目的 基于印度刺猬因子（Indian hedgehog，Ihh）-胶质瘤相关癌基因同源物（glioma-associated oncogene homolog，Gli）信号通路探讨乌头碱配伍芍药苷对膝骨关节炎（knee osteoarthritis，KOA）的干预作用及分子机制，并评价其给药安全性。方法 采用前交叉韧带横断手术构建大鼠KOA模型，设置假手术组、模型组、塞来昔布（24 mg/kg）组和乌头碱-芍药苷低（5＋50 μg/kg）、中（10＋100 μg/kg）、高（20＋200 μg/kg）剂量组，每组10只。通过行为学测试评价镇痛效果，采用苏木素-伊红（hematoxylin-eosin，HE）染色与显微计算机断层扫描技术（micro-computed tomography，Micro-CT）观察关节软骨形态及骨微结构变化；ELISA检测血清中基质金属蛋白酶13（matrix metalloproteinase 13，MMP13）和II型胶原蛋白（type Ⅱ collagen，Col Ⅱ）水平；免疫荧光染色和qRT-PCR检测膝关节组织中Ihh、Gli、斑驳蛋白1（patched 1，Ptch1）、MMP13的蛋白和基因表达；采用分子对接验证乌头碱、芍药苷与Ihh-Gli信号通路关键靶点的结合能力。通过检测血清天冬氨酸氨基转移酶活性及肌酐、血尿素氮、尿蛋白水平，并采用HE染色法对各组大鼠心脏、肝脏及肾脏组织进行形态学分析，以评价乌头碱配伍芍药苷的安全性。结果 给药期间各组大鼠未见死亡及中毒症状，血清肝、肾功能指标及心、肝、肾组织形态学均无显著异常，表明乌头碱配伍芍药苷具有较好的安全性。与假手术组比较，模型组大鼠机械痛阈值和热痛阈值显著降低（P＜0.01、0.001），关节软骨表面破坏、潮线不完整，软骨下骨丢失且骨小梁分布紊乱；血清中MMP13水平显著升高（P＜0.001），Col II水平显著降低（P＜0.001）；膝关节组织中Ihh、Gli、Ptch1、MMP13的蛋白和基因表达均显著上调（P＜0.001），提示Ihh-Gli通路在KOA状态下异常激活。与模型组比较，乌头碱配伍芍药苷能提高大鼠机械痛阈值和热痛阈值（P＜0.05、0.01、0.001），修复软骨基质，改善骨微结构，血清中MMP13升高、Col II水平降低（P＜0.05、0.01、0.001），膝关节组织中Ihh、Gli、Ptch1、MMP13的蛋白和基因表达均显著下调（P＜0.05、0.01、0.001）。分子对接结果显示，乌头碱、芍药苷与Ihh、Gli、含凝血酶敏感蛋白基序的解整合素金属蛋白酶5（a disintegrin and metalloproteinase with thrombospondin motifs 5，ADAMTS5）、MMP13的结合能均≤-5 kcal/mol，表明乌头碱、芍药苷与Ihh-Gli通路关键靶点具有较强的结合能力。结论 乌头碱配伍芍药苷可通过抑制Ihh-Gli信号通路异常激活，下调MMP13表达并上调Col II表达，抑制软骨基质降解，改善KOA大鼠关节功能与软骨病理损伤，且具有良好的安全性，为乌头汤核心药对治疗寒湿痹阻型KOA提供了实验依据。

Objective To investigate the therapeutic effect and molecular mechanism of aconitine combined with paeoniflorin on knee osteoarthritis (KOA) based on Indian hedgehog (Ihh)-glioma-associated oncogene homolog (Gli) signaling pathway, as well as to evaluate the safety of this combined administration. Methods Anterior cruciate ligament transection was performed to establish a rat KOA model. Sham group, model group, celecoxib (24 mg/kg) group, and aconitine-paeoniflorin low- (5 + 50 μg/kg), medium- (10 + 100 μg/kg), and high-(20 + 200 μg/kg) dose groups were set up, with 10 rats in each group. Behavioral tests were conducted to assess analgesic efficacy. Hematoxylin-eosin (HE) staining and micro-computed tomography (Micro-CT) were used to observe morphological changes of articular cartilage and bone microarchitecture. ELISA was used to detect levels of matrix metalloproteinase 13 (MMP13) and type Ⅱ collagen (Col Ⅱ) in serum. Immunofluorescence staining and qRT-PCR were applied to determine the protein and gene expressions of Ihh, Gli, patched 1 (Ptch1) and MMP13 in knee joint tissues. Molecular docking was performed to verify the binding affinity between aconitine, paeoniflorin and core targets of Ihh-Gli signaling pathway. Activity of aspartate aminotransferase and levels creatinine, blood urea nitrogen, urinary protein in serum were detected, and morphological analysis on heart, liver and kidney tissues of rats in each group were observed by HE staining to evaluate the safety of aconitine combined with paeoniflorin. Results During the administration period, no death or toxic symptoms were observed of rats in each group. There were no significant abnormalities in serum liver and kidney function indicators, as well as in the morphology of heart, liver and kidney tissues, indicating that the combination of aconitine and paeoniflorin had good safety. Compared with sham group, the mechanical pain threshold and thermal pain threshold of rats in model group were significantly reduced (P < 0.01, 0.001), the articular cartilage surface was damaged, the tidemark was incomplete, the subchondral bone was lost, and the distribution of bone trabeculae was disordered. The level of MMP13 in serum was significantly increased (P < 0.001), while the level of Col II was significantly decreased (P < 0.001). The protein and gene expressions of Ihh, Gli, Ptch1 and MMP13 in knee joint tissue were significantly upregulated (P < 0.001), indicating abnormal activation of Ihh-Gli pathway in KOA state. Compared with model group, the combination of aconitine and paeoniflorin could increase the mechanical pain threshold and thermal pain threshold of rats (P < 0.05, 0.01, 0.001), repair cartilage matrix, improve bone microstructure, increase MMP13 level and decrease Col II level in serum (P < 0.05, 0.01, 0.001), and significantly downregulate the protein and gene expressions of Ihh, Gli, Ptch1 and MMP13 in knee joint tissue (P < 0.05, 0.01, 0.001). The molecular docking results showed that the binding energies of aconitine and paeoniflorin to Ihh, Gli, a disintegrin and metalloprotease with thrombospondin motifs 5 (ADAMTS5) and MMP13 were all ≤ −5 kcal/mol, indicating that aconitine and paeoniflorin have strong binding abilities to key targets of Ihh-Gli pathway. Conclusion The combination of aconitine and paeoniflorin can inhibit abnormal activation of Ihh-Gli signaling pathway, downregulate MMP13 expression and upregulate Col II expression, inhibit cartilage matrix degradation, improve joint function and cartilage pathological damage in KOA rats, and has a good safety, providing experimental evidence for the treatment of cold dampness obstruction type KOA with the herb pair of Wutou Decoction.

R285.5

河北省国际合作专项（19392507D）；国家科技部对发展中国家科技援助项目（KY201904005）