Background

Dysregulated inflammation and osteoblast differentiation are implicated in osteoporosis. Exploring the activity of catalpol in inflammation and osteoblast differentiation deepens the understanding of osteoporosis pathogenesis.

Methods

LPS was used to treated hFOB1.19 cells to induce inflammation and repress osteoblast differentiation. FOB1.19 cells were induced in osteoblast differentiation medium and treated with LPS and catalpol. Cell viability was assessed using CCK-8. ALP and Alizarin red S staining were conducted for analyzing osteoblast differentiation. The levels of IL-1β, TNF-α and IL-6 were examined by ELISA. The methylation of TRAF6 promoter was examined through MS-PCR. The binding of miR-124–3p to DNMT3b and DNMT3b to TRAF6 promoter was determined with dual luciferase reporter and ChIP assays.

Results

LPS enhanced secretion of inflammatory cytokines and suppressed osteoblast differentiation. MiR-124–3p and TRAF6 were upregulated and DNMT3b was downregulated in LPS-induced hFOB1.19 cells. Catalpol protected hFOB1.19 cells against LPS via inhibiting inflammation and promoting osteoblast differentiation. MiR-124–3p targeted DNMT3b, and its overexpression abrogated catalpol-mediated protection in LPS-treated hFOB1.19 cells. In addition, DNMT3b methylated TRAF6 promoter to restrain its expression. Catalpol exerted protective effects through suppression of the miR-124–3p/DNMT3b/TRAF6 axis in hFOB1.19 cells.

Conclusion

Catalpol antagonizes LPS-mediated inflammation and suppressive osteoblast differentiation via controlling the miR-124–3p/DNMT3b/TRAF6 axis.

中文翻译：

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梓醇通过 miR-124-3p/DNMT3b/TRAF6 轴拮抗 LPS 介导的炎症并促进成骨细胞分化

背景

炎症失调和成骨细胞分化与骨质疏松症有关。探索梓醇在炎症和成骨细胞分化中的活性加深了对骨质疏松症发病机制的理解。

方法

LPS用于处理 hFOB1.19 细胞以诱导炎症并抑制成骨细胞分化。FOB1.19细胞在成骨细胞分化培养基中诱导并用LPS和梓醇处理。使用 CCK-8 评估细胞活力。进行ALP和茜素红S染色以分析成骨细胞分化。采用ELISA法检测IL-1β、TNF-α、IL-6水平。通过MS-PCR检查TRAF6启动子的甲基化。通过双荧光素酶报告基因和 ChIP 检测确定miR-124–3p 与DNMT3b以及 DNMT3b 与 TRAF6 启动子的结合。

结果

LPS 增强炎症细胞因子的分泌并抑制成骨细胞分化。在 LPS 诱导的 hFOB1.19 细胞中，MiR-124–3p 和 TRAF6 上调，DNMT3b 下调。梓醇通过抑制炎症和促进成骨细胞分化来保护 hFOB1.19 细胞免受 LPS 侵害。MiR-124–3p 靶向 DNMT3b，其过表达消除了 LPS 处理的 hFOB1.19 细胞中梓醇介导的保护。此外，DNMT3b甲基化TRAF6启动子以抑制其表达。Catalpol 通过抑制 hFOB1.19 细胞中的 miR-124–3p/DNMT3b/TRAF6 轴发挥保护作用。

结论

Catalpol 通过控制 miR-124–3p/DNMT3b/TRAF6 轴来拮抗 LPS 介导的炎症并抑制成骨细胞分化。