Department of Orthopaedics, the First Affiliated Hospital of Zhengzhou University, Zhengzhou University, 1 Jianshe Road, Zhengzhou, 450052, China.
Inflammation. 2013 Apr;36(2):272-8. doi: 10.1007/s10753-012-9543-5.
In the present study, we sought to explore the mechanism of quercetin-induced apoptosis in rheumatoid arthritis fibroblast-like synoviocytes (RAFLSs). DNA fragmentation assay was used to detect quercetin-induced apoptosis in RAFLSs. The cleavages of caspase-3 and caspase-9 and the accumulation of cytosolic cytochrome C were measured by western blot in quercetin-treated RAFLSs. Mitochondrial membrane potential was tested by flow cytometry. Small interfering RNAs were used to knock down the expression of protein 53 (p53) and analyze the role of p53 in quercetin-induced apoptosis in RAFLSs. DNA fragmentation assay showed that quercetin dose-dependently elevated the apoptosis of RAFLSs, accompanying with enhanced caspase-3 and caspase-9 cleavages. Moreover, quercetin caused a concentration-dependent loss of mitochondrial membrane potential and cytochrome c release to cytosol and also decreased Bcl-2/Bax ratio, indicating that quercetin-induced apoptosis is through mitochondrial pathway. Quercetin also elevated p53 phosphorylation at ser15. Pretreatment with pifithrin-α, a p53 inhibitor, significantly diminished p53 phosphorylation at the concentration of 30 μM and abrogated quercetin-induced apoptosis in a dose-dependent manner. Quercetin-induced apoptosis was also significantly blocked by p53 silencing, further suggesting the involvement of p53 in quercetin-induced apoptosis in RAFLSs. Our study indicated that quercetin-induced apoptosis of RAFLSs is through mitochondrial pathway, in which p53 plays an important role.
在本研究中，我们试图探讨槲皮素诱导类风湿关节炎成纤维样滑膜细胞（RAFLS）凋亡的机制。采用 DNA 片段化分析检测槲皮素诱导的 RAFLS 凋亡。用 Western blot 检测槲皮素处理的 RAFLS 中 caspase-3 和 caspase-9 的裂解以及细胞质细胞色素 C 的积累。用流式细胞术检测线粒体膜电位。用小干扰 RNA 敲低蛋白 53（p53）的表达，并分析 p53 在槲皮素诱导的 RAFLS 凋亡中的作用。DNA 片段化分析显示，槲皮素呈剂量依赖性地增加 RAFLS 的凋亡，同时增强 caspase-3 和 caspase-9 的裂解。此外，槲皮素导致线粒体膜电位和细胞色素 c 向细胞质释放的浓度依赖性丧失，并且降低了 Bcl-2/Bax 比值，表明槲皮素诱导的凋亡是通过线粒体途径发生的。槲皮素还使 p53 在丝氨酸 15 处磷酸化。p53 抑制剂 pifithrin-α预处理在 30μM 浓度下显著减少 p53 的磷酸化，并呈剂量依赖性地消除槲皮素诱导的凋亡。p53 沉默也显著阻断了槲皮素诱导的凋亡，进一步表明 p53 参与了槲皮素诱导的 RAFLS 凋亡。我们的研究表明，槲皮素诱导的 RAFLS 凋亡是通过线粒体途径发生的，其中 p53 发挥重要作用。
