仙灵骨葆通过雄激素信号通路增强骨骼肌功能的物质基础和作用机制研究

Decreased skeletal muscle function and low bone mineral density both increase the risk of fracture. At present, we have focused on bone mineral density for the drugs preventing bone fracture, but neglected the function on skeletal muscle. Traditional Chinese medicine Xian Ling Gu Bao (XLGB) including Epimedium was clinically approved effective on improving bone mineral density; our latest study found that XLGB was also able to increase the contractility of skeletal muscles, which is consistent with the theory of traditional Chinese Medicine for “improving both bone and muscle”. Skeletal muscle is the target organ of androgen, and serum testosterone is positively relevant to the function of skeletal muscle and low fracture rate. Therefore, we speculate that the substances which can stimulate androgen signaling pathway are the material basis of XLGB on enhancing the function of skeletal muscle. Firstly, we will adopt C2C12 cells and Hershberger assay to screen the compounds which can promote myogenesis and stimulate androgen signaling pathway; Secondly, we will use DXA, muscle electrical signal acquisition, grasping force measurement and muscle strength testing in the castrated rats model to investigate the effects of XLGB and the bioactive single compound(s) on enhancing the function of skeletal muscle; and finally we will use receptor binding affinity, the reporter gene et al to study the mechanism of the bioactive compound(s) on myogenesis by regulating androgen signaling pathway. This research aims to elucidate the bioactive substance(s) and mechanism of XLGB on enhancing the function of skeletal muscle, which will rich the theory of Chinese medicine on " improving both muscle and bone” and “the same therapy for different symptoms"; and we also expect to find phyto-androgens and provide novel structures for developing new drugs on prevention of fractures.

骨骼肌功能下降和骨密度降低均会增加骨折发生风险。目前预防骨折药物的评价重在关注骨密度，而忽略了骨骼肌功能。淫羊藿为主的中药仙灵骨葆临床疗效确切，能够提高骨密度；我们最新的研究发现仙灵骨葆还可以增加骨骼肌收缩力，符合中医“强肌健骨”治则。骨骼肌是雄激素作用的靶器官，并且血清睾酮与骨骼肌功能和低骨折率正相关，我们推测仙灵骨葆中能够调控雄激素信号通路的成分是增强骨骼肌功能的物质基础。本项目拟采用成肌细胞和赫斯伯格分析法筛选出具有成肌作用和雄激素样作用的成分，再使用去势大鼠模型、通过肌电信号采集、抓力测量和肌力测试等方法验证仙灵骨葆和活性成分增强骨骼肌功能的疗效，进而利用受体结合和报告基因等方法研究活性成分调控雄激素受体信号通路的作用机制。课题将阐明仙灵骨葆增强骨骼肌功能的药效物质和分子机制，丰富中医“强肌健骨，异病同治”的理论认识；并有望从中发现新型植物雄激素，为预防骨折的药物的研发提供新结构。

人口老龄化导致骨质疏松骨折带来巨大的经济社会负担，当前单一改善骨密度的药物预防骨折作用有限。基于中医“强肌健骨”的治则，复方中药能实现肌、骨同治，是预防骨折的新策略。在本项目支持下，我们进一步探索复方中药仙灵骨葆（XLGB）增加骨骼肌功能的药效物质基础和作用机制。第一部分：我们对仙灵骨葆中含量较高的17个化合物进行了促进成肌管作用的体外活性筛选，发现马钱苷酸、当药苷、淫羊藿苷和鼠李糖基淫羊藿次苷II能够促进C2C12细胞的成肌分化。第二部分：我们利用Hershberger分析方法，评价了包含了三种结构类型的5个化合物的雄激素类似物作用，发现均未表现雄激素激动或拮抗作用。第三部分：在大鼠实验中进一步确认了XLGB和活性成分淫羊藿苷能够增加骨骼肌最大强直力，宏基因组分析发现，XLGB和淫羊藿苷能够通过改善肠道菌群，首次揭示复方中药及单体成分调控肠-肌肉轴的重要功能。利用BaCl2诱导骨骼肌损伤动物模型，发现活性成分淫羊藿苷和鼠李糖基淫羊藿次苷II能够促进骨骼肌修复。进一步采用去睾丸大鼠（ORX）肌少症模型，发现淫羊藿苷可以增强大鼠的抓握能力，增强比目鱼肌和趾长伸肌的收缩能力，增加胫前肌和腓肠肌的肌纤维束的横截面积。第四部分：利用蛋白印迹和Q-PCR荧光定量的方法分析发现，淫羊藿苷主要通过抑制FOXO3a蛋白的表达和促进FOXO3a的磷酸化，下调FOXO3a/MuRF-1和Atrogin-1信号通路，抑制肌肉组织中蛋白的降解。.通过本项目的实施，确认了复方中药仙灵骨葆能够增加骨骼肌强功能，阐明了仙灵骨葆增强骨骼肌功能的药效物质主要是倍半萜类和异戊烯基黄酮类化合物；机制研究方面，发现淫羊藿苷并未调控雄激素信号通路，而是通过下调FOXO3a信号通路抑制骨骼肌降解，为开发治疗肌少症及预防骨质疏松骨折提供新的先导化合物结构类型。项目执行期间，共发表期刊论文9篇，申请发明专利3项，其中1项已授权，参加国内国际相关专业学术会议5次以上。