Intra-articular injection of Torin 1 reduces degeneration of articular cartilage in a rabbit osteoarthritis model

Abstract

Objectives: Recent studies have shown that systemic injection of rapamycin can prevent the development of osteoarthritis (OA)-like changes in human chondrocytes and reduce the severity of experimental OA. However, the systemic injection of rapamycin leads to many side effects. The purpose of this study was to determine the effects of intra-articular injection of Torin 1, which as a specific inhibitor of mTOR which can cause induction of autophagy, is similar to rapamycin, on articular cartilage degeneration in a rabbit osteoarthritis model and to investigate the mechanism of Torin 1's effects on experimental OA.

Methods: Collagenase (type II) was injected twice into both knees of three-month-old rabbits to induce OA, combined with two intra-articular injections of Torin 1 (400 nM). Degeneration of articular cartilage was evaluated by histology using the Mankin scoring system at eight weeks after injection. Chondrocyte degeneration and autophagosomes were observed by transmission electron microscopy. Matrix metallopeptidase-13 (MMP-13) and vascular endothelial growth factor (VEGF) expression were analysed by quantitative RT-PCR (qPCR).Beclin-1 and light chain 3 (LC3) expression were examined by Western blotting.

Results: Intra-articular injection of Torin 1 significantly reduced degeneration of the articular cartilage after induction of OA. Autophagosomes andBeclin-1 and LC3 expression were increased in the chondrocytes from Torin 1-treated rabbits. Torin 1 treatment also reduced MMP-13 and VEGF expression at eight weeks after collagenase injection.

Conclusion: Our results demonstrate that intra-articular injection of Torin 1 reduces degeneration of articular cartilage in collagenase-induced OA, at least partially by autophagy activation, suggesting a novel therapeutic approach for preventing cartilage degeneration and treating OA.Cite this article: N-T. Cheng, A. Guo, Y-P. Cui. Intra-articular injection of Torin 1 reduces degeneration of articular cartilage in a rabbit osteoarthritis model. Bone Joint Res 2016;5:218-224. DOI: 10.1302/2046-3758.56.BJR-2015-0001.

Keywords: Articular cartilage degradation; Autophagy; Intra-articular injection; Osteoarthritis; Torin 1.

Histological evaluation of osteoarthritis: (a) there were no degenerative changes of the cartilage in the knees from control rabbits that were treated with saline or Torin 1. The saline-treated rabbits had loss of cartilage surface, Safranin O staining, and had lesions extending into the radial zone at eight weeks after collagenase injection. In contrast, Torin 1-treated rabbits had abrogated loss of Safranin O staining and chondrocyte disappearance in the transitional zone at eight weeks after collagenase injection, and the cartilage surface was preserved. b) The Mankin scores showed that saline-treated rabbits had a significantly higher score than Torin 1-treated rabbits at eight weeks after induction of OA with collagenase injection. In saline-treated rabbits, the score of the collagenase-induced group was significantly higher than the control group. ***p < 0.001 (Magnification, ×100).

Fig. 2

TEM observation. Many more autophagic vacuoles were observed in chondrocytes from Torin 1-treated rabbits than from saline-treated rabbits. In control rabbits treated with saline or Torin 1, chondrocytes located in the lacunae contained autophagic vesicles and were not degenerated. In addition, Golgi apparatus and abundant rough endoplasmic reticulum (RER) were also observed. Chondrocytes from saline-treated rabbits were condensed with absent nuclei and several autophagosomes in the cytoplasm at eight weeks after collagenase injection, and some cell debris were seen in the lacunae. In contrast, chondrocytes from Torin 1-treated rabbits had nuclei and condensed chromatin at eight weeks after collagenase injection (Bar, 1μm).

MMP-13 and VEGF expression (a) and (b): the mRNA expression of MMP-13 and VEGF revealed a significant increase in the saline-treated rabbits at eight weeks after collagenase injection compared with control knees. Torin 1 treatment decreased the expression of MMP-13 and VEGF when compared with saline-treated rabbits at eight-week time points post collagenase injection ***p < 0.001.

Beclin-1 and LC3 expression: a) the expression of Beclin-1 and LC3 evaluated by Western blotting (WB), (b) and (c). The expression of Beclin-1 and LC3 was significantly decreased in saline-treated rabbits at eight weeks post-collagenase injection compared with saline-treated rabbits of the control group. Torin 1 treatment increased Beclin-1 and LC3 expression in control rabbits compared with those treated with saline at eight weeks. Similarly, the expression of Beclin-1 and LC3 was increased in Torin 1-treated rabbits compared with those treated with saline at eight weeks after collagenase injection. **p < 0.01, ***p < 0.001. The WB data were quantified by GEL – DOC 2000 UV GEL imaging system.