Young In-Chi, Chuang Sung-Ting, Hsu Chia-Hsien, Sun Yu-Jun, Liu Hwa-Chang, Chen Yo-Shen, Lin Feng-Huei
Institute of Biomedical Engineering, National Taiwan University, No. 49, Fanglan Rd, Taipei, 10672, Taiwan.
Institute of Pharmacology, College of Medicine, National Taiwan University, No. 1, Sec. 1, Ren-Ai Rd, Taipei, 10051, Taiwan.
BMC Complement Altern Med. 2017 Feb 2;17(1):91. doi: 10.1186/s12906-017-1581-y.
BACKGROUND: During the onset of osteoarthritis (OA), certain biochemical events have been shown to accelerate cartilage degradation, including the dysregulation of cartilage ECM anabolism, abnormal generation of reactive oxygen species (ROS) and overproduction of proteolytic enzymes and inflammatory cytokines. The potency of aucubin in protecting cellular components against oxidative stress, inflammation and apoptosis effects are well documented, which makes it a potential candidate for OA treatment. In this study, we aimed to evaluate the protective benefits of aucubin against OA using HO and compression induced OA-like chondrocyte models. METHODS: The effects of aucubin were studied in porcine chondrocytes after 1 mM HO stimulation for 30 min or sustained compression for 24 h. Effects of aucubin on cell proliferation and cytotoxicity of chondrocytes were measured with WST-1 and LDH assays. ROS production was evaluated by the Total ROS/Superoxide Detection Kit. Caspase-3 activity was evaluated by the CaspACE assay system. The levels of apoptosis were evaluated by the Annexin V-FITC apoptosis detection kit. OA-related gene expression was measured by reverse transcription quantitative polymerase chain reaction (RT-qPCR). Total DNA quantification was evaluated by the DNeasy Blood and Tissue kit. Sulfated-glycosaminoglycans (sGAGs) production and content were evaluated by DMMB assay and Alcian blue staining. RESULTS: The results showed that the ROS scavenge effects of aucubin appeared after 1 h of pretreatment. Aucubin could reduce the caspase-3 activity induced by HO, and reduced the apoptosis cell population in flowcytometry. In RT-qPCR results, aucubin could maintain ACAN and COL2A1 gene expressions, and prevent IL6 and MMP13 gene up-regulation induced by HO and compression stimulations. In the DMMB assay and Alcian blue staining, aucubin could maintain the sGAG content and protect chondrocytes against compressive stress, but not oxidative stress from HO. CONCLUSIONS: These results indicated that aucubin has protective effects in an osteoarthritic chondrocyte model induced by HO and mechanical stimulus.
背景：在骨关节炎（OA）发病过程中，某些生化事件已被证明会加速软骨降解，包括软骨细胞外基质（ECM）合成代谢失调、活性氧（ROS）异常生成以及蛋白水解酶和炎性细胞因子过度产生。桃叶珊瑚苷在保护细胞成分免受氧化应激、炎症和凋亡影响方面的功效已有充分记录，这使其成为OA治疗的潜在候选药物。在本研究中，我们旨在使用过氧化氢（HO）和压缩诱导的OA样软骨细胞模型评估桃叶珊瑚苷对OA的保护作用。 方法：在1 mM HO刺激30分钟或持续压缩24小时后，研究桃叶珊瑚苷对猪软骨细胞的影响。用WST-1和乳酸脱氢酶（LDH）测定法检测桃叶珊瑚苷对软骨细胞增殖和细胞毒性的影响。通过总ROS/超氧化物检测试剂盒评估ROS生成。通过CaspACE检测系统评估半胱天冬酶-3活性。通过膜联蛋白V-异硫氰酸荧光素（Annexin V-FITC）凋亡检测试剂盒评估凋亡水平。通过逆转录定量聚合酶链反应（RT-qPCR）测量OA相关基因表达。通过DNeasy血液和组织试剂盒评估总DNA定量。通过二甲基甲酰胺亚甲基蓝（DMMB）测定法和阿尔新蓝染色评估硫酸化糖胺聚糖（sGAGs）的产生和含量。 结果：结果表明，桃叶珊瑚苷的ROS清除作用在预处理1小时后出现。桃叶珊瑚苷可降低HO诱导的半胱天冬酶-3活性，并减少流式细胞术中的凋亡细胞群体。在RT-qPCR结果中，桃叶珊瑚苷可维持聚集蛋白聚糖（ACAN）和Ⅱ型胶原α1链（COL2A1）基因表达，并防止HO和压缩刺激诱导的白细胞介素6（IL6）和基质金属蛋白酶13（MMP13）基因上调。在DMMB测定法和阿尔新蓝染色中，桃叶珊瑚苷可维持sGAG含量并保护软骨细胞免受压缩应力，但不能抵抗HO引起的氧化应激。 结论：这些结果表明，桃叶珊瑚苷在HO和机械刺激诱导的骨关节炎软骨细胞模型中具有保护作用。
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