尿石素B对骨髓来源巨噬细胞向破骨细胞分化的调控机制

Regulatory mechanism of urolithin B in osteoclastic differentiation of bone marrow-derived macrophages

背景:尿石素B在机体免疫应答中起重要调节作用,具备抗炎、抗氧化和抗癌的特性,并能抑制Raw 264.7细胞向破骨细胞分化,但其对于骨髓来源的巨噬细胞向破骨细胞分化的具体作用及机制尚未阐明。系统性研究破骨细胞过度活化的调控机制,有助于探索新的治疗靶点,筛选研发更安全、有效的治疗药物,为阻断破骨细胞过度活化提供新思路。目的:利用骨髓来源的巨噬细胞建立体外破骨细胞分化模型,探究尿石素B对核因子κB受体活化因子配体介导破骨细胞分化的影响,并系统性分析其作用机制。方法:(1)采用CCK-8法筛选尿石素B干预骨髓来源巨噬细胞的安全工作浓度;(2)用不同浓度(0,12.5,25,50μmol/L)尿石素B干预骨髓来源的巨噬细胞向破骨细胞分化,进行抗酒石酸酸性磷酸酶染色观察破骨细胞的数目及面积大小;(3)不同浓度(0,12.5,25,50μmol/L)尿石素B干预骨髓来源巨噬细胞的破骨分化,通过实时荧光定量PCR检测破骨特异性基因的相对表达水平;(4)蛋白印迹实验观察尿石素B对骨髓来源巨噬细胞P65、ERK信号通路的影响;(5)蛋白印迹实验检测尿石素B对骨髓来源巨噬细胞的破骨分化关键转录因子活化T细胞核因子1和c-Fos的影响。结果与结论:(1)50μmol/L及以下浓度的尿石素B对骨髓来源巨噬细胞的增殖无影响,能显著抑制骨髓来源巨噬细胞的破骨分化;(2)尿石素B主要在破骨形成前中期抑制骨髓来源巨噬细胞的破骨分化;(3)尿石素B可下调骨髓来源巨噬细胞中破骨特异性基因的相对表达水平;(4)50μmol/L的尿石素B抑制骨髓来源巨噬细胞的P65和ERK磷酸化水平,进而抑制破骨细胞形成;(5)50μmol/L的尿石素B抑制骨髓来源的巨噬细胞中破骨分化关键转录因子活化T细胞核因子1和c-Fos的表达;(6)提示尿石素B通过P65/ERK信号轴下调破骨关键转录因子活化T细胞核因子1、c-Fos的表达,抑制下游破骨特异性基因的表达,从而抑制骨髓来源的巨噬细胞向破骨细胞分化。

BACKGROUND:Urolithin B plays an important role in regulating the body’s immune response and has antioxidant,anti-cancer and anti-inflammatory properties.Notably,urolithin B has been reported to have inhibitory effects on osteoclast differentiation of Raw 264.7 cells.However,a more comprehensive understanding of its specific mechanism of action in the context of osteoclast differentiation in bone is worth exploring.Systematic research on the regulatory mechanisms of osteoclast overactivation can help to explore new therapeutic targets,screen and develop safer and more effective therapeutic drugs,and provide new ideas to block the overactivation of osteoclasts.OBJECTIVE:By establishing an in vitro osteoclast differentiation model using bone marrow-derived macrophages,to investigate the effect of urolithin B on nuclear factor-κB receptor-activating factor ligand-mediated osteoclast differentiation and to systematically analyze its mechanism of action.METHODS:(1)The safe working concentration of urolithin B was screened by cell counting kit-8 method.(2)Different concentrations of urolithin B(0,12.5,25,and 50μmol/L)were used to intervene with the osteoclast differentiation of bone marrow-derived macrophages,and the number of osteoclasts and the size of osteocytes were observed using tartrate-resistant acid phosphatase staining.(3)Different concentrations of urolithin B(0,12.5,25,and 50μmol/L)were used to intervene with the osteoclast differentiation of bone marrow-derived macrophages,and the relative expression levels of osteoclast-specific genes were detected using real-time fluorescence quantitative PCR.(4)The effect of urolithin B on the P65 and ERK signaling pathways of bone marrow-derived macrophages was observed by western blot.(5)The effect of urolithin B on the key transcription factors nuclear factor of activated T cells 1 and c-Fos in the osteoclastic differentiation of bone marrow-derived macrophages was detected by western blot.RESULTS AND CONCLUSION:50μmol/L or lower concentration of urolithin B had no effect on the proliferation of bone marrow-derived macrophages but significantly inhibited osteoclastic differentiation of bone marrow-derived macrophages.Urolithin B mainly inhibited osteoclastic differentiation of bone marrow-derived macrophages in pre-medium term.Urolithin B down-regulated the relative expression levels of osteoclast specific genes in bone marrow-derived macrophages.50μmol/L urolithin B inhibited the phosphorylation levels of P65 and ERK in bone marrow-derived macrophages,which led to the inhibition of osteoclast formation.50μmol/L urolithin B inhibited the expression of key transcription factors nuclear factor of activated T cells 1 and c-Fos in bone marrow-derived macrophages into osteoclasts.To conclude,urolithin B inhibits bone marrow-derived macrophages from differentiating into osteoclasts by suppressing the expression of downstream osteoclastogenic-related signature genes and downregulating the expression of the important osteoclastogenic transcription factors,nuclear factor of activated T cells 1 and c-Fos,via the P65/ERK signaling axis.