Lee Do Ik, Jang Su Kil, Park Da Woon, Kim Seung Tae, Park Jun Sub, Jo Bo Ram, Park Jung Youl, Park Hee Yong, Joo Seong Soo
College of Pharmacy, Chung-Ang University.
College of Life Science, Gangneung-Wonju National University.
Biol Pharm Bull. 2017;40(5):630-637. doi: 10.1248/bpb.b16-00876.
The aim of the present study was to examine the inhibitory roles and mechanisms of hirsutenone (HTN) in the regulation of osteoclastogenesis. Gene levels were compared to assure the effects of HTN on osteoclastogenesis in mouse splenocytes/CD4 T cells, mouse macrophage-like cell line RAW264.7 (preosteoclast), MG63 (osteoblast), and RPMI1788 (B cell) cells. The mechanism by which HTN regulates the degradation of tumor necrosis factor receptor-associated factor 6 (TRAF6) and inhibits inhibitor of kappaB (IκB) and nuclear factor-kappaB (NF-κB) signaling was examined by Western blotting and luciferase reporter assays. Our results demonstrated that HTN effectively downregulated the expression of interferon γ (IFNγ), interleukin-22 (IL-22), IL-1β, and tartrate-resistant acid phosphatase (TRAP) in splenocyte-/CD4-RAW264.7 co-culture system. Moreover, receptor activator of nuclear factor-κB ligand (RANKL) and CD25 expression were also significantly inhibited in MG63 and CD4 single culture system, suggesting an additional independent effect of HTN on osteoclastogenesis. Notably, TRAF6 was markedly degraded along with a decrease in nuclear factor of activated T-cells (NFATc) and NF-κB activities in RAW264.7 cells. Finally, we concluded that HTN directly or indirectly inhibits osteoclastogenesis via the inhibition of NF-κB signaling by promoting TRAF6 degradation, and plays a crucial role in suppressing the expression of RANKL and cytokines expressed in IFNγ-producing T-helper 1 (Th1) cells. These findings suggest that HTN may be a promising therapeutic candidate for diseases resulting from bone loss.
本研究的目的是探讨毛兰素（HTN）在破骨细胞生成调控中的抑制作用及机制。比较基因水平以确定HTN对小鼠脾细胞/CD4 T细胞、小鼠巨噬细胞样细胞系RAW264.7（前破骨细胞）、MG63（成骨细胞）和RPMI1788（B细胞）细胞中破骨细胞生成的影响。通过蛋白质免疫印迹法和荧光素酶报告基因检测，研究HTN调节肿瘤坏死因子受体相关因子6（TRAF6）降解、抑制κB抑制蛋白（IκB）和核因子κB（NF-κB）信号传导的机制。我们的结果表明，在脾细胞/CD4-RAW264.7共培养体系中，HTN有效下调了干扰素γ（IFNγ）、白细胞介素-22（IL-22）、IL-1β和抗酒石酸酸性磷酸酶（TRAP）的表达。此外，在MG63和CD4单一培养体系中，核因子κB受体激活剂配体（RANKL）和CD25的表达也受到显著抑制，提示HTN对破骨细胞生成有额外的独立作用。值得注意的是，在RAW264.7细胞中，TRAF6明显降解，同时活化T细胞核因子（NFATc）和NF-κB活性降低。最后，我们得出结论，HTN通过促进TRAF6降解抑制NF-κB信号传导，直接或间接抑制破骨细胞生成，并在抑制RANKL和在产生IFNγ的辅助性T细胞1（Th1）中表达的细胞因子方面发挥关键作用。这些发现表明，HTN可能是治疗骨质流失相关疾病的有前景的候选药物。
