Effects of bisphosphonates on osteoclastogenesis in RAW264.7 cells
Effects of bisphosphonates on osteoclastogenesis in RAW264.7 cells
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DOI：
10.3892/ijmm.2012.952
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发表时间：
2012-06-01
影响因子：
5.4
通讯作者：
Kitagawa, Yoshimasa
中科院分区：
文献类型：
--
作者：
Abe, Keigo;Yoshimura, Yoshitaka;Kitagawa, Yoshimasa
二膦酸盐用作治疗剂，用于管理骨质疏松症和其他骨疾病。然而，双膦酸盐对破骨细胞生成的确切作用和机制尚不清楚，因为先前的研究报告了相互矛盾的结果，并且没有研究详细评估双膦酸盐对破骨细胞生成的作用。因此，本研究的目的是确定双膦酸盐对RAW 264.7（RAW）细胞中破骨细胞生成的影响。为了检测双膦酸盐通过核因子-κ B受体激活因子（RANK）配体（RANKL）对破骨细胞分化的直接作用，将RAW细胞与双膦酸盐一起培养。向RAW细胞中添加双膦酸盐导致破骨细胞和大破骨细胞（>= 8个核）的数量以双膦酸盐浓度依赖性和时间依赖性方式显著减少。不含氮的双膦酸盐的细胞毒性是特定的破骨细胞，而含氮的双膦酸盐的细胞毒性和诱导细胞死亡的破骨细胞和RAW细胞。再吸收活性显着减少治疗与双磷酸盐，从而证实，双磷酸盐损害破骨细胞的吸收活性。我们还研究了双膦酸盐对与破骨细胞生成，破骨细胞特异性标志物和骨化相关基因的mRNA表达的影响，使用定量实时PCR。结果表明，双膦酸盐抑制破骨细胞分化和灌注，并诱导破骨细胞凋亡。对于双膦酸盐诱导的破骨细胞凋亡，我们进一步研究了DNA片段化和Caspase-Glo 3/7检测。DNA断裂后，证实与二膦酸盐治疗，而caspase-3/7活性显着增加时，与对照组相比。总之，二膦酸盐直接抑制RANKL刺激的破骨细胞分化和RAW细胞融合。已证实双膦酸盐损害破骨细胞吸收活性并诱导细胞凋亡。不含氮的双膦酸盐的作用也特异于破骨细胞，而含氮的双膦酸盐具有细胞毒性，并在破骨细胞和RAW细胞中诱导细胞死亡。
Bisphosphonates are used as therapeutic agents for the management of osteoporosis and other bone diseases. However, the precise effects and mechanisms of bisphosphonates on osteoclastogenesis are unclear, as previous studies have reported contradictory findings and no studies have circumstantially assessed the effects of bisphosphonates on osteoclastogenesis. Therefore, the aim of this study was to determine the effects of bisphosphonates on osteoclastogenesis in RAW264.7 (RAW) cells. To examine the direct effects of bisphosphonates on osteoclast differentiation via receptor activator of nuclear factor-kappa B (RANK) ligand (RANKL), RAW cells were cultured with bisphosphonates. Addition of bisphosphonates to RAW cells led to a significant decrease in the number of osteoclasts and large osteoclasts (>= 8 nuclei) in a bisphosphonate concentration-dependent and time-dependent manner. The cytotoxicity of non-nitrogen-containing bisphosphonates was specific to osteoclasts, while nitrogen-containing bisphosphonates were cytotoxic and induced cell death in both osteoclasts and RAW cells. Resorption activity was significantly diminished by treatment with bisphosphonates, thus confirming that bisphosphonates impair the absorptive activity of osteoclasts. We also investigated the effects of bisphosphonates on the mRNA expression of genes associated with osteoclastogenesis, osteoclast-specific markers and apoptosis-related genes using quantitative real-time PCR. The results suggest that bisphosphonates suppress osteoclast differentiation and infusion, and induce osteoclast apoptosis. With regard to osteoclast apoptosis induced by bisphosphonates, we further investigated the detection of DNA fragmentation and Caspase-Glo 3/7 assay. DNA fragmentation was confirmed after treatment with bisphosphonates, while caspase-3/7 activity increased significantly when compared with controls. In conclusion, bisphosphonates directly inhibited RANKL-stimulated osteoclast differentiation and fusion in RAW cells. It was confirmed that bisphosphonates impair osteoclast resorption activity and induce apoptosis. The effects of non-nitrogen-containing bisphosphonates were also specific to osteoclasts, while nitrogen-containing bisphosphonates were cytotoxic and induced cell death in both osteoclasts and RAW cells.