-骨"中医理论指引下，基于MRI功能成像(包括磁共振波谱技术、Dixon水-脂分离成像技术)及GC-MS联用技术的代谢组学研究方法对ICA干预去势SD大鼠OP模型骨髓脂肪酸进行定量分析，并对胫骨及椎体行显微CT扫描、生物力学试验、骨代谢血清学检测、骨髓病理组织学检查脂肪细胞定量分析、骨髓RT-PCT及Western blot检查(Runx2、PPARgamma2、Tracp)。. 本实验项目在按照原定技术路线基础上进行了研究，基本上完成了课题的初期设想和实验目标。发现骨髓不饱和脂肪酸对维持骨质量表现出正性作用，而骨髓饱和脂肪酸则对骨质量表现为脂毒性作用。早期ICA干预OP动物模型，ICA对骨表现出双重作用，即抑制骨吸收（血清CTX-I水平降低，Tracp mRNA及其蛋白表达低下）、促进骨形成（血清BALP水平增高，Runx2mRNA及其蛋白高表达）；其次，ICA在保护雌激素缺乏所致的骨丢失同时还表现出抑制雌激素缺乏所致的骨髓白色脂肪组织增多（ICA降低OVX动物模型骨髓脂肪细胞密度、脂肪细胞直径及脂肪细胞面积百分比，降低骨髓间充质干细胞特异性成脂转录因子PPARgamma2 mRNA及其蛋白的表达）。. 该项目证实了OP归因于“骨的肥胖”，骨髓脂肪代谢紊乱是OP新的致病机制，为OP的防治提供了新的靶点。项目也证明了ICA抗OP的机制除了抑制骨吸收、促进骨形成外还可通过调控骨髓腔的脂肪含量起作用，即验证了ICA抗骨髓白色脂肪过剩是其防治OP的新机制的假说，其结果将对ICA防治OP的机理研究有创新性突破，从新的视角为OP防治提供新的理论基础及临床应用前景。