基于三维M-Act/Tox协同评价的抗骨质疏松中药筛选新方法的建立与应用

Hepatotoxicity of bone-strengthening traditional medicine has aroused increasing attention. The action (Act) / toxicity (Tox) of Traditional Chinese medicine (TCM) are closely related with in vivo metabolism (Metabolism, M), so it is very important for considering M-Act/Tox in anti-osteoporosis screening of TCM, but we need to break through the technology bottleneck of integrative evaluation on M-Act/Tox of micro-amount components with in vivo and high efficient manner..In previous foundations, we estabolished drug metabolism model and anti- osteoporosis screening model using zebrafish suitable for micro-amount components, and we found that zebrafish make drug metabolism, and produce activity or company toxicity. So that, we presumed that if these three links of metabolism, anti-osteoporosis bioactivity and toxicity were detected after zebrafish dosed respectively, then integrative evaluation on M-Act/Tox by in vivo and high efficient manner can be realized. In addition, zebrafish, the current in vivo and in vitro models has one’s own advantages and disadvantages, if these three models bombined organicly, then the three dimensional M-Act/Tox evaluation method with collaborative and synergetic characteristic was developted. .Juvenile zebrafish were separated into healthy veichle group and osteoporosis model group, after exposed to test compounds (icariin for example) in micro plates, the zebrafish were divided into three parts for detection: activity assay on mineralization and markers of zebrafish sketal,metabolites detection with LC/MS, and toxicity assay of LC50 and main organ toxicity of fish with microscopic detection, thus the integrative M-Act/Tox of micro-amount components were realized high efficiently using zebrafish for preliminary anti-osteoporosis screening, in addition, metabolism, anti-osteoporosis activity and toxicity of the object were studied futhurly and complementary using current in vivo and in vitro models, thus collaborative evaluation method of three dimensional M-Act/Tox were developted. This novel method will be applied for screening the relative Act/Tox material basis of representive bone-strengthening formula/herb (Zhuangguguanjie pill / Epimediifolium.), which will provide novel ideas and methods for effective screening in vivo and in vitro anti-osteoporosis components of TCM with considering both effectiveness and safety.

壮骨中药肝毒已受重视。中药效(Act)/毒(Tox)与在体代谢(M)密切相关，故抗骨质疏松中药筛选关注M-Act/Tox至关重要，但需突破量微成分三者一体化在体、高效评价瓶颈。.前期基金建立量微成分斑马鱼代谢和抗骨质疏松筛选法，发现鱼使药物代谢、产生效应或伴毒性。故推测，给鱼测试药后分各环节检测，可实现M-Act/Tox一体化在体、高效评价。另斑马鱼与现有体内、外模型各具优势与不足，有机联合，达三维M-Act/Tox协同共进。.将斑马幼鱼置微板造模、给药,分别检测鱼骨矿化与标志物；代谢物LC/MS分析；鱼LC50与脏器毒显微检测，实现量微成分抗骨质疏松M-Act/Tox高效初筛，再用现有体内、外模型完善深入目标物M-Act/Tox，即成三维M-Act/Tox协同评价法。将其用于代表方/药（壮骨关节丸/淫羊藿）的效/毒筛选，为兼顾体内、外成分有效性与安全性的抗骨质疏松中药筛选提供思路方法。

针对量微成分无法高效进行在体代谢、抗骨质疏松活性及毒性评价的难点与瓶颈，建立斑马鱼M-Act/Tox一体化评价新方法，进一步基于斑马鱼、体外和体内模型的各自优势与局限，将三者有机联合，形成三维M-Act/Tox协同评价新方法。应用三维M-Act/Tox协同评价法筛选壮骨关节丸的效/毒药味：斑马鱼Act/Tox评价发现：补骨脂、淫羊藿、续断、骨碎补、狗脊、鸡血藤、熟地黄能够对抗泼尼松龙诱导的斑马鱼骨质疏松。补骨脂、续断、乳香没药、独活和木香，鸡血藤和狗脊（醇提液），3dpf鱼卵黄囊肿大、变形或变黑，提示与肝毒相关，6dpf鱼的LC50值较小（33.2~392.7µg生药/mL），提示毒性较大，淫羊藿毒性次之，桑寄生、熟地黄和骨碎补的安全性较好，与文献报道具一致性。进一步用三维M-Act/Tox协同评价法筛选淫羊藿的抗骨质疏松效/毒物质基础：（1）斑马鱼的代谢研究发现：淫羊藿黄酮成分具共同的脱糖基代谢规律，易于脱去葡萄糖基和木糖基，难于脱去鼠李糖基，多种黄酮在体内转化成共含鼠李糖基的箭藿苷C或宝藿苷I。（2）斑马鱼骨质疏松模型评价发现：不同品种、产地淫羊藿均有抗骨质疏松活性；淫羊藿总黄酮（含8个代表黄酮）是抗骨质疏松主要活性成分，主要作用机制为下调RANKL基因表达，抑制骨吸收，与现有体内外模型报道结论一致；有机酸类成分（如绿原酸）具有一定的抗骨质疏松活性，而生物碱和多糖没有活性。（3）斑马鱼毒性评价发现：不同品种、产地淫羊藿对斑马鱼的毒性具有差异。不同组分安全性具差异：淫羊藿生物碱、黄酮组分及部分黄酮成分对斑马鱼有一定毒性，有机酸类，多糖成分安全性最好；淫羊藿黄酮单体的安全性不同：毒性由大到小为箭藿苷B，箭藿苷C >朝藿定C，箭藿苷A >淫羊藿苷，宝藿苷I，朝藿定A和朝藿定B。绿原酸、新绿原酸、隐绿原酸及木兰花碱毒性较低。斑马鱼M-Act/Tox评价是关注体内、外成分，兼顾有效性与安全性的抗骨质疏松筛选新方法，条件简单，化合物用量少，高效率，低成本，高灵敏。斑马鱼与体内、外评价方法优势互补、有机呼应。