目的 探讨乌头汤通过调控印度刺猬因子（Indian hedgehog，Ihh）-胶质瘤相关癌基因同源物（glioma-associated oncogene homolog，Gli）信号通路治疗膝骨关节炎（knee osteoarthritis，KOA）的作用机制。方法SD雄性大鼠随机分为假手术组、模型组、塞来昔布（24 mg/kg）组和乌头汤低、中、高剂量（1.05、2.10、4.20 g/kg）组，每组10只。采用改良前交叉韧带切除技术建立KOA模型，给予药物干预8周后，测定机械痛阈值、热痛阈值及爬坡能力；ELISA检测血清中基质金属蛋白酶-3（matrix metalloproteinase-3，MMP-3）、MMP-13和II型胶原蛋白（type Ⅱ collagen，Col Ⅱ）水平；采用苏木素-伊红（hematoxylin-eosin，HE）染色观察膝关节的形态学变化；采用显微计算机断层扫描技术（micro-computed tomography，Micro-CT）分析膝关节的骨微结构变化；采用Western blotting检测膝关节Ihh、斑驳蛋白1（patched 1，Ptch1）、Gli和MMP-13蛋白表达。结果 与假手术组比较，模型组大鼠软骨表层缺损、变薄，细胞减少且排列紊乱，膝关节横截面和冠状面骨小梁减小，分布稀疏，骨密度降低，热痛阈值和机械痛阈值显著降低（P＜0.001），斜坡角度变小（P＜0.001），血清中MMP-3、MMP-13水平显著升高（P＜0.05、0.01），Col Ⅱ水平显著降低（P＜0.05），膝关节Ihh-Gli信号通路相关蛋白表达水平显著升高（P＜0.001）。与模型组比较，乌头汤组大鼠软骨表面平整，细胞数量增多且形态完整，膝关节横截面和冠状面骨小梁增多，分布密集，热痛阈值和机械痛阈值显著升高（P＜0.05、0.001），斜坡角度变大（P＜0.01、0.001），血清中MMP-3、MMP-13水平显著降低（P＜0.05），Col Ⅱ水平显著升高（P＜0.05），膝关节Ihh-Gli信号通路相关蛋白表达水平显著降低（P＜0.05、0.001）。结论 乌头汤通过抑制Ihh-Gli信号通路过度激活，减少软骨细胞异常增殖与分化，从而保护关节软骨。

Objective To explore the mechanism of Wutou Decoction (乌头汤) in treating knee osteoarthritis (KOA) by regulating Indian hedgehog factor (Ihh)-glioma associated oncogene homolog (Gli) signaling pathway. Methods Male SD rats were randomly divided into sham group, model group, celecoxib (24 mg/kg) group, Wutou Decoction low-, medium-, and high-dose (1.05, 2.10, 4.20 g/kg) groups, with 10 rats in each group. The improved anterior cruciate ligament resection technique was used to establish a KOA model, after eight weeks of drug intervention, mechanical pain threshold, thermal pain threshold and climbing ability were measured. ELISA was used to detect the levels of matrix metalloproteinase-3 (MMP-3), MMP-13 and type II collagen (Col II) in serum. The morphological changes of knee joint were observed by hematoxylin-eosin (HE) staining. Micro computed tomography (Micro-CT) technology was used to analyze the changes in bone microstructure of knee joint. Western blotting was used to detect the expressions of Ihh, patched 1 (Ptch1), Gli and MMP-13 proteins in knee joint. Results Compared with sham group, rats in model group showed defects and thinning of cartilage surface layer, decreased and disordered cell arrangement, decreased cross-sectional and coronal bone trabeculae, sparse distribution, decreased bone density, thermal pain threshold and mechanical pain threshold were significantly reduced (P < 0.001), slope angle was decreased (P < 0.001), levels of MMP-3 and MMP-13 in serum were significantly increased (P < 0.05, 0.01), Col II level was significantly decreased (P < 0.05), and expression levels of Ihh-Gli signaling pathway related proteins in knee joint were significantly increased (P < 0.001). Compared with model group, the cartilage surface of rats in Wutou Decoction group was smooth, the number of cells was increased, and the morphology was intact. The knee joint cross-section and coronal bone trabeculae were increased, and the distribution was dense. The thermal pain threshold and mechanical pain threshold were significantly increased (P < 0.05, 0.001), slope angle was increased (P < 0.01, 0.001), levels of MMP-3 and MMP-13 in serum were significantly reduced (P < 0.05), level of Col II was significantly increased (P < 0.05), and expression levels of Ihh-Gli signaling pathway related proteins in knee joint were significantly reduced (P < 0.05, 0.001). Conclusion Wutou Decoction protects articular cartilage by inhibiting excessive activation of Ihh-Gli signaling pathway, reducing abnormal proliferation and differentiation of chondrocytes.

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）