Affiliated Hospital of Nanjing University of Chinese Medicine, Nanjing 210029, China; Jiangsu Province Hospital of Chinese Medicine, Nanjing 210029, China; Key Laboratory for Metabolic Diseases in Chinese Medicine, First College of Clinical Medicine, Nanjing University of Chinese Medicine, Nanjing 210023, China.
Affiliated Hospital of Nanjing University of Chinese Medicine, Nanjing 210029, China; Jiangsu Province Hospital of Chinese Medicine, Nanjing 210029, China.
Life Sci. 2019 Jul 1;228:158-166. doi: 10.1016/j.lfs.2019.05.003. Epub 2019 May 2.
Chondrocyte apoptosis is the most common pathological feature of cartilage in osteoarthritis (OA). Excessive mechanical stress can induce chondrocyte apoptosis and destroy cartilage tissue. Transient receptor potential channel vanilloid 4 (TRPV4) is a mechanosensitive ion channel that mediates chondrocyte response to mechanical stress. Here, we investigated the potential role of TRPV4 in chondrocyte apoptosis induced by excessive mechanical stress.
Using a rat OA anterior cruciate-ligament transection (ALCT) model, we detected immunolocalization of calmodulin protein and mRNA and protein levels of TRPV4, calmodulin, and cleaved caspase-8 in articular cartilage. Primary chondrocytes were isolated and cultured in vitro, and Fluo-4AM staining was used to assess intracellular Ca levels in order to evaluate TRPV4-mediated Ca influx. Flow cytometry and western blot were performed to detect apoptosis and apoptosis-related protein levels in chondrocytes, respectively.
TRPV4 was upregulated in ALCT-induced OA articular cartilage, and we found that administration of a TRPV4 inhibitor attenuated cartilage degeneration. Additionally, TRPV4 specifically mediated extracellular Ca influx, leading to chondrocyte apoptosis in vitro, which was inhibited by transfection of TRPV4 small-interfering RNA or administration of a TRPV4 inhibitor. Moreover, increased Ca influx triggered apoptosis by upregulating FAS-associated protein with death domain and cleaved caspase-3, -6, -7, and -8 levels, with these effects abolished by TRPV4 knockdown or TRPV4 inhibition.
These results indicated that TRPV4 was upregulated in OA articular cartilage, and that excessive mechanical stress might induce chondrocyte apoptosis via TRPV4-mediated Ca influx, suggesting TRPV4 as a potential drug target in OA.
软骨细胞凋亡是骨关节炎（OA）中软骨最常见的病理特征。过度的机械应力可诱导软骨细胞凋亡并破坏软骨组织。瞬时受体电位通道香草素 4（TRPV4）是一种机械敏感的离子通道，介导软骨细胞对机械应激的反应。在这里，我们研究了 TRPV4 在过度机械应力诱导的软骨细胞凋亡中的潜在作用。
使用大鼠 OA 前交叉韧带切断（ALCT）模型，我们检测了关节软骨中钙调蛋白蛋白和 mRNA 的免疫定位以及 TRPV4、钙调蛋白和裂解 caspase-8 的蛋白水平。分离并体外培养原代软骨细胞，并用 Fluo-4AM 染色评估细胞内 Ca 水平，以评估 TRPV4 介导的 Ca 内流。通过流式细胞术和 Western blot 分别检测软骨细胞的凋亡和凋亡相关蛋白水平。
TRPV4 在 ALCT 诱导的 OA 关节软骨中上调，我们发现 TRPV4 抑制剂的给药可减轻软骨退变。此外，TRPV4 特异性介导细胞外 Ca 内流，导致体外软骨细胞凋亡，而转染 TRPV4 小干扰 RNA 或给予 TRPV4 抑制剂可抑制该作用。此外，增加的 Ca 内流通过上调 Fas 相关死亡结构域蛋白和裂解 caspase-3、-6、-7 和 -8 的水平引发凋亡，这些作用可通过 TRPV4 敲低或 TRPV4 抑制消除。
这些结果表明 TRPV4 在 OA 关节软骨中上调，过度的机械应力可能通过 TRPV4 介导的 Ca 内流诱导软骨细胞凋亡，提示 TRPV4 可能成为 OA 的潜在药物靶点。
