Takayama Koji, Kawakami Yohei, Kobayashi Makoto, Greco Nick, Cummins James H, Matsushita Takehiko, Kuroda Ryosuke, Kurosaka Masahiro, Fu Freddie H, Huard Johnny
Arthritis Res Ther. 2014 Nov 17;16(6):482. doi: 10.1186/s13075-014-0482-4.
Recent studies have revealed that rapamycin activates autophagy in human chondrocytes preventing the development of osteoarthritis (OA) like changes in vitro, while the systemic injection of rapamycin reduces the severity of experimental osteoarthritis in a murine model of OA in vivo. Since the systemic use of rapamycin is associated with numerous side effects, the goal of the current study was to examine the beneficial effect of local intra-articular injection of rapamycin in a murine model of OA and to elucidate the mechanism of action of rapamycin on articular cartilage.
Destabilization of the medial meniscus (DMM) was performed on 10-week-old male mice to induce OA. Intra-articular injections of 10 μl of rapamycin (10 μM) were administered twice weekly for 8 weeks. Articular cartilage damage was analyzed by histology using a semi-quantitative scoring system at 8 and 12 weeks after surgery. Mammalian target of rapamycin (mTOR), light chain 3 (LC3), vascular endothelial growth factor (VEGF), collagen, type X alpha 1 (COL10A1), and matrix metallopeptidase 13 (MMP13) expressions were analyzed by immunohistochemistry. VEGF, COL10A1, and MMP13 expressions were further examined via quantitative RT-PCR (qPCR).
Intra-articular injection of rapamycin significantly reduced the severity of articular cartilage degradation at 8 and 12 weeks after DMM surgery. A reduction in mTOR expression and the activation of LC3 (an autophagy marker) in the chondrocytes was observed in the rapamycin treated mice. Rapamycin treatment also reduced VEGF, COL10A1, and MMP13 expressions at 8 and 12 weeks after DMM surgery.
These results demonstrate that the intra-articular injection of rapamycin could reduce mTOR expression, leading to a delay in articular cartilage degradation in our OA murine model. Our observations suggest that local intra-articular injection of rapamycin could represent a potential therapeutic approach to prevent OA.
最近的研究表明，雷帕霉素可激活人软骨细胞中的自噬，在体外预防骨关节炎（OA）样改变的发生，而在体内OA小鼠模型中全身注射雷帕霉素可减轻实验性骨关节炎的严重程度。由于全身使用雷帕霉素会产生许多副作用，因此本研究的目的是在OA小鼠模型中研究关节内局部注射雷帕霉素的有益作用，并阐明雷帕霉素对关节软骨的作用机制。
对10周龄雄性小鼠进行内侧半月板失稳（DMM）手术以诱导OA。每周两次关节内注射10μl雷帕霉素（10μM），持续8周。在术后8周和12周，使用半定量评分系统通过组织学分析关节软骨损伤情况。通过免疫组织化学分析雷帕霉素靶蛋白（mTOR）、微管相关蛋白1轻链3（LC3）、血管内皮生长因子（VEGF）、X型胶原α1链（COL10A1）和基质金属蛋白酶13（MMP13）的表达。通过定量逆转录聚合酶链反应（qPCR）进一步检测VEGF、COL10A1和MMP13的表达。
关节内注射雷帕霉素可显著降低DMM手术后8周和12周时关节软骨降解的严重程度。在接受雷帕霉素治疗的小鼠中，观察到软骨细胞中mTOR表达降低以及LC3（自噬标志物）激活。雷帕霉素治疗还降低了DMM手术后8周和12周时VEGF、COL10A1和MMP13的表达。
这些结果表明，在我们的OA小鼠模型中，关节内注射雷帕霉素可降低mTOR表达，从而延缓关节软骨降解。我们的观察结果表明，局部关节内注射雷帕霉素可能是预防OA的一种潜在治疗方法。
