蜂毒素通过抑制白细胞介素 6/sIL-6R 复合物诱导的 NF-κB 和 STAT3 激活以及 Bcl-2 表达来增强人成纤维样滑膜细胞在类风湿关节炎中的凋亡。
Melittin enhances apoptosis through suppression of IL-6/sIL-6R complex-induced NF-κB and STAT3 activation and Bcl-2 expression for human fibroblast-like synoviocytes in rheumatoid arthritis.
机构信息
Department of Internal Medicine, Catholic University of Daegu School of Medicine, 3056-6 Daemyung 4-Dong, Namgu, Daegu 705-718, Republic of Korea.
出版信息
Joint Bone Spine. 2011 Oct;78(5):471-7. doi: 10.1016/j.jbspin.2011.01.004. Epub 2011 Feb 26.
OBJECTIVE
Resistance to apoptosis of fibroblast-like synoviocytes (FLS) is considered as a major characteristic in RA. This study was designed to identify whether melittin has a pro-apoptotic effect in IL-6/sIL6R-stimulated human FLS by investigating the expression of mitochondrial apoptosis-related genes, nuclear factor-κB (NF-κB), and signal transducer and activators of transcription (STAT) activation.
METHODS
Cell viability was determined using a MTT assay after melittin treatment. Expressions of STAT3 and mitochondrial apoptosis-related genes induced by the IL-6/sIL-6R complex were determined by real time-polymerase chain reaction and western blotting. The expression of NF-κB p65 following IL-6 stimulation was determined by western blot analysis. The effects of melittin on the expression of apoptosis-related genes and the transcription factors NF-κB p65 and STAT3 were assessed in FLS. Apoptosis of FLS was determined by TUNEL-labeling to detect DNA strand breaks and DNA fragmentation assays. Caspase-3 activity was determined by a colorimetric assay.
RESULTS
IL-6/sIL-6R induced the activation of the transcription factors, STAT3, NF-κB p65 (nucleus), and Bcl-2. Melittin increased the expression of pro-apoptosis-related molecules, namely caspase-3, caspase-9, Apaf-1, and cytosolic cytochrome c, in a dose-dependent manner after treatment with IL-6/sIL-6R. Melittin inhibited STAT3 activation, translocation of NF-κB p65 into the nucleus, and expression of anti-apoptotic genes such as Bcl-2 and mitochondrial cytochrome c.
CONCLUSIONS
The pro-apoptotic effects of melittin likely result from inhibition of the activation of the transcription factors, STAT3 and NF-κB p65, and regulation of mitochondrial apoptosis-related genes. Melittin is thus a promising therapeutic option for RA as it induces apoptosis in apoptosis-resistant synoviocytes.
目的
成纤维样滑膜细胞（FLS）抵抗细胞凋亡被认为是类风湿关节炎（RA）的主要特征。本研究旨在通过研究线粒体凋亡相关基因、核因子-κB（NF-κB）和信号转导和转录激活因子（STAT）的激活，确定蜂毒素（melittin）是否对白细胞介素-6（IL-6）/可溶性白细胞介素-6 受体（sIL-6R）刺激的人 FLS 具有促凋亡作用。
方法
用 MTT 法检测蜂毒素处理后细胞活力。用实时聚合酶链反应和蛋白质印迹法检测由 IL-6/sIL-6R 复合物诱导的 STAT3 和线粒体凋亡相关基因的表达。用蛋白质印迹法检测 IL-6 刺激后 NF-κB p65 的表达。用蜂毒素评估 FLS 中凋亡相关基因以及转录因子 NF-κB p65 和 STAT3 的表达。用 TUNEL 标记检测 DNA 链断裂和 DNA 片段化测定法检测 FLS 的凋亡。用比色法测定半胱天冬酶-3 活性。
结果
IL-6/sIL-6R 诱导转录因子 STAT3、NF-κB p65（核）和 Bcl-2 的激活。蜂毒素在 IL-6/sIL-6R 处理后呈剂量依赖性增加促凋亡相关分子的表达，即 caspase-3、caspase-9、Apaf-1 和胞浆细胞色素 c。蜂毒素抑制 STAT3 激活、NF-κB p65 向核内易位以及抗凋亡基因如 Bcl-2 和线粒体细胞色素 c 的表达。
结论
蜂毒素的促凋亡作用可能是由于抑制转录因子 STAT3 和 NF-κB p65 的激活以及调节线粒体凋亡相关基因所致。因此，蜂毒素有望成为治疗 RA 的一种方法，因为它可诱导抗凋亡滑膜细胞凋亡。
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