背景:骨关节炎(OA)是老年人群普遍存在的退行性疾病,可导致严重的关节功能障碍。研究已经揭示了多种药理活性的薯片美素,包括抗OA功效。本研究进一步研究了其对白细胞介素(IL)-1β诱导的软骨细胞OA的影响。
方法:从幼鼠中分离原代软骨细胞,用IL-1β(10ng/mL)刺激,然后用二恶英素(10和20μM)预处理以进行体外测定。CCK-8测定评估了二十美素的细胞毒性,而炎症因子的水平(PGE2,亚硝酸盐,TNF-α,通过ELISA评估匀浆细胞中的IL-6)。炎症细胞因子的水平,细胞外基质(ECM)含量,和信号相关蛋白(Nrf2,HO-1和NF-κBp65)通过蛋白质印迹法进行评估。胶原II的表达,通过免疫荧光染色证实了软骨细胞中的p65和Nrf2。用Nrf2敲低质粒(si-Nrf2)转染IL-1β和薯不上素处理的软骨细胞,以研究Nrf2的作用。通过手术使内侧半月板(DMM)不稳定来诱导体内OA小鼠模型。进行SafraninO染色以评估膝关节组织中的OA严重程度。
结果:Diosmetin抑制iNOS的表达,COX-2,PGE2,亚硝酸盐,TNF-α,IL-1β在软骨细胞中诱导的IL-6、MMP-13和ADAMTS-5。p-p65,p-IκBα的表达,在IL-1β处理的软骨细胞中,通过diosmetin处理,核p65降低,而Nrf2和HO-1的增加。通过siRNA敲除Nrf2逆转了二十美素对IL-1β诱导的培养软骨细胞ECM蛋白和炎症因子降解的抑制作用。在DMM诱导的OA模型中,Diosmetin减轻软骨退变并降低骨关节炎研究协会国际评分。C:发明内容:通过激活Nrf2/HO-1信号传导途径,通过失活NF-κB信号传导改善培养的鼠软骨细胞中炎症生物标志物的表达和ECM大分子的降解。
BACKGROUND: Osteoarthritis (OA) is a prevailing degenerative disease in elderly population and can lead to severe joint dysfunction. Studies have revealed various pharmacological activities of
diosmetin, including the anti-OA efficacy. The present study further investigated its effect on interleukin (IL)-1β-induced OA in chondrocytes.
METHODS: Primary chondrocytes were isolated from young mice, stimulated with IL-1β (10 ng/mL), and pretreated with diosmetin (10 and 20 μM) to conduct the in vitro assays. CCK-8 assay assessed the cytotoxicity of
diosmetin whereas the levels of inflammatory factors (PGE2, nitrite, TNF-α, and IL-6) in homogenized cells were evaluated by ELISA. The levels of inflammatory cytokines, content of extracellular matrix (ECM), and signaling-related proteins (Nrf2, HO-1, and NF-κB p65) were assessed by western blotting. Expression of collagen II, p65, and Nrf2 in the chondrocytes was confirmed by immunofluorescence staining. The chondrocytes treated with IL-1β and
diosmetin were transfected with Nrf2 knockdown plasmid (si-Nrf2) to investigate the role of Nrf2. In vivo OA mouse model was induced by surgically destabilizing the medial meniscus (DMM). Safranin O staining was conducted to assess the OA severity in the knee-joint tissue.
RESULTS: Diosmetin suppressed the expression of iNOS, COX-2, PGE2, nitrite, TNF-α, IL-6, MMP-13, and ADAMTS-5 induced by IL-1β in chondrocytes. The expression of p-p65, p-IκBα, and nuclear p65 was decreased whereas that of Nrf2 and HO-1 increased by diosmetin treatment in IL-1β-treated chondrocytes. Nrf2 knockdown by siRNA reversed the inhibitory effect of
diosmetin on IL-1β-induced degradation of ECM proteins and inflammatory factors in cultured chondrocytes. In the DMM-induced model of OA, diosmetin alleviated cartilage degeneration and decreased the Osteoarthritis Research Society International score.
CONCLUSIONS: Diosmetin ameliorates expression of inflammation biomarkers and ECM macromolecules degradation in cultured murine chondrocytes via inactivation of NF-κB signaling by activating Nrf2/HO-1 signaling pathway.