This study aims to investigate the effects of Jiawei Duhuo Jisheng Mixture on mitochondrial autophagy in the cartilage of rabbits with knee osteoarthritis(KOA) based on the PTEN-induced kinase 1(PINK1)/Parkinson protein(Parkin) pathway and explore its potential mechanism in improving cartilage lesions. A KOA model was established by fixing a high-molecular resin plaster bandage on the right hind limb of the rabbits for six weeks. After successful modeling, the modeling group was randomly divided into a model group, a celecoxib group, and low-and high-dose groups of Jiawei Duhuo Jisheng Mixture, with eight rabbits in each group. The celecoxib group was administered celecoxib by gavage at a single dose of 0.009 3 g·kg~(-1). The low-and high-dose groups of Jiawei Duhuo Jisheng Mixture were given Jiawei Duhuo Jisheng Mixture at single doses of 6.8 mL·kg~(-1)(4.515 2 g·kg~(-1)) and 27.2 mL·kg~(-1)(18.060 8 g·kg~(-1)), respectively. Administered once daily for six weeks, the rabbits in each group then underwent behavioral testing. After sample collection, the gross morphological changes of the knee articular cartilage were observed with the naked eye. Hematoxylin-eosin(HE) staining was used to detect pathological changes in cartilage tissue, which were quantitatively evaluated by using the Lequesne MG score, Pelletier score, and Mankin score. Transmission electron microscopy was used to observe the ultrastructural changes of chondrocyte mitochondria. Flow cytometry was used to detect the mitochondrial membrane potential(Δψm) and the average fluorescence intensity of reactive oxygen species(ROS) in chondrocytes and calculate the percentage of cells with low Δψm. Western blot was used to detect the expression level of mitochondrial autophagy-related proteins in cartilage tissue, including PINK1, Parkin, selective autophagy adapter protein 62(P62), light chain 3(LC3)Ⅱ/LCⅠ, mitochondrial outer membrane translocase 20(TOM20), collagen type Ⅱ alpha 1(COL2A1), aggrecan(ACAN), matrix metalloproteinase(MMP)-9, and MMP-13. Immunohistochemistry(IHC) was used to detect the expression of PINK1, Parkin, and LC3B in cartilage tissue. The results showed that, compared with the blank group, the model group exhibited marked knee joint swelling and damage, tissue fibrosis, sparse chondrocyte distribution, and indistinct and incomplete tide marks. The Lequesne MG, Pelletier, and Mankin scores increased significantly. Autophagosomes were reduced, and mitochondria were morphologically abnormal. The percentage of chondrocytes with low Δψm, ROS average fluorescence intensity, and the expression of P62, TOM20, MMP-9, and MMP-13 proteins in cartilage tissue rose significantly, while the expression of PINK1, Parkin, LC3Ⅱ/LCⅠ, COL2A1, and ACAN proteins in cartilage tissue decreased significantly. Compared with the model group, the celecoxib and both Jiawei Duhuo Jisheng Mixture groups showed improved knee articular cartilage surface, relatively intact tide marks, and denser chondrocytes. Their Lequesne MG, Pelletier, and Mankin scores dropped significantly. Autophagosomes increased, and mitochondrial swelling and damage eased. The percentage of chondrocytes with low Δψm, ROS average fluorescence intensity, and the expression of P62, TOM20, MMP-9, and MMP-13 proteins in cartilage tissue decreased significantly, while the expression of PINK1, Parkin, LC3Ⅱ/LCⅠ, COL2A1, and ACAN proteins in cartilage tissue increased significantly. In conclusion, Jiawei Duhuo Jisheng Mixture can effectively activate the PINK1/Parkin pathway to promote mitochondrial autophagy and alleviate articular cartilage damage in rabbits with KOA, thus slowing the progression of KOA.
