基于整合药理学的蒙药方剂塔布森-2治疗骨质疏松症的药效物质基础和分子作用机制研究

With the acceleration of aging process in China, the incidence of osteoporosis has become the third of the chronic diseases. The clinical symptoms of osteoporosis were found to be similar to that of "Guku disease" in traditional Mongolian medicine. The classic Mongolian prescription Tabusen-2 used for the treatment of bone disease was reported to be effective on osteoporosis in clinic. Tabusen-2 is composed of “Lancitou” (inflorescences of Echinops latifolius) and “Duzhong” (barks of Eucommia ulmoides). It was shown effective on osteoporosis model animals. But the effective substances and molecular mechanism of the prescription have not been clarified, and the scientific nature of the similarity between osteoporosis and “Guku disease” has not been disclosed. In this project, these scientific problems will be systematically elucidated based on integrated pharmacology. Firstly, the effective substances and their targets will be predicted by network pharmacological study, and the fingerprints of the prescription in vitro will be established on the basis of the prediction. Then, Caco-2 cell model, everted intestinal sac model and fingerprints of simulated intestinal adsorption solution of Tabusen-2 will be established for determining the absorption ingredients of Tabusen-2 and studying the absorption character of Tabusen-2, and OB-OC cell model will be used to evaluate anti-osteoporosis activity in vitro of the simulated intestinal absorption solution. Furthermore, ovariectomized rats model and metabolic fingerprints will be built for characterizing the blood migrating components and the urine metabolic profiling of Tabusen-2, so then determining effective substances in vivo of Tabusen-2 and the biomarker of the model rats. Afterwards, pharmacokinetic parameters of representative effective compounds of Tabusen-2 will be determined in vivo. Finally, the molecular mechanism of representative effective components and Tabusen-2 will be verified by investigating the regulation of target protein expression in OB-OC cell model and in ovariectomized rats model, respectively. The findings of this project not only provide the modern scientific evidence for the therapeutic effect of Tabusen-2 on osteoporosis, but also open up new ideas and methods for modernized research of Mongolian prescription.

随着我国人口老龄化进程加速，骨质疏松症发病率已居慢性病第三位。当代蒙医发现其与蒙医 “骨枯症”临床症候相似，以塔布森-2治之疗效确切。该方由蓝刺头与杜仲组成，对骨质疏松动物有治疗作用，但药效物质基础、作用机制尚无报道，和 “骨枯症”的联系亦未阐明。本课题基于整合药理学策略解析上述科学问题。先以网络药理学预测本方药效物质群及靶点，以此为据建立体外指纹图谱。后利用Caco-2细胞、外翻肠囊模型和指纹图谱技术，研究本方肠吸收动态，并以OB-OC模型评价肠吸收液的体外抗骨质疏松活性，确定体外效应物质群。同时，利用去卵巢大鼠模型和指纹图谱技术，研究血中移行成分和尿液代谢轮廓，确定体内效应物质群和生物标志物，完善代表成分的药动学参数。最后，利用上述细胞和动物模型，验证代表成分和整方对预测靶蛋白表达量的调节作用。本研究既为塔布森-2治疗骨质疏松提供科学依据，又为蒙医方剂的现代研究提供新思路和新方法。

骨质疏松是一种全身代谢性疾病，随着世界人口老龄化加剧，骨质疏松对人类健康的影响日益严重，成为人类健康的一大难题。当代蒙医发现其与蒙医 “骨枯症”临床症候相似，以塔布森-2治之疗效确切。该方由蓝刺头与杜仲组成，对骨质疏松动物有治疗作用，但药效物质基础、作用机制尚无报道，和 “骨枯症”的联系亦未阐明。本课题采用整合药理学研究策略，整合网络药理学、中药分析学、血清药物化学、药代动力学、药理学、代谢组学、分子生物学等多学科的方法和技术，对塔布森-2治疗骨质疏松症的药效物质基础和分子作用机制进行了系统研究。主要结果如下：（1）基于网络药理学,预测出塔布森-2治疗骨质疏松的关键作用通路3条，潜在关键靶点3个；（2）建立了塔布森-2最佳水提工艺，建立了水提物的HPLC指纹图谱和9种有效候选成分的一测多评含量测定方法；（3）借助肠囊外翻和Caco-2细胞模型，采用LC-MS/MS技术，分别筛选塔布森-2肠吸收原型成分46个和24个，最佳吸收部位为空肠，吸收方式为主动转运和被动扩散；（4）基于血清药物化学方法，采用LC-MS/MS技术，筛选塔布森-2入血原型成分29个；建立了塔布森-2含药血浆中16种成分的LC-MS/MS定量分析方法，确定了相应的药动学参数；（5）方中5个具有良好吸收特性的单体成分可以不同程度增加骨质疏松斑马鱼的椎体矿化面积以及脊椎数，具有显著的抗骨质疏松作用；方中18个具有良好吸收特性的成分对破骨细胞的形成具有不用程度的体外抑制作用。（6）塔布森-2对骨质疏松大鼠的骨密度降低、骨微结构病理改变均具有纠正作用。同时，可下调OVX大鼠血清中VDR、CYP19A1的表达，与网络药理学预测靶点一致。此外，可上调D-gal老年大鼠血清磷和骨钙素含量、下调碱性磷酸酶活性。（7）血清和粪便代谢组学研究均表明维生素B6代谢为塔布森-2调节的主要物质代谢途径，尿液代谢组学分析表明组氨酸代谢为其调节的主要物质代谢途径。结论：塔布森-2治疗骨质疏松的药效物质基础主要为方中部分苯丙素、黄酮、环烯醚萜、木脂素类成分；通过下调VDR、CYP19A1在血液中的表达、干预维生素B6代谢是其抗骨质疏松的主要作用机制。本项目很大程度上阐明了塔布森-2的药效物质基础和作用机制，为本方的临床推广和该起点研发注入了科学内涵，并提供了科学依据，并为蒙医方剂的现代研究提供新思路和新方法。