Background: Osteoarthritis (OA) is characterized by degeneration of articular cartilage. Studies have found that enhancement of autophagy, an intracellular catabolic process, may limit the pathologic progression of OA. Chloramphenicol is a potent activator of autophagy; however, the effects of chloramphenicol on articular cartilage are unknown.
Questions/purposes: Using human OA knee chondrocytes in vitro, we asked, does chloramphenicol (1) activate autophagy in chondrocytes; (2) protect chondrocytes from IL-1β-induced apoptosis; and (3) reduce the expression of matrix metallopeptidase (MMP)-13 and IL-6 (markers associated with articular cartilage degradation and joint inflammation). Using an in vivo rabbit model of OA, we asked, does an intra-articular injection of chloramphenicol in the knee (4) induce autophagy; (5) reduce OA severity; and (6) reduce MMP-13 expression?
Methods: Human chondrocytes were extracted from 10 men with OA undergoing TKA. After treatment with 25 μg/mL, 50 μg/mL, or 100μg/mL chloramphenicol, the autophagy of chondrocytes was detected with Western blotting, transmission electron microscopy, or an autophagy detection kit. There were four groups in our study: one group was untreated, one was treated with 100 μg/mL chloramphenicol, another was treated with 10 ng/mL of IL-1β, and the final group was treated with 10 ng/mL of IL-1β and 100 μg/mL of chloramphenicol. All groups were treated for 48 hours; cell apoptosis was detected with Western blotting and flow cytometry. Inflammation marker IL-6 in the cell culture supernatant was detected with an ELISA. Articular cartilage degradation-related enzyme MMP-13 was analyzed with Western blotting. A rabbit model of OA was induced by intra-articular injection of type II collagenase in 20 male 3-month-old New Zealand White rabbits' right hind leg knees; the left hind leg knees served as controls. Rabbits were treated by intra-articular injection of saline or chloramphenicol once a week for 8 weeks. Autophagy of the articular cartilage was detected with Western blotting and transmission electron microscopy. Degeneration of articular cartilage was analyzed with Safranin O-fast green staining and the semi-quantitative index Osteoarthritis Research Society International (OARSI) grading system. Degeneration of articular cartilage was evaluated using the OARSI grading system. The expression of MMP-13 in articular cartilage was detected with immunohistochemistry.
Results: Chloramphenicol activated autophagy in vitro in the chondrocytes of humans with OA and in an in vivo rabbit model of OA. Chloramphenicol inhibited IL-1-induced apoptosis (flow cytometry results with chloramphenicol, 25.33 ± 3.51%, and without chloramphenicol, 44.00 ± 3.61%, mean difference, 18.67% [95% CI 10.60 to 26.73]; p = 0.003) and the production of proinflammatory cytokine IL-6 (ELISA results, with chloramphenicol, 720.00 ± 96.44 pg/mL, without chloramphenicol, 966.67 ± 85.05 pg/mL; mean difference 74.24 pg/mL [95% CI 39.28 to 454.06]; p = 0.029) in chondrocytes. After chloramphenicol treatment, the severity of cartilage degradation was reduced in the treatment group (OARSI 6.80 ± 2.71) compared with the control group (12.30 ± 2.77), (mean difference 5.50 [95% CI 1.50 to 9.50]; p = 0.013). Furthermore, chloramphenicol treatment also decreased the production of MMP-13 in vitro and in vivo.
Conclusions: Chloramphenicol reduced the severity of cartilage degradation in a type II collagen-induced rabbit model of OA, which may be related to induction of autophagy and inhibition of MMP-13 and IL-6.
Clinical relevance: Our study suggests that an intra-articular injection of chloramphenicol may reduce degeneration of articular cartilage and that induction of autophagy may be a method for treating OA. The animal model we used was type II collagen-induced OA, which was different from idiopathic OA and post-traumatic OA. Therefore, we need to use other types of OA models (idiopathic OA or a surgically induced OA model) to further verify its effect, and the side effects of chloramphenicol also need to be considered, such as myelosuppression.