基于“肾髓系统”理论——补肾益髓法探讨淫羊藿总黄酮棕色化骨髓白色脂肪防治骨质疏松的机制研究

Previous studies regarding the mechanisms of total flavonoids of epimedium (TFE) as anti-osteoporotic agents only focused on the osteoblast and/or osteoclast itself, which in turn gets us into trouble. Our previous studies suggest that metabolic abnormalities of marrow fat play vital roles on bone deterioration. Osteoporosis may be attributable to “obesity of the bone”. We explored further and found that icariin has the potential to reverse marrow adiposity. However, the underlying mechanisms remain elusive. In the preliminary experiments, we found that TFE upregulates expressions of brown adipocytes in the osteoporotic bone marrow and improves bone quality. The interchangeable characteristics between brown and white adipocytes tempts us to propose the hypothesis that browning marrow white adipocytes may represent a new therapeutic target for the prevention and treatment of osteoporosis. Therefore, we hypothesize that enhancing white-to-brown adipocyte phenotype in bone marrow is a new anti-osteoporotic mechanism of TFE. To prove this hypothesis, we test the effects of TFE on marrow white-to-brown fat in a rat model of osteoporosis and 3T3-L1 adipocytes model using by functional MRI, micro-CT, biomechanical test, RT-PCR, Western blot, and bone histomorphometry. Additionally, we induce the molecular mechanisms of TFE for osteoporosis treatment by using target prediction technology combined with bioinformatics tools to build drug ingredient-core target-disease network and performing enrichment analysis of signaling pathways based on traditional Chinese medicine "Kidney-Marrow System". It’s possible to make innovative breakthroughs to get better insight into other potential mechanisms of TFE for the prevention and treatment of osteoporosis.

淫羊藿总黄酮(TFE)抗骨质疏松(OP)机理研究集中在以成骨细胞和/或破骨细胞为研究对象，难免陷入“就骨论骨”困境。我们前期研究提出骨髓脂质代谢失衡是骨衰败的重要病理基础，骨髓白色脂肪过剩是OP病机之一。我们进一步发现淫羊藿具有抑制骨髓白色脂肪的作用，但具体的调控机理尚未解决。预实验中，我们发现TFE可诱导OP模型骨髓棕色脂肪表达增多，改善骨质量。白色/棕色脂肪互换特性引导我们进一步提出棕色化骨髓白色脂肪是防治OP的假说，推测TFE诱导骨髓白色脂肪“棕色化”是其抗OP的新机制。项目以中医“肾髓系统”补肾益髓法为基石，复制OP动物模型和细胞模型并行TFE干预，应用MRI功能成像、显微CT、生物力学、PCR、免疫印迹、骨组织形态计量学等方法，采用靶点预测技术，结合生物信息学手段，构建药物—靶点—疾病网络，并行信号通路富集分析，归纳TFE治疗OP的分子机制，从功能基因及其作用靶点证明我们的假说。

淫羊藿总黄酮抗骨质疏松机理研究集中在以成骨细胞或破骨细胞为研究对象，难免陷入“就骨论骨”困境。前期研究提出骨髓脂质代谢失衡是骨衰败的重要病理基础，骨髓白色脂肪过剩是骨质疏松病机之一。骨髓脂肪组织具有棕色脂肪特征，进一步研究发现淫羊藿具有抑制骨髓白色脂肪的作用。然而，淫羊藿总黄酮对骨质疏松骨髓棕色脂肪的影响尚不清楚。本研究拟解决这个问题，希望为骨质疏松症预防和治疗提供一个新的靶点。骨发育成熟雌性Sprague-Dawley大鼠随机分为假手术对照组、去势对照组、去势+雌激素阳性对照组、去势+淫羊藿总黄酮干预组。在基线、去势后6周和12周进行化学移位编码磁共振检查以检测股骨的骨髓脂肪分数。采用双能X射线吸收法测量腰椎和股骨的骨密度。ELISA测定血清骨生物标志物，显微CT测定胫骨近端小梁骨微结构，HE染色定量骨髓脂肪细胞参数。实时定量PCR和免疫组化、免疫荧光检测整个胫骨骨髓脂肪细胞基因和蛋白表达谱。发现淫羊藿总黄酮治疗可以降低去势大鼠的骨转换率，改善骨密度和骨小梁微结构。去势导致骨髓脂肪增多，表现为骨髓脂肪分数增加、骨髓脂肪细胞尺寸增大、脂肪细胞数量和脂肪细胞面积百分比增加，骨髓白色脂肪细胞基因和蛋白包括PPARgamma2和FABP4表达增加。淫羊藿总黄酮治疗可恢复雌激素缺乏引起的这些病理变化。淫羊藿总黄酮还增加整个胫骨中棕色脂肪细胞转录因子Ucp1和Prdm16的表达。综上所述，从功能基因及其作用靶点水平揭示雌激素缺乏导致骨质疏松新的病理分子机制，为骨质疏松的防治（骨髓白色脂肪“棕色化”）提供科学的理论依据。淫羊藿总黄酮对去势大鼠骨髓肥胖的调节至少部分是通过维持白色和棕色脂肪组织的相互作用来介导的。明确淫羊藿总黄酮诱导骨髓白色脂肪细胞“棕色化”是其抗原发性骨质疏松的新机制，丰富中医“肾髓系统”理论——补肾益髓法干预骨质疏松的科学内涵，具有明朗的应用前景。