杜仲籽粕治疗绝经后骨质疏松的药效物质和免疫调节机制研究

It has been demonstrated that the immune dysfunction is much related with the occurance of postmenopausal osteoporosis (PMOP). Eucommia ulmoides Oliv. is the relic plant of the tertiary period survived only in China, its bark was used as superior traditional Chinese medicine that possesses explicit therapeutic action on PMOP. Results from applicant's group recently reported that the draff of Eucommia ulmoides Oliv. seeds (EUSD) after the extracting of the oils and gums contains abundant active compound aucubin, and shows anti-inflammatory effects and significant improvement of bone quality and quantity in osteoporosis mice induced by glucocorticoid. Thus, the applicant hypothesizes that EUSD targeting on immunological modulation might be considered as a novel potential source of therapeutic drug in treatment of PMOP. Accordingly, the study of the current application conducted in PMOP rats will be focused on 1) the extracting,seperating and identifying of the secondary metabolites from EUSD through comparison with reference substances; 2) investigating the effects of the total extracts and main component on growth of osteoblasts and mRNA/protein expressions of osteoproteger; 3) analyzing its inhibitory action on bone losing via immune modulating function in OVX-rats, by monitoring the dynamic levels of E2, IL-6, TNF-a, IGF-1, INF-γ ,ALP and BMD, detecting the levels of serum immunoglobulin and complement, analyzing the cellular immunity through the level of CD4+CD25+Treg T cell. Aiming at systemic, cellular and molecular levels from angels of skeleton, blood and humour, the current application will throughly explores the basis and protential mechanisms involving immune way of Du-zhong seeds draff on PMOP, for the scientific evaluation of the value of EUSD as a new potential drug in prevention and treatment of PMOP. Furthermore, the interrelation of active compounds of traditional Chinese medicine and the kidneytonifying effect, strengthening bone, strengthening and consolidating body will be initially revealed.

最新研究显示免疫功能下降与绝经后骨质疏松症（PMOP）的发病密切相关，杜仲可补益肝肾、强筋骨，常用于治疗PMOP。课题组前期实验发现杜仲籽粕总提取物能够抗炎和改善糖皮质激素所致骨质疏松小鼠骨生物学性质。据此申请者提出杜仲籽粕作为新药物资源可通过免疫调节途径防治PMOP的全新设想。本课题将对籽粕进行系统的提取分离分析；再考察总提取物及各类成分对成骨细胞的生长及骨保护素mRNA和蛋白表达的影响；重点研究它们对PMOP模型大鼠骨流失的抑制，动态监控E2、IL-6、TNF-α、IGF-1、INF-γ、ALP和BMD等指标，检测血清免疫球蛋白和补体含量，对脾脏CD4+CD25+Treg 细胞进行细胞免疫测试。从骨骼、脏器、血液、体液多角度，在系统、细胞、分子多水平上分析籽粕通过免疫途径抗PMOP的药效物质和作用机制，探讨中药补肾-壮骨-扶正固本的内在关系，科学评价籽粕作为新资源的价值。

为了合理利用药材资源，课题组按照计划完成杜仲籽粕治疗绝经后骨质疏松症(PMOP)的药效物质基础和免疫调节机制进行的全面研究。1. 对杜仲籽粕进行了系统的提取分离分析，提取物（ESE）主要含有以桃叶珊瑚苷（AU）为主的环烯醚萜类化合物和少量糖，经优化工艺后AU的提取率达到70%。2. ESE能够促进体外原代培养的成骨细胞增殖并能提高成骨细胞ALP分泌量，促进成骨细胞矿化结节的形成，AU是主要起效成分，干预成骨细胞48h时促进增殖的作用最强，呈剂量依赖关系。3. 利用RealTime-PCR和Western blot法考察了AU对成骨细胞的骨保护素（OPG）及核因子κ B受体活化因子配体（RANKL ）的基因与蛋白表达， AU通过上调OPG的表达、下调RANKL的表达，使OPG竞争性结合RANKL，抑制了RANKL与RANK的结合，从而抑制破骨细胞的活性。4.连续给予双侧摘卵巢术(OVX)大鼠AU 28周，AU可以显著提高OVX大鼠的骨密度，降低机体钙磷流失，增加股骨骨折力和股压碎力，显著性的提升股骨骨小梁数量、增加骨小梁厚度，改善骨微结构，对血清雌激素水平没有影响。5. 采用各组大鼠的含药血清考察AU对小鼠腹腔巨噬细胞、脾淋巴细胞及胸腺细胞增殖的影响，发现OVX组大鼠的含药血清对腹腔巨噬细胞、Con A、LPS诱导的脾淋巴细胞的增殖促进作用明显低于Sham组，各剂量的给药组血清对腹腔巨噬细胞、Con A、LPS诱导的脾淋巴细胞增殖均有促进作用，其中高剂量作用最强；对胸腺细胞的增殖无影响。6. 通过体外实验、流式细胞术、ELISA法、免疫比浊法测定发现AU可以显著升高OVX大鼠脾脏指数，升高免疫球蛋白IgG和IgM 、补体C3和C4 水平，增加脾脏中CD4+CD25+Foxp3+ Treg的表达及CD4+/CD8+，提高IGF-1 含量，降TNF-α及IL-6含量。课题从骨骼、脏器、血液、体液多角度，在系统、细胞、分子多水平上分析籽粕通过免疫途径抗PMOP的药效物质和作用机制，表明AU是杜仲籽粕改善PMOP模型大鼠骨质的关键药效物质，AU不但能够促进成骨细胞增殖、抑制破骨细胞活性，促进骨形成减少骨吸收，还可以提高PMOP模型大鼠免疫力和免疫监控能力，维持免疫系统平衡，说明中药补肾—壮骨—扶正固本的存在一定相关性，杜仲籽粕可作为药用新资源用于PMOP的防治。