去毒附子治疗骨性关节炎的体内药效物质基础及其调控软骨细胞TGF-β信号通路的分子机制研究

Osteoarthritis (OA) belongs to the TCM syndrome ″Gu Bi″ which is caused by liver and kidney deficiency-induced wind-cold damp pathogen. It is commonly occurred in clinic with no effective treatment available. Fuzi with warming and invigorating kidney-yang as well as coldness-and-analgesia expelling effects is suitable for the OA syndrome treatment, but its toxicity has restricted its efficacy. In our preliminary study, we have successfully developed a detoxication strategy for Fuzi and demonstrated the anti-OA effect of detoxicated Fuzi with chondroprotective, analgesia and MMP-13 inhibitory activity. However, the medicinal material basis and mechanism of action of the detoxicated Fuzi remain unclear to date. It is known that MMP-13-induced cartilage erosion is the main pathogenesis of OA, which can be regulated by TGF-β signaling pathway. Accordingly, we proposed a scientific hypothesis that the detoxicated Fuzi contains many blood-migrating anti-OA components which can act on various receptor targets of TGF-β signaling pathway with multi-component and multi-target based mechanism of action. To test such hypothesis, this study plans to screen out effective extracts of detoxicated Fuzi by using transgenetic OA model; to separate and collect the identified single component migrated in blood via metabonomic and serum pharmacochemical methods; and to clarify the TGF-β signaling regulation-based molecular mechanism of each component using RNAi-treated chondrocyte model. This study will reveal the in vivo medicinal material basis and mechanism of action of the detoxicated Fuzi, thereby supporting the development of anti-OA TCMs.

骨性关节炎（OA）属中医"骨痹"，乃肝肾亏虚引风寒湿邪合入所致，缺少有效治疗手段。附子能温补肾阳、祛风散寒，适用于OA证的治疗，但其毒性制约了疗效发挥。本项目组前期建立了附子去毒工艺，首次证明去毒附子能通过保护软骨、缓解疼痛治疗OA，并抑制MMP-13活性，但其药效物质基础和作用机制仍不清楚。已知MMP-13引起的软骨退变是OA的主要病机，受TGF-β信号通路调节。我们由此提出科学假说：去毒附子的血中移行成分能调控TGF-β信号通路各受体靶点，通过多成分作用多靶点的协同机制改善软骨代谢、治疗OA。为验证该假说，本项目拟应用转基因OA模型筛选出去毒附子的有效提取物；以代谢组学和血清药化学的方法对其血中移行组分进行分离、鉴定、纯化和收集；再应用RNAi的软骨细胞模型检测各单一成分调控TGF-β信号的分子机制。通过本研究将揭示去毒附子的体内药效物质基础和作用机制，为抗OA中药的开发研究奠定基础。

附子能温补肾阳、祛风散寒，适用于骨性关节炎（OA）的治疗，但其毒性制约了疗效发挥。本项目组前期建立了附子去毒工艺，首次提出去毒附子能通过保护软骨、缓解疼痛治疗OA科学假说。本项目通过：1）应用高效液相色谱法得到附子不同煎煮时间下三种主要毒性二萜双酯型生物碱成分（乌头碱、中乌头碱、次乌头碱）的性质与含量变化，进而选用ICR小鼠开展急性毒性实验，明确不同煎煮时间对附子急性毒性的影响，验证去毒附子工艺；2）对去毒附子不同提取物/有效部位进行体外药效筛选，明确去毒附子水提物对软骨细胞的促增殖作用最显著，同时应用QTOF-MS飞行时间质谱法解析其主要成分；3）选用SD大鼠建立经典的OA模型，通过小动物成像X-ray、疼痛行为学（von Frey test、自主活动检测）、膝关节组织病理学等方法评价附子久煎去毒后的药效作用，明确了去毒附子可抑制OA大鼠膝关节的破坏作用，有效阻止OA的进展，行为学实验亦揭示去毒附子镇痛、改善疼痛行为的作用，体外实验表明去毒附子含药血清对软骨细胞具有明显的促增值作用，并能显著抑制MIA对软骨细胞的破坏作用，起到软骨保护的作用，其作用依赖浓度和时间的变化，以10%去毒附子含药血清浓度作用最为显著；4）应用LC-MS代谢组学方法分析去毒附子入血代谢成分，并与QTOF-MS分析得到的已知成分进行关联分析可知，去毒附子已知成分与其入血成分之间并未发现直接关联，推断去毒附子并非通过已知成分或其衍生物发挥保护关节软骨的作用；5）应用PCR芯片技术对去毒附子干预前后软骨细胞TGF-β信号通路下游基因的表达情况进行分析，发现去毒附子促进软骨细胞增殖、改善软骨代谢的分子机制可能与TGF-β信号通路无直接相关，仍有待进一步研究。本项目研究将为附子的去毒炮制和临床应用奠定基础，为骨关节病的治疗提供新的途径。本项目成果共发表论文8篇，其中SCI收录3篇，申请发明专利3项，2项授权，培养2名硕士研究生毕业。