RAS双轴ACE/ANG II/AT1R、ACE2/ANG(1-7)/MAS在高糖致骨代谢异常中的生物学作用及补肾中药活性成分干预研究

Diabetic osteoporosis (DOP) was regarded as the concept of wasting-thirst and atrophic debility of bones. Currently it is necessary to deeply understand DOP from the view of traditional medicine by applying modern biological approach and provide theoretical basis for the prevention and treatment of DOP by integrated Traditional and Western Medicine. The applicant proposed to study the biological effects of ACE/ANG II/AT1R and ACE2/ANG(1-7)/MAS, bi-axis within renin-angiotensin system (RAS), in bone metabolism disorder induced by high glucose level according to the personal serial fundamental studies on DOP, the tissue RAS, and RAS regulatory network. This study will investigate the effects of RAS active peptides, angiotensin II (Ang II) and angiotensin (1-7), on the function of osteoblasts, osteoclasts and the co-culturing system in vitro condition with high glucose level. The potential active compound targeting to inhibit the activity of renin, the rate-limiting step in RAS cascade, will be obtained in kidney-tonifying traditional Chinese medicine (TCM) via combination of virtual screening and in vitro activity tests. Finally the protective effects of potential active compounds through regulating RAS against osteoporosis induced by hyperglycemia will be identified. This project will provide theoretical basis for further clarifying the pathological mechanism of DOP, explore the potential treatment targets for researching and developing novel TCM for anti-DOP, and provide the experimental basis for clinical application of the relevant TCM.

糖尿病性骨质疏松症（DOP）属于“消渴”、“骨痿”范畴。运用现代生物学手段深入理解传统医学对DOP的认识，为中西医结合防治DOP提供理论基础是当务之急。申请者根据自身在DOP、组织肾素-血管紧张素系统（RAS）、RAS调控网络等方面所开展的系列基础研究，提出以RAS双臂ACE/ANGII/AT1R轴、ACE2/ANG(1-7)/MAS轴为基础，探讨其在高糖致骨代谢异常中的生物学作用。主要研究RAS活性肽血管紧张素II、血管紧张素（1-7）对体外高糖培养的成骨、破骨细胞以及共培养体系骨细胞功能的影响；进而，通过虚拟筛选并结合体外活性测试获得潜在的具有抑制RAS级联限速步肾素活性的补肾中药活性成分；最后，评价潜在活性单体的通过调控RAS而抗高血糖诱导骨质疏松症的作用。本研究课题为进一步掌握DOP的病理机制提供理论基础，为研发抗DOP创新中药提供潜在的治疗靶标，为该类中药的临床应用提供实验基础。

骨质疏松与糖尿病是常见的、并发的慢性疾病。本课题探讨肾素-血管紧张素系统（RAS）双轴活性肽Ang II、Ang(1-7)在高糖条件下对骨代谢的调控作用。在高糖环境下（30 mM），Ang II能够抑制小鼠源成骨细胞MC3T3E1矿化结节的生成，并被AT1R受体拮抗剂奥美沙坦（OLM）阻断；Ang(1-7)能够促进成骨细胞矿化结节的生成，并被其受体MAS拮抗剂A779所阻断。在高糖环境下，Ang II、Ang(1-7)分别显著降低、提高MC3T3E1细胞碱性磷酸酶（ALP）活性，并能够被各自的受体拮抗剂OLM、A779所阻断。Ang II、Ang(1-7)分别明显提高、减少原代培养小鼠骨髓巨噬细胞（BMM）经RANKL诱导分化为成熟破骨细胞的数量，Ang II提高破骨侵蚀面积，而Ang(1-7)减少破骨侵蚀面积。Ang II提高小鼠源单核巨噬细胞RAW264.7破骨吸收因子MMP-9、Cathepsin K蛋白表达，而Ang(1-7)抑制这些破骨因子的表达。进一步针对Akt、p38、ERK、JNK四个信号通路的调控作用研究，发现高糖条件下，Ang II在15 min主要激活破骨细胞Akt和p38通路，而在30 min激活JNK通路，而Ang(1-7)显著抑制p38 MAPK和ERK磷酸化。统计文献报道防治糖尿病性骨质疏松的中药使用频次，锁定单味中药丹参、黄芪；再根据两味中药所含单体的主要化学结构并结合已报道的中药单体活性，共选择9个单体化合物进行体外活性筛选。发现丹参酮IIA和槲皮素处理HEK-293T细胞24小时后，呈剂量依赖性抑制Renin活性，且Ang II的蛋白表达显著降低。与糖尿病小鼠相比，丹参酮IIA、槲皮素显著降低糖尿病小鼠血清Ang II含量，两个药物的高剂量组都能明显提高糖尿病小鼠胫骨BMD、BV/TV。HE和TRAP组织形态学染色发现，丹参酮 IIA、槲皮素能够改善糖尿病小鼠股骨松质骨的骨小梁微结构损伤，减少股骨远端破骨细胞阳性染色数量。该项研究进一步明确RAS在糖尿病性骨质疏松中的病理生理作用，初步揭示丹参、黄芪及其有效成分通过作用于RAS改善高糖致骨代谢紊乱的作用，为后续的药物研发提供实验基础。