Abstract: BACKGROUND: Modified Du Huo Ji Sheng Decoction has clinical advantages in the treatment of knee osteoarthritis. However, the specific mechanism by which it ameliorates cartilage degeneration through regulating the Hedgehog signaling pathway remains unclear.
OBJECTIVE: To investigate the protective effects of modified Du Huo Ji Sheng Decoction on cartilage degeneration in a mouse model of knee osteoarthritis.
METHODS: Fifty male C57BL/6J mice were randomly divided into five groups: sham-operated group, model group, low-dose modified Du Huo Ji Sheng Decoction group, high-dose modified Du Huo Ji Sheng Decoction group, and celecoxib group (positive control). Mice in the sham-operated group underwent only skin incision and joint cavity exposure as a simulated surgical procedure, while mice in the other groups underwent medial meniscus destabilization surgery to establish a knee osteoarthritis model. Fourteen days after modeling, intragastric administration was initiated. The low-dose and high-dose modified Du Huo Ji Sheng Decoction groups received doses of 1.34 and 4.02 g/kg, respectively, and the celecoxib group received 26 mg/(kg·d) celecoxib. All treatments were administered once daily for 8 weeks. Histopathological staining, Micro-CT scanning, immunohistochemistry, real-time quantitative PCR, and western blot assay were employed to assess the degree of cartilage degeneration, subchondral bone microstructure, cartilage matrix metabolism-related proteins (type II collagen and matrix metalloproteinase 13), and the expression levels of key Hedgehog signaling pathway genes and proteins (Patched 1, Indian hedgehog, and Smoothened).
RESULTS AND CONCLUSION: (1) Toluidine blue staining results showed that, compared with the sham-operated group, the model group exhibited erosive damage on the cartilage surface, disordered tissue structure, and an elevated Osteoarthritis Research Society International score. Compared with the model group, each dosage group of modified Du Huo Ji Sheng Decoction showed restored cartilage surface smoothness, progressively regular cartilage arrangement, and reduced Osteoarthritis Research Society International scores. (2) Micro-CT three-dimensional reconstruction of the knee joint subchondral bone showed that, compared with the sham-operated group, the model group exhibited significantly reduced medullary cavity area in the subchondral bone region, presenting a hardened and dense subchondral bone structure, with increased bone volume fraction and trabecular thickness, and decreased trabecular number and trabecular separation. Compared with the model group, each dosage group of modified Du Huo Ji Sheng Decoction showed increased medullary cavity area in the subchondral bone, reduced subchondral bone sclerosis, decreased bone volume fraction and trabecular thickness, and restoration of trabecular number and trabecular separation to near-normal levels. (3) Immunohistochemical results showed that type II collagen expression was decreased and matrix metalloproteinase 13 expression was increased in the cartilage of the model group. After intervention with modified Du Huo Ji Sheng Decoction, their expression was restored. (4) TUNEL staining results showed that the number of positive cells in the cartilage of the model group was increased, and after intervention with modified Du Huo Ji Sheng Decoction, the number of positive cells was significantly reduced. (5) Real-time quantitative PCR results showed that the expression of Hedgehog signaling pathway-related genes Patched 1, Indian hedgehog, and Smoothened was increased in the cartilage of the model group. After intervention with modified Du Huo Ji Sheng Decoction, these showed a dose-dependent decrease. (6) Western blot results showed that the expression of Patched 1, Indian hedgehog, Smoothened, and matrix metalloproteinase 13 was increased, and type II collagen expression was decreased in the cartilage of the model group. After intervention with modified Du Huo Ji Sheng Decoction, this trend was significantly reversed. These findings indicate that modified Du Huo Ji Sheng Decoction can restore the balance between synthesis and catabolism of the cartilage matrix, inhibit chondrocyte apoptosis, and delay cartilage degeneration in knee osteoarthritis by regulating the Hedgehog signaling pathway.

Key words: modified Du Huo Ji Sheng Decoction, knee osteoarthritis, cartilage degeneration, Hedgehog signaling pathway