Department of Orthopaedics, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, China.
Respiratory and Thoracic Surgery Ward, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, China.
Osteoarthritis Cartilage. 2017 Nov;25(11):1912-1921. doi: 10.1016/j.joca.2017.08.003. Epub 2017 Aug 18.
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.
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.
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-α, IL-1β 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.
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.
利用慢病毒载体介导的短发夹 RNA（LV-shRNA）下调软骨细胞中瘦素受体功能性同工型（Ob-Rb）的表达，并确定其对软骨退变的影响。
在体外，采用实时定量聚合酶链反应（qRT-PCR）和蛋白质印迹法筛选最佳 Ob-Rb LV-shRNA（LV-shRNA3），并检测其对培养的大鼠软骨细胞中 9 种与 OA 相关的介质的影响。在体内，通过手术诱导大鼠右膝关节 OA 模型，并向关节内注射 LV-shRNA3、慢病毒载体介导的非靶向对照序列（LV-NTC）或磷酸盐缓冲液。采用骨关节炎研究协会国际评分（OARSI）评估软骨退变，并对上述各组 OA 相关介质的表达进行免疫组织化学染色。
LV-shRNA3 可显著下调培养软骨细胞中的 Ob-Rb 表达。伴随着 Ob-Rb 的下调，促炎介质（TNF-α、IL-1β 和 IL-6）和分解代谢介质（ADAMTS-5、MMP-9、NOS-2 和 COX-2）的表达水平也显著降低，而合成代谢型 II 型胶原的表达水平显著增加。体内研究结果显示，LV-shRNA3 可显著降低 OARSI 评分。免疫组织化学分析表明，LV-shRNA3 可显著下调软骨细胞中的 Ob-Rb 表达。伴随着 Ob-Rb 的下调，ADAMTS-5 和 MMP-9 的表达水平也显著降低，而 II 型胶原的表达水平增加。
这些结果表明，LV-shRNA3 介导的软骨细胞 Ob-Rb 下调抑制了 OA 大鼠模型中的软骨退变，提示 Ob-Rb 可能是 OA 治疗的新靶点。
