肉苁蓉中抑制Cathepsin K活性成分筛选及抗骨质疏松作用机制研究

Cistanche deserticola Y. C. Ma, an important invigorant herb in traditional Chinese medicine (TCM), has been used for treatment of antiosteoporotic diseases with other TCM for many years. In our previous study, the antiosteoporotic activity of this herb has been proved both in vitro and in vivo. Furthermore, the cathepsin K, a key enzyme responsible for osteoclast-mediated bone resorption, was identified as the antiosteoporotic targets of this herb by molecular docking technology. In present study, the antiosteoporotic active compounds will be isolated, screened and identified from Cistanche deserticola based on the inhibition of osteoclast cathepsin K activity in vitro; the HPLC fingerprint of those bioactive constituents will be established; the key proteins (p-ERK、p-PI3K、RANK、TRAF6 and AP-1) and transcription factors (RANK、TRAF6、AP-1、NFATc1 and cFos), which are related to antiosteoporotic targets in two signal pathway (ERK1/2/MAPK and PI3K/Akt), will be expressed by using western-blot and real-time PCR techniques; the ovariectomized rats will be used to evaluate the antiosteoporotic properties of the active compounds and fractions, the biochemical and bone metabolic markers as well as bone mineral density, mechanics and histomorphometry will be measured using samples of urine, serum, femur, femur and tibia, respectively. Finally, the antiosteoporotic mechanism of Cistanche deserticola will be interpreted from the results of the above tests.

肉苁蓉是传统补肾类中药，常以君臣类药组方治疗骨质疏松。前期我们已通过体内外实验初步证实肉苁蓉具有抗骨质疏松作用，并以分子对接筛选出其作用靶点为骨吸收过程的关键酶-组织蛋白酶K。本课题中我们以体外抑制破骨细胞组织蛋白酶K活性为导向进行肉苁蓉抗骨质疏松有效成分群的分离和鉴定，并建立有效部位HPLC指纹图谱；采用Western-blot和RT-PCR技术对与骨质疏松靶点相关的2条信号通路{MAPK（ERK1/2）和PI3K／Akt}上的关键蛋白(p-ERK、p-PI3K、RANK、TRAF6和AP-1)及转录因子(RANK、TRAF6、AP-1、NFATc1和cFos)进行表达；以去卵巢致骨质疏松大鼠为模型动物进行药效学研究,检测实验动物血清和尿液生化指标、骨吸收代谢相关指标以及骨密度、骨机械力学和骨组织形态计量学等指标；综合动物、细胞、蛋白和基因分子水平结果阐述肉苁蓉抗骨质疏松作用机制。

肉苁蓉是传统补肾类中药，依据“肾主骨”理论和肉苁蓉常以君臣类药组方治疗骨质疏松的文献基础，项目以体外抑制骨吸收指标组织蛋白酶K活性为导向，采用树脂、硅胶、凝胶和制备HPLC等手段，进行了肉苁蓉抗骨质疏松有效成分群的分离和鉴定，共分离40个化合物，鉴定了37个化合物，其中苯乙醇苷类化合物9个，重量在克以上的化合物有5个。采用AB-8大孔吸附树脂，以水、20%乙醇、50%乙醇梯度洗脱，收集40%和50%乙醇洗脱部位进行二次洗脱，最终收集40%洗脱部位为肉苁蓉总苯乙醇有效部位，建立了其HPLC特征图谱。采用新生SD乳鼠成骨细胞和RAW264.7诱导破骨细胞为筛选介质，对分离得到9个肉苁蓉苯乙醇苷类化合物进行体外抗骨质疏松活性筛选；针对毛蕊花糖苷、肉苁蓉苷A、紫丁香苷、2-乙酰毛蕊花糖和6-乙酰毛蕊花糖骨质疏松大鼠和小鼠骨组织的RANK、TRAF6、NFKBIA、RANKL、OPG、PI3K和Akt关键蛋白酶表达进行了研究，阐明了肉苁蓉苷A、紫丁香苷等化合物是通过抑制TRAF6蛋白介导的NF-kappaB信号通路和PI3K/Akt信号通路以及提高OPG/RANKL比值起抗骨质疏松药效作用的。对肉苁蓉提取物、肉苁蓉总苯乙醇苷、毛蕊花糖苷、肉苁蓉苷A、紫丁香苷、2-乙酰毛蕊花糖和6-乙酰毛蕊花糖进行了系统的大鼠和小鼠去卵巢抗骨质疏松作用研究。通过micro-CT、骨机械力学、骨形成和骨吸收指标等的测定，确定了肉苁蓉提取物、总苯乙醇苷部位和各化合物具有抗骨质疏松的药效作用，具体表现为各药物改善了骨小梁疏松度、增加了骨密度、抑制了骨吸收指标TRAP、Cathepsin K和DPD/Cr的活性，但对骨形成指标ALP和BGP并显著作用，即以上各药物是通过抑制骨吸收起抗骨质疏松的药效作用。.通过本项目的研究，确定了肉苁蓉提取物、总苯乙醇苷有效部位、毛蕊花糖苷、肉苁蓉苷A、紫丁香苷、2-乙酰毛蕊花糖和6-乙酰毛蕊花糖抗骨质疏松药效作用；以去卵巢大鼠、破骨和成骨细胞、RANK等关键蛋白酶等结果阐明了肉苁蓉抗骨质疏松分子机制。以上结果为肉苁蓉及其有效成分抗骨质疏松的成药性研究提供了实验数据。本项目形成研究论文4篇，其中SCI论文2篇；申请发明专利6项。