Yu Seon-Mi, Kim Song-Ja
Department of Biological Sciences, Kongju National University, Gongju 314-701, Republic of Korea.
Int J Mol Med. 2015 Feb;35(2):325-32. doi: 10.3892/ijmm.2014.2014. Epub 2014 Nov 27.
Dedifferentiation and inflammation are major features of cartilage degeneration during the pathogenesis of osteoarthritis (OA). Thymoquinone (TQ) is the major compound of black seed oil isolated from Nigella sativa with various beneficial or harmful effects on several diseases; however, its effects on the dedifferentiation and inflammation of chondrocytes have not yet been characterized. In the present study, we investigated whether TQ regulates the dedifferentiation and inflammation of rabbit articular chondrocytes, focusing on the production of reactive oxygen species (ROS) in rabbit articular chondrocytes. TQ induced the generation of ROS in a dose-dependent manner, as shown by staining with the fluorescent probe, 2'-7'-dichlorofluorescein diacetate. We confirmed that TQ induced dedifferentiation by measuring the loss of type II collagen and the reduction in chondroitin sulfate proteoglycan levels. TQ also caused inflammation by inducing the expression of cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2). The antioxidant, N-acetyl cysteine (NAC), prevented the dedifferentiation and inflammation which was generated by the TQ-induced production of ROS. Furthermore, TQ caused a dose-dependent increase in p38, phosphorylated extracellular signal-regulated kinase (p-ERK) and phosphoinositide 3-kinase (PI3K) expression. NAC abrogated this effect and attenuated the dedifferentiation and inflammation which was generated by the TQ-induced production of ROS. To identify the ROS-regulated pathways, we treated the chondrocytes with the p38 inhibitor, SB203580, the MEK inhibitor, PD98059, and the PI3K inhibitor, LY294002. PD98059 inhibited the TQ-induced dedifferentiation and SB203580 and LY294002 prevented the TQ-induced inflammation. These findings suggest that the TQ-induced production of ROS causes dedifferentiation through the ERK pathway and inflammation through the PI3K and p38 pathways in rabbit articular chondrocytes.
去分化和炎症是骨关节炎（OA）发病机制中软骨退变的主要特征。百里醌（TQ）是从黑种草中分离出的黑种草籽油的主要成分，对多种疾病具有各种有益或有害作用；然而，其对软骨细胞去分化和炎症的影响尚未得到明确阐述。在本研究中，我们研究了TQ是否调节兔关节软骨细胞的去分化和炎症，重点关注兔关节软骨细胞中活性氧（ROS）的产生。如用荧光探针2′,7′-二氯荧光素二乙酸酯染色所示，TQ以剂量依赖性方式诱导ROS生成。我们通过测量II型胶原蛋白的丢失和硫酸软骨素蛋白聚糖水平的降低，证实TQ诱导了去分化。TQ还通过诱导环氧合酶-2（COX-2）和前列腺素E2（PGE2）的表达引发炎症。抗氧化剂N-乙酰半胱氨酸（NAC）可预防由TQ诱导的ROS产生所引发的去分化和炎症。此外，TQ导致p38、磷酸化细胞外信号调节激酶（p-ERK）和磷酸肌醇3-激酶（PI3K）表达呈剂量依赖性增加。NAC消除了这种作用，并减轻了由TQ诱导的ROS产生所引发的去分化和炎症。为了确定ROS调节的信号通路，我们用p38抑制剂SB203580、MEK抑制剂PD98059和PI3K抑制剂LY294002处理软骨细胞。PD98059抑制了TQ诱导的去分化，SB203580和LY294002预防了TQ诱导的炎症。这些发现表明，TQ诱导的ROS产生通过ERK信号通路导致兔关节软骨细胞去分化，并通过PI3K和p38信号通路导致炎症。
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