Department of Trauma Center, The First Affiliated Hospital of Hainan Medical College, Haikou, China.
School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, China.
J Cell Biochem. 2018 Nov;119(11):8922-8936. doi: 10.1002/jcb.27145. Epub 2018 Jun 28.
Accumulating evidence suggests that autophagy plays a protective role in chondrocytes and prevents cartilage degeneration in osteoarthritis (OA). The objective of this study was to investigate the effect of diazoxide on chondrocyte death and cartilage degeneration and to determine whether these effects are correlated to autophagy in experimental OA. In this study, a cellular OA model was established by stimulating SW1353 cells with interleukin 1β. A rat OA model was generated by transecting the anterior cruciate ligament combined with the resection of the medial menisci, followed by treatment with diazoxide or diazoxide combination with 3-methyladenine. The percentage of viable cells was evaluated using calcein-acetoxymethyl/propidium iodide double staining. The messenger RNA expression levels of collagen type II alpha 1 chain (COL2A1), matrix metalloproteinase 13 (MMP-13), TIMP metallopeptidase inhibitor 1 (TIMP-1), and a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5) were determined using quantitative real-time polymerase chain reaction. The cartilage thickness and joint space were evaluated using ultrasound. SW1353 cell degeneration and autophagosomes were observed using transmission electron microscopy. The expression levels of microtubule-associated protein 1 light chain 3 (LC3), beclin-1, P62, COL2A1, and MMP-13 were evaluated using immunofluorescence staining and Western blot analysis. Diazoxide significantly attenuated articular cartilage degeneration and SW1353 cell death in experimental OA. The restoration of autophagy was observed in the diazoxide-treated group. The beneficial effects of diazoxide were markedly blocked by 3-methyladenine. Diazoxide treatment also modulated the expression levels of OA-related biomarkers. These results demonstrated that diazoxide exerted a chondroprotective effect and attenuated cartilage degeneration by restoring autophagy via modulation of OA-related biomarkers in experimental OA. Diazoxide treatment might be a promising therapeutic approach to prevent the development of OA.
越来越多的证据表明自噬在软骨细胞中发挥保护作用，可防止骨关节炎（OA）中的软骨退化。本研究旨在探讨二氮嗪对软骨细胞死亡和软骨退变的影响，并确定这些作用是否与实验性 OA 中的自噬有关。在这项研究中，通过用白细胞介素 1β刺激 SW1353 细胞建立了细胞性 OA 模型。通过前交叉韧带切断术结合内侧半月板切除术建立大鼠 OA 模型，然后用二氮嗪或二氮嗪联合 3-甲基腺嘌呤进行治疗。通过 calcein-acetoxymethyl/propidium iodide 双重染色评估活细胞的百分比。使用定量实时聚合酶链反应测定胶原类型 II 阿尔法 1 链（COL2A1）、基质金属蛋白酶 13（MMP-13）、基质金属蛋白酶抑制剂 1（TIMP-1）和解整合素金属蛋白酶与凝血酶样 5（ADAMTS5）的信使 RNA 表达水平。使用超声评估软骨厚度和关节间隙。通过透射电子显微镜观察 SW1353 细胞变性和自噬体。使用免疫荧光染色和 Western blot 分析评估微管相关蛋白 1 轻链 3（LC3）、beclin-1、P62、COL2A1 和 MMP-13 的表达水平。二氮嗪显著减轻了实验性 OA 中的关节软骨退变和 SW1353 细胞死亡。在二氮嗪治疗组观察到自噬的恢复。3-甲基腺嘌呤明显阻断了二氮嗪的有益作用。二氮嗪处理还调节了 OA 相关生物标志物的表达水平。这些结果表明，二氮嗪通过调节 OA 相关生物标志物在实验性 OA 中发挥软骨保护作用并减轻软骨退变，从而恢复自噬。二氮嗪治疗可能是预防 OA 发展的一种有前途的治疗方法。
