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

doi:10.12307/2025.368

Abstract

Abstract: 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 50 μmol/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 50 μmol/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: 50 μmol/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. 50 μmol/L urolithin B inhibited the phosphorylation levels of P65 and ERK in bone marrow-derived macrophages, which led to the inhibition of osteoclast formation. 50 μmol/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.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Key words: urolithin B, P65, ERK, nuclear factor of activated T cells 1, c-Fos, bone marrow-derived macrophage, osteoclastic differentiation

CLC Number:

Luo Xi, Chen Jianquan. Regulatory mechanism of urolithin B in osteoclastic differentiation of bone marrow-derived macrophages[J]. Chinese Journal of Tissue Engineering Research, 2025, 29(11): 2201-2209.

Figures/Tables 6

2.1 尿石素B对骨髓来源巨噬细胞增殖的影响首先利用CCK-8法来评估不同浓度尿石素B(0，12.5，25，50，100和150 μmol/L)尿石素B对骨髓来源巨噬细胞增殖的影响。用不同浓度尿石素B处理24，48，72 h，CCK-8结果显示在尿石素B剂量在50 μmol/L及以内时，对骨髓来源巨噬细胞增殖无明显影响，高浓度尿石素B(100，150 μmol/L)与对照组(0 μmol/L)相比差异有显著性意义(P < 0.05)，对骨髓来源巨噬细胞增殖有抑制作用，具体结果见图1。因此在后续实验中，选取50 μmol/L及以下浓度探究尿石素B对骨髓来源巨噬细胞破骨分化的影响。 2.2 尿石素B抑制RANKL诱导骨髓来源巨噬细胞的破骨分化 CCK-8检测结果显示50 μmol/L及以下浓度的尿石素B对骨髓来源巨噬细胞增殖无明显抑制作用，为了进一步探究尿石素B对骨髓来源巨噬细胞破骨分化的影响，此次研究在体外细胞实验使用不同浓度(0，12.5，25和50 μmol/L)的尿石素B干预RANKL刺激骨髓来源巨噬细胞破骨分化过程。RANKL刺激5 d后，观察到RANKL阳性对照组出现较多密且大的多核破骨细胞。抗酒石酸酸性磷酸酶染色结果显示，当尿石素B为12.5 μmol/L时骨髓来源巨噬细胞破骨分化受到明显抑制，破骨细胞数量及大小明显减少，增加到50 μmol/L几乎完全抑制了破骨细胞的生成，差异有显著性意义(P < 0.01)，见图2。这一结果表明尿石素B能够抑制体外RANKL诱导的骨髓来源巨噬细胞破骨分化，并呈浓度依赖性。 2.3 尿石素B主要在骨髓来源巨噬细胞的破骨分化前中期产生抑制作用在RANKL诱导骨髓来源巨噬细胞过程中，诱导第二三天时，骨髓来源巨噬细胞会出现部分细胞逐渐有融合的趋势；诱导的第4天，出现小块细胞融合区域，破骨细胞开始形成，呈圆形或类圆形；到第5天出现大的边界清晰的呈煎饼状的成熟破骨细胞。为了检测尿石素B对RANKL诱导的破骨细胞形成的主要影响阶段，此次研究使用50 μmol/L尿石素B对RANKL诱导的骨髓来源巨噬细胞进行了不同时间段的干预。分别在RANKL刺激的早期(0-2 d)、中期(2-4 d)、晚期(4-5 d)、全程(0-5 d)分别给予50 μmol/L浓度的尿石素B干预骨髓来源巨噬细胞破骨分化过程。抗酒石酸酸性磷酸酶染色结果表明，在破骨细胞形成前中期加入尿石素B较显著地减少了破骨细胞的数量和大小，见图3。 2.4 尿石素B抑制破骨细胞分化相关特征基因的mRNA表达为了进一步探讨尿石素B对破骨细胞分化的抑制作用，此次研究通过荧光定量PCR评估在体外其对Nfatc1、Oscar、Ctsk、Mmp9、Acp5和Atp6V0d2等破骨分化特征基因表达的影响。在mRNA水平上检测尿石素B对RANKL诱导5 d骨髓来源巨噬细胞后破骨分化特征基因的表达情况，以18s表达量为标准对照。实时定量荧光PCR结果显示：经尿石素B干预上述破骨分化特征基因表达均有所下降，50 μmol/L浓度的尿石素B处理组与对照组相比，破骨分化基因表达差异有显著性意义(P < 0.01)，见图4。这说明尿石素B通过下调破骨分化特征基因的转录激活与表达，抑制了破骨细胞的形成与成熟。 2.5 尿石素B抑制RANKL介导的P65、ERK信号通路激活此次研究采用抑制作用最强的浓度50 μmol/L来研究尿石素B对RANKL诱导的骨髓来源巨噬细胞中核因子κB(P65)、MAPK(ERK)信号通路的影响。用RANKL在0，5，15，30，60 min不同时间梯度来刺激骨髓来源巨噬细胞。结果表明，RANKL刺激显著提高了p-P65、p-ERK的表达水平，呈现出先升高后降低的趋势，p-P65、p-ERK在5 min达到峰值。与RANKL阳性对照组对比，尿石素B处理组主要在5 min降低了磷酸化ERK相对于其总蛋白的表达水平，在各时间点均抑制了磷酸化P65相对于其总蛋白的表达水平。这表明尿石素B抑制了RANKL诱导的P65和ERK信号通路的磷酸化情况，从而干预破骨细胞分化，见图5。 2.6 骨髓来源巨噬细胞破骨分化过程中NFATc1、c-Fos表达变化及尿石素B对其的抑制作用此次研究采用抑制作用最强的浓度50 μmol/L来研究尿石素B对骨髓来源巨噬细胞破骨分化过程中破骨关键转录因子NFATc1、c-Fos表达的影响。在RANKL诱导0，1，3或5 d，收集了阳性对照组和尿石素B干预组的骨髓来源巨噬细胞。通过蛋白印迹实验发现，在RANKL处理骨髓来源巨噬细胞破骨分化过程中，NFATc1和c-Fos蛋白表达水平逐渐上升，在第5天达到峰值。在50 μmol/L浓度尿石素B处理后这些蛋白表达水平受到明显抑制，尤其是第3，5天。由此可知，尿石素B抑制了NFATc1和c-Fos的活性，从而影响RANKL诱导的骨髓来源巨噬细胞破骨分化过程，见图6。"

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