Lentiviral vector-mediated shRNAs targeting a functional isoform of the leptin receptor (Ob-Rb) inhibit cartilage degeneration in a rat model of osteoarthritis

Objective: To downregulate the expression of leptin receptor functional isoform (Ob-Rb) on chondrocytes using lentiviral vector-mediated short-hairpin RNA (LV-shRNA) and to determine its effects on cartilage degeneration. Method: In vitro, quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting were performed to select an optimal Ob-Rb LV-shRNA (LV-shRNA3) and to determine its effects on nine OA-related mediators in cultured rat chondrocytes. In vivo, an OA model was surgically induced in the right knees of rats, and LV-shRNA3, lentiviral vector-mediated non-targeting control sequence (LV-NTC) or phosphate buffered saline was injected into the joints. Osteoarthritis Research Society International (OARSI) scoring was performed to assess cartilage degeneration, and immunohistochemistry was performed to evaluate OA-related mediator expression in the above groups. Results: Ob-Rb expression was significantly downregulated by LV-shRNA3 in cultured chondrocytes. In conjunction with Ob-Rb downregulation, the expression levels of pro-inflammatory mediators (TNF-alpha, IL-1 beta and IL-6) and catabolic mediators (ADAMTS-5, MMP-9, NOS-2 and COX-2) were also significantly decreased, and the expression levels of anabolic type II collagen were significantly increased. The in vivo study results showed that OARSI scores were significantly decreased by LV-shRNA3. Immunohistochemistry analysis demonstrated that Ob-Rb expression levels on chondrocytes were significantly downregulated by LV-shRNA3. In conjunction with Ob-Rb downregulation, ADAMTS-5 and MMP-9 expression levels were also significantly decreased, and type II collagen expression levels were increased. Conclusion: These results indicate that LV-shRNA3-mediated Ob-Rb downregulation on chondrocytes inhibits cartilage degeneration in a rat model of OA, suggesting that Ob-Rb may be a novel target in the treatment of OA. (C) 2017 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.