[Simvastatin Promotes Murine Osteoclasts Apoptosis in vitro Through NFATc1 Pathway]

Nan Fang Yi Ke Da Xue Xue Bao. 2019 Jun 30;39(6):672-678. doi: 10.12122/j.issn.1673-4254.2019.06.07.
[Article in Chinese]

Abstract

Objective: To explore the mechanism by which simvastatin (SIM) regulates osteoclast apoptosis.

Methods: Murine macrophage RAW264.7 cells were divided into 5 groups, namely group A (control group), group B (sRANKL+ M-CSF), group C (SIM+sRANKL+M-CSF), group D (VIVIT peptide+sRANKL+ M-CSF), and group E (SIM+VIVIT peptide+sRANKL+M-CSF). WST-1 assay was used to assess the effects of simvastatin on the proliferation activity of the osteoclasts, and flow cytometry was performed to analyze the effects of SIM and VIVIVIT peptide (a NFATc1 pathway inhibitor) on apoptosis of the osteoclasts. The translocation of NFATc1 into the nucleus was investigated using immunofluorescence assay, and Western blotting was employed to assess the effect of SIM on the phosphorylation of NFATc1 in the nucleus.

Results: WST-1 assay showed that SIM (1×10-6 mol/L) treatment for 24 and 48 h significantly inhibited the proliferation of the osteoclasts (P=0.039 and 0.022, respectively). Compared with the control group, the SIM-treated osteoclasts exhibited significantly reduced cell percentage in G0/G1 phase (P=0.041) and increased cells in sub-G1 phase (P=0.028) with obvious cell apoptosis. DAPI staining and flow cytometry showed that both SIM and VIVIVIT peptide alone significantly promoted osteoclast apoptosis (P=0.002 and 0.015, respectively), and their combination produced a similar pro-apoptosis effect (P=0.08). Immunofluorescence and Western blotting showed that SIM significantly inhibited the intranuclear translocation of NFATc1 and the phosphorylation of NFATc1 pathway protein (P=0.013).

Conclusions: SIM promotes osteoclast apoptosis through NFATc1 signaling pathway.

目的: 探索辛伐他汀(SIM)调节破骨细胞凋亡机制的实验研究。

方法: 细胞分A组(Control组)、B组(sRANKL+M-CSF组)、C组(SIM+sRANKL+M-CSF组)、D组(VIVIT peptide+sRANKL+M-CSF组)、E组(SIM+VIVIT peptide+sRANKL+ M-CSF组);WST-1检测不同浓度的辛伐他汀对破骨细胞增殖活性影响;流式细胞仪检测SIM及加入NFATc1通路抑制剂VIVIT peptide后对破骨细胞凋亡的影响;免疫荧光检测NFATc1向细胞核的转位情况;Western blot检测SIM对细胞核内NFATc1磷酸化的影响。

结果: WST-1显示,与对照组相比,SIM(10-6mol/L)作用24 h、48 h明显抑制破骨细胞的增殖活性(P=0.039<0.05,P=0.022<0.05)。与对照组相比,SIM处理后破骨细胞G0/G1期受到抑制(P=0.041<0.05),SIM增加了Sub-G1期的细胞数(P=0.028<0.05)。倒置显微镜显示SIM促进破骨细胞凋亡,凋亡的细胞漂浮于培养基中。细胞核的DAPI染色和流式细胞仪检测结果显示SIM促进破骨细胞凋亡(P=0.002<0.01),VIVIT peptide单独处理组(P=0.015<0.05),或SIM+VIVIT peptide组的细胞凋亡数与SIM组相似(P=0.08>0.05)。免疫荧光显示SIM抑制NFATc1的细胞核内转位,免疫印迹结果显示,SIM抑制NFATc1通路蛋白的磷酸化(P=0.013<0.05)。

结论: SIM通过NFATc1信号通路促进破骨细胞凋亡。

Keywords: NFATc1; apoptosis; osteoclasts; simvastatin.

MeSH terms

  • Animals
  • Apoptosis
  • Cell Differentiation
  • Mice
  • NFATC Transcription Factors
  • Osteoclasts*
  • RANK Ligand
  • Simvastatin

Substances

  • NFATC Transcription Factors
  • Nfatc1 protein, mouse
  • RANK Ligand
  • Simvastatin

Grant support

国家自然科学基金(81503477);辽宁省教育厅科学技术研究项目(L201621);辽宁省自然科学基金重点项目(20170540597);沈阳市科技局科技人才应用技术研究计划(18-014-4-47)