去毒附子汤对证干预阳虚寒湿型骨关节炎的网络分子机制研究

Osteoarthritis (OA) belongs to the TCM syndrome "Gu Bi". The intrinsic pathogenesis of OA is yang-deficiency, and its manifestation is cold-damp blockage. The syndrome yang-deficiency and cold-damp is commonly seen in OA patients. Fuzi decoction could warm yang and dispelcold, which is suitable for the treatment of such OA syndrome. However, the toxicity of Fuzi (Aconitum carmichaeli) has hindered its clinical application. In our preliminary study, we have established a detoxication method for Fuzi and achieved its detoxication with saving efficacy. By using detoxicated Fuzi, a detoxicated Fuzi decoction could be made with its detoxication with saving efficacy could also be achieved. It is known that multi-pathway modulated inflammatory degeneration of synovium and cartilage is the major character of OA. Therefore, we proposed a scientific hypothesis: detoxicated Fuzi decoction can be safely and specifically used for treating the syndrome yang-deficiency and cold-damp of OA, and its action mechanism is related to the modulation on a multi-target and multi-pathway based molecular network. To verify such hypothesis, this study plans to establish a rat OA model with syndrome yang-deficiency and cold-damp to reveal the molecular pathogenesis of OA and to verify the syndrome-specific efficacy and advantage of detoxicated Fuzi decoction. Then we suppose to establish a methodology of "metabolic network pharmacology" to reveal the multi-herb and multi-component based targets and pathways, and to clarify the syndrome-specific molecular network mechanism of detoxicated Fuzi decoction based on its "multi-component to multi-target to multi-pathway" character. This study would provide new ideas for study of OA syndrome and TCM formula, and also provide scientific evidence for clinical use and secondary development of detoxicated Fuzi decoction.

骨关节炎（OA）属中医“骨痹”，以阳虚为本，寒湿痹阻为标，多见阳虚寒湿证。附子汤能温阳散寒，适用于阳虚寒湿证的治疗，但附子毒性影响了其临床应用。项目组前期建立附子去毒工艺，实现附子的去毒存效。选用去毒附子组成去毒附子汤，能同样使附子汤去毒存效。已知滑膜和软骨的炎性病变是OA主要特征，受多通路调控。在上述基础上，我们提出科学假说：去毒附子汤适用于阳虚寒湿型OA的对证治疗，且安全有效，其作用机制与多靶点多通路的分子网络调控有关。为验证该假说，本项目拟建立大鼠阳虚寒湿OA模型，在揭示其分子病机的同时，验证去毒附子汤的对证功效和安全优势，进而建立“代谢网络药理学”方法揭示该方多药多成分对应的作用靶点和通路，基于“多成分→多靶点→多通路”阐明该方对证干预OA的网络分子机制。本项目将为OA中医证候及中药复方的研究提供新的思路、为去毒附子汤的临床应用和二次开发提供科学依据。

骨关节炎（OA）属中医“骨痹”多见阳虚寒湿证。附子汤能温阳散寒，适用于阳虚寒湿证的治疗，但附子毒性影响了其临床应用.项目组前期建立附子去毒工艺，实现附子的去毒存效。已知滑膜和软骨的炎性病变是OA主要特征，受多通路调控。项目组提出科学假说:去毒附子汤适用于阳虚寒湿型OA的对证治疗，且安全有效，其作用机制与多靶点多通路的分子网络调控有关。本项目结果显示,结果显示利用大鼠OA模型,通过HE,TB和ABH染色显示附子汤逆转软骨明显退变，软骨细胞减少和凋亡，胶原蛋白大量破坏，基质紊乱，明确了去毒附子汤干预阳虚寒湿型 OA 的药效作用。进一步通过网络药理学分析和RNA测序结果，得到附子汤靶基因与筛选的骨关节炎靶点的65 个交叉基因以及靶向PI3K/Akt 信号通路。最终通过细胞实验和分子实验验证附子汤基于PI3K/Akt信号通路调控Col2,Col10,MMP9和MMP13等基因和蛋白表达。进一步探讨附子对 OA 的潜在作用机制，我们研究了附子的化学成分乌头碱、新乌头碱、次乌头碱、苯甲酰乌头碱、苯甲酰新乌头碱、苯甲酰次乌头碱对软骨细胞的影响和分子作用，结果表明苯甲酰乌头碱和苯甲酰次乌头碱可能是附子发挥保护 OA 作用的主要成分，而苯甲酰新乌头碱则对附子保护 OA 有不利的影响。为去毒附子汤的临床应用和二次开发提供科学依据。