Leptin and ROS are implicated in the regulation of inflammatory pathways including NLRP3-inflammasome. We investigated the functional link between leptin, ROS and NLRP3-inflammasome formation/activation in osteoarthritis (OA), an age-related disease. We found that inflammasome components’ (NLRP3, ASC, Caspase-1 and cleaved Caspase-1) protein expression were increased in OA cartilage biopsies and chondrocytes compared to healthy cartilage and chondrocytes. Immunofluorescence showed increased co-localization of NLRP3/ASC and NLRP3/Caspase-1, ASC-specks formation and ROS levels in OA compared to normal chondrocytes. NOX4 mRNA expression and IL-1β/IL-18 secretion levels were also elevated in OA chondrocytes. Furthermore, NLRP3-siRNA in OA chondrocytes revealed significant MMP-9/MMP-13 downregulation. To elucidate leptin/ROS/NLRP3-inflammasome interactions, OA chondrocytes were treated with ROS-inhibitor NAC, NOXs-inhibitor DPI, NOX4-inhibitor GLX351322 and leptin-siRNA, while normal chondrocytes were incubated with leptin with or without DPI or GLX351322. We observed attenuated ROS levels and NLRP3-inflammasome formation/activation in NAC-, DPI- or GLX351322-treated OA chondrocytes, while the same effect was shown after transfection with leptin-siRNA. Furthermore, incubation of normal chondrocytes with leptin enhanced ROS production and inflammasome formation/activation, while pretreatment with DPI or GLX351322 abolished leptin’s stimulatory effects confirming leptin-NOX4-ROS-inflammasome regulatory axis. Overall, our findings provide novel evidence indicating that leptin-induced NLRP3-inflammasome formation/activation in OA chondrocytes is mediated by NOX4-dependent ROS production.

瘦素和 ROS 参与炎症通路的调节，包括 NLRP3 炎性体。我们调查了瘦素、ROS 和 NLRP3 炎性体形成/激活在骨关节炎 (OA) 中的功能联系，骨关节炎是一种与年龄相关的疾病。我们发现与健康的软骨和软骨细胞相比，OA 软骨活检组织和软骨细胞中的炎性体成分（NLRP3、ASC、Caspase-1 和裂解的 Caspase-1）蛋白表达增加。免疫荧光显示与正常软骨细胞相比，OA 中 NLRP3/ASC 和 NLRP3/Caspase-1 的共定位、ASC 斑点形成和 ROS 水平增加。OA 软骨细胞中 NOX4 mRNA 表达和 IL-1β/IL-18 分泌水平也升高。此外，OA 软骨细胞中的 NLRP3-siRNA 显示出显着的 MMP-9/MMP-13 下调。为了阐明瘦素/ROS/NLRP3-炎性体相互作用，OA 软骨细胞用 ROS-抑制剂 NAC、NOXs-抑制剂 DPI、NOX4-抑制剂 GLX351322 和瘦素-siRNA 处理，而正常软骨细胞与瘦素一起孵育，有或没有 DPI 或 GLX351322。我们在 NAC、DPI 或 GLX351322 处理的 OA 软骨细胞中观察到 ROS 水平和 NLRP3-炎性体形成/激活减弱，而在转染瘦素-siRNA 后显示出相同的效果。此外，正常软骨细胞与瘦素的孵育增强了 ROS 的产生和炎性体的形成/激活，而用 DPI 或 GLX351322 预处理消除了瘦素的刺激作用，证实了瘦素-NOX4-ROS-炎性体调节轴。全面的，