从彝药斯赤列中寻找具有NOX4抑制活性的抗骨质疏松活性化合物

Increasement of NOX4 expression induced the boost of ROS, which was the important factor to destroy the balance of osteoblast and osteoclast differentiation. Sichilie was used to treat traumatic injury, bone fractures, joint pain, et al. As an ethnic and folk herbal medicines, the raw material resources of the plant is abundant. We investigated the chemical constituents and the activities for promoting bone fracture healing of the extracts of Sichilie in previous research. Some triterpenoids and other chemical constituents were isolated and identified. Although the effective phytochemical constituents were not discovered, it’s very interesting that the extracts of Sichilie was active to preventing osteoporosis by inhibiting the upregulation of NOX4. Base on the previous studies and abundent valuable references, the research methods of phytochemical compositions combining activities were projecting to know the active principles for preventing osteoporosis by inhibiting the upregulation of NOX4. Chromatograph and spectrascopy methods would be utilized to isolated and identified the active chemical constituents. By the means of the overexpression of NOX4 in HEK293 cell, the activies of compounds inhibiting NOX4 would be investigated. Furthermore, osteoclast precursors cells and osteoblast cells from the aging rats model with BMSCs would play a role finding the active compounds which could stimulate osteoblast differentiation and inhibit osteoclast differentiation. Then repeat-dose oral studies of these effective compounds were done in senescene accelarated mice (SAMP6) to find the valuable compounds and demonstrate the phytochemical structure-efficacy relationship. To improve the quality standard of the herbal material, HPLC and LC-MS would be used to analyse the fingerprint chromatograms. Above all, the project was significant to find the compounds inhibiting NOX4 from the extracts of Sichilie, which could set an example for the systematic study of the Yi People’s herbal mdedicine.

衰老造成的NOX4表达上升诱导ROS大量生成是导致成骨与破骨分化失衡的重要原因。斯赤列是彝族传统用药，广泛用于治疗骨质疏松、骨折等疾病。我们前期研究发现斯赤列提取物中含有大量三萜类化合物，且具有较好的促进成骨细胞分化、改善骨质疏松的作用并显示出一定的NOX4抑制活性，但具体作用物质基础与作用机制不明。因此，本项目拟建立高表达NOX4的HEK293细胞模型并结合色谱方法，系统分离彝药斯赤列抑制NOX4活性的化合物，以氧化应激致大鼠BMSCs衰老模型成骨细胞和破骨前体细胞模型进一步筛选出具有调节成骨和破骨功能的活性化合物，再以自发性老年骨质疏松SAMP6小鼠模型考察其体内活性，筛选出活性优良的化合物，并进行构效关系分析，采用HPLC、LC-MS法建立药材的指纹图谱。通过本项目研究，有望从彝药斯赤列中筛选出以NOX4为靶点的抗骨质疏松活性化合物，为我省丰富的彝药资源提供系统的基础研究示范。

“从彝药斯赤列中寻找具有NOX4抑制活性的抗骨质疏松活性化合物” (项目编号：81660716)课题组按任务计划(2017.1-2020.12)，合理使用实验经费，开展了相应实验。.利用硅胶等多种色谱方法，分别从斯赤列各萃取物中分离得到40个化合物。综合利用各种谱图解析方法，鉴定其中38个化合物结构，20个为三萜类，8个为甾体类，10个为其他类型化合物，如黄酮类、木脂素类等。其中11个化合物为首次从该植物中分离得到。.分别利用相似度评价、主成分分析和聚类分析三种方法对不同斯赤列样品的UPLC指纹图谱进行评价，不同产地和不同采收期的斯赤列样品中11个指标成分的含量存在一定差异，说明产地和采收期是影响其质量的重要因素，但产地对其质量的影响相对更大；LC-MS研究结果提示，选择分两个方法采集比较可行：采用1 mM甲酸铵-乙腈为流动相，在ESI-模式下和ESI+模式下各分析了3个指标化合物。.课题组利用ORAC方法开展体外抗氧化能力，其挥发油效果相对较好。体外细胞实验和整体动物实验分别标明：斯赤列2种提取物均可提高成骨细胞的增殖能力、促进碱性磷酸酯酶和骨矿化结节的形成，成骨细胞分化基因RUNX2、BSP、OCN和COL1A1相对表达量均有上升，均可刺激成骨细胞的增殖与分化。且XMC-B的活性强于XMC-A的。斯赤列提取物对Ⅱ型胶原蛋白诱导的大鼠类风湿性关节炎模型具有明显的对抗作用，可减轻大鼠足趾肿胀度，显著减少血清中炎症因子IL-6的含量，有降低血清中炎症因子TNF-α含量趋势。通过比较7种化合物的6个浓度的体外抗炎作用发现，均具有一定的抗炎作用，且抗炎作用随化合物浓度的增加而增加。骨质疏松整体动物实验，因疫情和实验周期问题，该部分实验还需后续实验研究进行完善。.综合分析，斯赤列提取物中的三萜类可能是促进骨细胞生长，从而发挥改善骨质疏松作用的成分类型。.已发表论文3篇，其中1篇为SCI收录,1篇为中文核心期刊收录；中文核心期刊已录取1篇待出版；培养硕士研究生2名；2人晋升职称；申请与该项目相关专利2篇，未获得授权。