Lee Sung Won, Song Yeon Suk, Shin Sang Hwa, Kim Kyung Taek, Park Young Chul, Park Bong Soo, Yun Il, Kim Kunhong, Lee Sang Yeob, Chung Won Tae, Lee Hye Jeong, Yoo Young Hyun
Dong-A University, Seo-Gu, Busan, South Korea.
Arthritis Rheum. 2008 Mar;58(3):790-800. doi: 10.1002/art.23220.
To examine whether cilostazol, a selective phosphodiesterase type III inhibitor, protects rat articular chondrocytes against nitric oxide (NO)-induced apoptosis and prevents cartilage destruction in mono-iodoacetate-induced osteoarthritis (OA) in a rat model in which inducible nitric oxide synthase (iNOS) is expressed.
The NO donor sodium nitroprusside was administered to rat articular chondrocytes that had been pretreated with cilostazol. Induction of apoptosis was evaluated by DNA electrophoresis and pulsed-field gel electrophoresis. The expression level and the subcellular location of apoptosis-associated factors were examined by Western blot analysis and confocal microscopy, respectively. Protein kinase CK2 (PKCK2) activity was also assayed. To examine whether orally administered cilostazol prevents cartilage destruction in vivo, cartilage samples obtained from rats with experimentally induced OA were subjected to hematoxylin and eosin, Safranin O, and TUNEL staining and immunohistochemical analysis of iNOS expression.
Cilostazol prevented NO-induced reduction in viability, in a dose-dependent manner. It also prevented the up-regulation of phosphorylated p53 and p38, the down-regulation of heme oxygenase 1, the subcellular translocation of apoptosis-inducing factor and cytochrome c, and the activation of caspases 3, 7, and 8 induced by NO treatment, indicating that cilostazol prevented NO-induced cell death by blocking apoptosis. In addition, cilostazol prevented NO-induced translocation of cleaved Bid onto mitochondria, and caused phosphorylated Bid to accumulate in the nucleus and cytosol. Cilostazol prevented the down-regulation of PKCK2 and the reduction in PKCK2 activity induced by NO, indicating that its apoptosis-preventing activity was mediated via PKCK2. It also prevented chondrocyte apoptosis and cartilage destruction in a rat model of experimentally induced OA.
Our findings indicate that cilostazol prevents NO-induced apoptosis of chondrocytes via PKCK2 in vitro and prevents cartilage destruction in a rat model of OA.
研究选择性Ⅲ型磷酸二酯酶抑制剂西洛他唑是否能保护大鼠关节软骨细胞免受一氧化氮（NO）诱导的凋亡，并在诱导型一氧化氮合酶（iNOS）表达的大鼠单碘乙酸盐诱导骨关节炎（OA）模型中预防软骨破坏。
将NO供体硝普钠给予经西洛他唑预处理的大鼠关节软骨细胞。通过DNA电泳和脉冲场凝胶电泳评估凋亡诱导情况。分别通过蛋白质印迹分析和共聚焦显微镜检查凋亡相关因子的表达水平和亚细胞定位。还测定了蛋白激酶CK2（PKCK2）活性。为了研究口服西洛他唑是否能在体内预防软骨破坏，对实验性诱导OA大鼠的软骨样本进行苏木精和伊红染色、番红O染色、TUNEL染色以及iNOS表达的免疫组织化学分析。
西洛他唑以剂量依赖的方式预防了NO诱导的活力降低。它还预防了NO处理诱导的磷酸化p53和p38上调、血红素加氧酶1下调、凋亡诱导因子和细胞色素c的亚细胞易位以及半胱天冬酶3、7和8的激活，表明西洛他唑通过阻断凋亡预防了NO诱导的细胞死亡。此外，西洛他唑预防了NO诱导的裂解型Bid转位至线粒体，并导致磷酸化Bid在细胞核和细胞质中积累。西洛他唑预防了NO诱导的PKCK2下调和PKCK2活性降低，表明其抗凋亡活性是通过PKCK2介导的。它还在实验性诱导OA的大鼠模型中预防了软骨细胞凋亡和软骨破坏。
我们的研究结果表明，西洛他唑在体外通过PKCK2预防NO诱导的软骨细胞凋亡，并在OA大鼠模型中预防软骨破坏。
