Rheumatoid arthritis (RA) is a chronic, symmetric, systemic autoimmune disease. Because insufficient apoptosis of fibroblast-like synoviocytes (FLS) is an important characteristic of RA, promoting apoptosis is considered a potential therapeutic tool for treating RA. We have previously found that daphnetin (7,8-dihydroxycoumarin, DAP) has a pro-apoptotic effect on fibroblast-like synoviocytes from collagen-induced arthritis (CIA) rats. In the present study, we further investigated the mechanisms of DAP-induced apoptosis in CIA-FLS. CIA-FLS were incubated with DAP for 48 h in the presence or absence of caspase inhibitors, including inhibitors of caspase-3, caspase-8, or caspase-9 or a pan-caspase inhibitor; then, a series of experiments were performed to evaluate the mechanisms of DAP-induced apoptosis. Our results showed that DAP markedly decreased cell viability and induced the apoptosis of CIA-FLS along with typical morphological and ultrastructural changes; moreover, DAP increased FasL, cytochrome c (Cyt-c), Bax, caspase-3, caspase-8, and caspase-9 mRNA expression and Bax, caspase-3, caspase-8, and caspase-9 protein expression. In contrast, DAP decreased Bcl-2 mRNA and protein expression and promoted the release of Cyt-c from the mitochondria into the cytosol; these effects were attenuated to varying degrees by pre-treatment with caspase inhibitors, especially with caspase-3 or caspase-9 inhibitors or a pan-caspase inhibitor. In conclusion, the current findings demonstrate that the DAP-induced apoptosis of CIA-FLS occurred mainly via a caspase-dependent pathway, in particular the mitochondrial pathway, and that the Bax/Bcl-2 ratio was involved in this process. Thus, DAP may be a potential therapeutic agent for RA.

中文翻译：

---

瑞香素主要通过线粒体途径诱导胶原诱导关节炎大鼠成纤维细胞样滑膜细胞凋亡

类风湿性关节炎 (RA) 是一种慢性、对称性、全身性自身免疫性疾病。由于成纤维细胞样滑膜细胞 (FLS) 的凋亡不足是 RA 的一个重要特征，因此促进细胞凋亡被认为是治疗 RA 的潜在治疗工具。我们之前发现，瑞香素（7,8-二羟基香豆素，DAP）对来自胶原诱导性关节炎 (CIA) 大鼠的成纤维细胞样滑膜细胞具有促凋亡作用。在本研究中，我们进一步研究了 DAP 诱导 CIA-FLS 细胞凋亡的机制。在存在或不存在 caspase-3、caspase-8 或 caspase-9 抑制剂或泛 caspase 抑制剂的情况下，CIA-FLS 与 DAP 一起孵育 48 小时；然后，进行了一系列实验来评估 DAP 诱导细胞凋亡的机制。我们的研究结果表明，DAP 显着降低细胞活力并诱导 CIA-FLS 细胞凋亡，并伴有典型的形态学和超微结构变化；此外，DAP 增加 FasL、细胞色素 c (Cyt-c)、Bax、caspase-3、caspase-8 和 caspase-9 mRNA 表达以及 Bax、caspase-3、caspase-8 和 caspase-9 蛋白表达。相反，DAP 降低 Bcl-2 mRNA 和蛋白表达，促进 Cyt-c 从线粒体释放到细胞质中；通过使用 caspase 抑制剂，特别是使用 caspase-3 或 caspase-9 抑制剂或泛 caspase 抑制剂进行预处理，这些影响在不同程度上减弱。总之，目前的研究结果表明，DAP 诱导的 CIA-FLS 细胞凋亡主要通过半胱天冬酶依赖性途径发生，特别是线粒体途径，并且 Bax/Bcl-2 比率参与了这个过程。因此，DAP 可能是 RA 的潜在治疗剂。