Zoledronate and lipopolysaccharide suppress osteoblast differentiation through downregulating phosphorylation of Smad in pre-osteoblastic MC3T3-E1 cells
Zoledronate and lipopolysaccharide suppress osteoblast differentiation through downregulating phosphorylation of Smad in pre-osteoblastic MC3T3-E1 cells
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DOI：
10.1016/j.ajoms.2022.01.007
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发表时间：
2022-06-07
影响因子：
0.4
通讯作者：
Yoshioka, Izumi
中科院分区：
文献类型：
--
作者：
Amamoto, Shinsuke;Yoshiga, Daigo;Yoshioka, Izumi
目的：治疗双磷酸盐相关颌骨坏死（BRONJ）这一严重问题至关重要；然而，其发病机制尚不清楚。我们之前曾在体内报道过，细菌感染或脂多糖（LPS）的施用会加重大鼠的 BRONJ 症状。为了阐明 BRONJ 的详细机制，我们研究了 LPS 和唑来膦酸 (ZOL) 的共同给药是否会加剧前成骨细胞 MC3T3-E1 细胞的体外分化。方法：用 ZOL、LPS 或两种药物按浓度梯度处理 MC3T3-E1 细胞指定的时间，然后使用 WST-8 测定法测量细胞活力。使用碱性磷酸酶（ALP）染色、ALP 活性测定和茜素红 S 染色评估成骨分化。通过实时定量逆转录聚合酶链反应检测相关基因的表达。进行Western blotting检测Smad1/5/8的磷酸化水平。结果：在成骨细胞分化的早期阶段，与单次施用 LPS 或 ZOL 相比，同时施用 LPS 和 ZOL 更能抑制 ALP 活性和 ALP 染色。与单次施用 LPS 或 ZOL 相比，同时施用 LPS 和 ZOL 还可以抑制成骨细胞分化标志物 Runx2、Col I、ALP 和 BSP 的表达，以及 Smad1/5/8 的磷酸化。结论：我们的数据表明，ZOL 和 LPS 通过 BMP2-Smad 信号传导对成骨细胞分化具有抑制作用。这种效应可能导致体外矿化减少，这表明在接受 ZOL 治疗的患者中，ZOL 会导致口腔骨坏死，而 LPS 普遍由细菌产生。
Objective: It is crucial to treat Bisphosphonate-related osteonecrosis of the jaw (BRONJ), a serious problem; however, its pathogenesis is unclear. We have previously reported in vivo that bacterial infection or administration of lipopolysaccharide (LPS) aggravates BRONJ in rats. To elucidate the detailed mechanism of BRONJ, we investigated whether co-administration of LPS and zoledronate (ZOL) exacerbated the differentiation of preosteoblastic MC3T3-E1 cells in vitro. Methods: MC3T3-E1 cells were treated with ZOL, LPS, or both agents at a concentration gradient for the indicated times, after which cell viability was measured using the WST-8 assay. Osteogenic differentiation was assessed using alkaline phosphatase (ALP) staining, ALP activity assay, and Alizarin Red S staining. The expression of relevant genes was detected by quantitative real-time reverse-transcriptase polymerase chain reaction. Western blotting was performed to examine the phosphorylation levels of Smad1/5/8. Results: Co-administration of LPS and ZOL inhibited ALP activity and ALP staining more than a single administration of LPS or ZOL from the early stage of osteoblast differentiation. Co-administration of LPS and ZOL also inhibited the expression of osteoblast differentiation markers, Runx2, Col I, ALP, and BSP, as well as phosphorylation of Smad1/5/8 more than a single administration of LPS or ZOL. Conclusions: Our data suggest that ZOL and LPS have an inhibitory effect on osteoblast differentiation through BMP2-Smad signaling. This effect may have led to a decrease in mineralization in vitro, suggesting that in patients treated with ZOL, ZOL causes osteonecrosis in their oral cavities, where LPS is universally produced by bacteria.