The Role Of Autophagy In Knee Osteoarthritis Of Rabbit

Objective: The purpose of this study was to determine the changes of autophagy in the progress of osteoarthritis(OA) and the specific role of autophagy in OA, and to investigate the therapeutic effects and mechanism of Torin 1 on rabbit OA model by intra-articular injection. Methods: 1. A rabbit OA model was established by intra-articular injection of collagenase(type II), followed by treatment with an autophagy specific inhibitor, 3-Methyladenine(3-MA). Six animals from each group were sacrificed at two, four, six, and eight weeks after the initial injection. Cartilage degeneration was examined by light microscope, autophagosomes and chondrocytes degeneration were observed by transmission electron microscopy. Expression of Beclin-1 and light chain 3B(LC3B) was evaluated by western blotting. 2. A rabbit OA model was established by intra-articular injection of collagenase, followed by treatment with Torin 1 or 3-MA. All animals were sacrificed at the end of 8 weeks after the initiation of experiment. The caitilage degeneration was examined by histological evaluation, chondrocytes degeneration and autophagosomes were observed by transmission electron microscopy. Expression levels of Beclin-1 and LC3 B were evaluated by western blotting. 3. Collagenase was injected twice into both knees of 3-month-old rabbits to induce OA, combined with twice intra–articular injection of Torin 1. Degeneration of articular cartilage was evaluated by histology using the Mankin scoring system at 8 weeks after injection. Chondrocytes degeneration and autophagosomes were observed by transmission electron microscopy. Matrix metallopeptidase-13(MMP-13) and vascular endothelial growth factor(VEGF) expressions were analyzed by quantitative real time PCR(q PCR). Beclin-1 and LC3 B expressions were examined by Western blotting. Results: 1. The autophagy was enhanced in the early stages and weakened in the late stages of experimental OA, and inhibition of autophagy by 3-MA significantly aggravated the degeneration of chondrocytes and cartilage in experimental OA. 2. The degeneration of chondrocytes and cartilage was closely related to the loss of autophagy in the late stage of experimental OA. Autophagy inhibition by 3-MA increased the severity of chondrocytes and cartilage degeneration, while Torin 1 reduced that by autophagy activation. 3. Intra-articular injection of Torin 1 significantly reduced degeneration of articular cartilage and chondrocytes after induction of OA with collagenase injection. The autophagosomes, Beclin-1 and LC3 B expression were promoted in chondrocytes from Torin 1-treated rabbits. Torin 1 treatment also reduced MMP-13 and VEGF expressions at 8 weeks after collagenase injection. Conclusion: 1. Our results determine the changes of autophagy in different stages of OA and the role of impaired autophagy in the development of OA, suggesting that regulation of autophagy may be a potential therapeutic strategy to ameliorate OA. 2. The loss of autophagy is closely linked with the late stage of experimental OA and autophagy plays a protective role in the pathogenesis of it. Treatment of Torin 1 is effective to inhibit the pathological progression of OA and it may be a useful therapeutic drug for OA. 3. Intra-articular injection of Torin 1 reduces degeneration of articular cartilage in collagenase-induced OA by autophagy activation, suggesting a useful tip for clinical treatment of OA.