异甘草素通过自噬途径抑制破骨细胞形成
首发时间:2017-05-17
摘要:目的:探讨自噬在异甘草素抑制 RANKL 诱导的破骨细胞分化中的作用,并进一步分析在此过程中 NF-κB (Nuclear factor-kappa B) 信号通路对自噬的调节作用。方法:不同浓度异甘草素作用于 RAW 264.7 细胞。抗酒石酸酸性磷酸酶( tartrate-resistant acid phosphatase, TRAP)染色、鬼笔环肽染色和骨吸收陷窝实验评价破骨细胞的分化和功能。 CCK-8 (Cell Counting Kit-8) 试剂盒检测细胞活性。 MDC 染色、免疫荧光、透射电镜观察、 western blot和实时定量 PCR( quantitative real-time PCR)检测自噬特异性反应。通过 TRAP、鬼笔环肽观察破骨细胞形成及功能,实时定量 PCR 检测破骨细胞分化相关因子表达。结果:异甘草素可减少破骨细胞数目,抑制自噬标志蛋白 LC3-II 和 Beclin 1 的聚集,以及酸性泡和自噬体形成。同时异甘草素处理后的细胞自噬相关基因及蛋白表达水平明显减弱。结论: 阻断自噬途径很有可能是异甘草素抑制体外破骨细胞分化和功能的重要机制。
关键词: 异甘草素;自噬;破骨细胞形成
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Effects of ISL on autophagy during anti-osteoclastogenesis in vitro
Abstract:Objective: This study aimed to evaluate the potential alterations of autophagy during anti-osteoclastogenic effects by ISL in vitro. Methods: RAW 264.7 cells were treated with RANKL plus varying concentrations of ISL. RANKL-induced osteoclast differentiation and function were assessed with TRAP staining, F-actin staining, and SEM. Cytotoxicity of material extracts was evaluated with Cell Counting Kit-8 assay. MDC staining, immunofluorescence, TEM, western blot, and quantitative real-time PCR (Real Time-PCR) were used to detect the specific features for autophagy. Then RAW 264.7Effects of ISL on autophagy during anti-osteoclastogenesis in vitro cells were preincubation with rapamycin, and then treated with RANKL and ISL. TRAP and F-actin staining was used to assess osteoclastogenesis. The mRNA expressions associated with osteoclast differentiation were detected with Real Time-PCR. Results: We observed that ISL inhibited RANKL-induced osteoclastogenesis and suppressed autophagic LC3-II and Beclin 1 accumulation. ISL treatment resulted in the interruption of several specific features for autophagy in osteoclast precursors, including acidic vesicular organelle formation, LC3-II accumulation, and appearance of autophagic vacuoles. The RANKL-stimulated expression levels of autophagy-related genes and proteins also diminished in ISL-treated osteoclast precursors. Conclusion: ISL inhibits osteoclast differentiation and function in an autophagic way in vitro.
Keywords: Isoliquiritigenin autophagy osteoclastogenesis
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No.4734171119949814****
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