PDF(Pubmed)
Abstract：
Senile osteoporosis may be caused by an imbalance in intestinal flora and oxidative stress. Trimethylamine-N-oxide (TMAO), a metabolite of dietary choline dependent on gut microbes, has been found to be significantly increased in osteoporosis. However, the role of TMAO in bone loss during osteoporosis remains poorly understood. In this study, we examined the impact of TMAO on osteoclast differentiation and bone resorption in an in vitro setting.
Osteoclast differentiation was induced by incubating RAW 264.7 cells in the presence of Receptor Activator for Nuclear Factor-κB Ligand (RANKL) and macrophage-stimulating factor (M-CSF). Flow cytometry, TRAP staining assay, CCK-8, and ELISA were employed to investigate the impact of TMAO on osteoclast differentiation and bone resorption activity in vitro. For mechanistic exploration, RT-PCR and Western blotting were utilized to assess the activation of the NF-κB pathway. Additionally, protein levels of secreted cytokines and growth factors were determined using suspension array technology.
Our findings demonstrate that TMAO enhances RANKL and M-CSF-induced osteoclast formation and bone resorption in a dose-dependent manner. Mechanistically, TMAO triggers the upregulation of the NF-κB pathway and osteoclast-related genes (NFATc1, c-Fos, NF-κB p65, Traf6, and Cathepsin K). Furthermore, TMAO markedly elevated the levels of oxidative stress and inflammatory factors.
In conclusion, TMAO enhances RANKL and M-CSF-induced osteoclast differentiation and inflammation in RAW 264.7 cells by activating the NF-κB signaling pathway. These findings offer a new rationale for further academic and clinical research on osteoporosis treatment.
Osteoclast differentiation was induced by incubating RAW 264.7 cells in the presence of Receptor Activator for Nuclear Factor-κB Ligand (RANKL) and macrophage-stimulating factor (M-CSF). Flow cytometry, TRAP staining assay, CCK-8, and ELISA were employed to investigate the impact of TMAO on osteoclast differentiation and bone resorption activity in vitro. For mechanistic exploration, RT-PCR and Western blotting were utilized to assess the activation of the NF-κB pathway. Additionally, protein levels of secreted cytokines and growth factors were determined using suspension array technology.
Our findings demonstrate that TMAO enhances RANKL and M-CSF-induced osteoclast formation and bone resorption in a dose-dependent manner. Mechanistically, TMAO triggers the upregulation of the NF-κB pathway and osteoclast-related genes (NFATc1, c-Fos, NF-κB p65, Traf6, and Cathepsin K). Furthermore, TMAO markedly elevated the levels of oxidative stress and inflammatory factors.
In conclusion, TMAO enhances RANKL and M-CSF-induced osteoclast differentiation and inflammation in RAW 264.7 cells by activating the NF-κB signaling pathway. These findings offer a new rationale for further academic and clinical research on osteoporosis treatment.
摘要：
背景：老年骨质疏松症可能是由肠道菌群失衡和氧化应激引起的。三甲胺-N-氧化物(TMAO),依赖肠道微生物的膳食胆碱代谢产物，已发现骨质疏松症显著增加。然而,TMAO在骨质疏松过程中骨丢失的作用仍然知之甚少。在这项研究中,我们检查了TMAO在体外条件下对破骨细胞分化和骨吸收的影响。
方法：在存在核因子-κB配体（RANKL）和巨噬细胞刺激因子（M-CSF）的情况下，通过孵育RAW264.7细胞来诱导破骨细胞分化。流式细胞术,TRAP染色测定，CCK-8和ELISA用于研究TMAO对体外破骨细胞分化和骨吸收活性的影响。对于机械勘探，RT-PCR和Western印迹用于评估NF-κB途径的激活。此外,使用悬浮阵列技术测定分泌的细胞因子和生长因子的蛋白质水平。
结果：我们的发现表明，TMAO以剂量依赖性方式增强RANKL和M-CSF诱导的破骨细胞形成和骨吸收。机械上,TMAO触发NF-κB途径和破骨细胞相关基因的上调（NFATc1，c-Fos，NF-κBp65、Traf6和组织蛋白酶K)。此外，TMAO显著升高氧化应激和炎症因子水平。
结论：结论：TMAO通过激活NF-κB信号通路增强RANKL和M-CSF诱导的RAW264.7细胞破骨细胞分化和炎症反应。这些发现为骨质疏松症治疗的进一步学术和临床研究提供了新的理论基础。
方法：在存在核因子-κB配体（RANKL）和巨噬细胞刺激因子（M-CSF）的情况下，通过孵育RAW264.7细胞来诱导破骨细胞分化。流式细胞术,TRAP染色测定，CCK-8和ELISA用于研究TMAO对体外破骨细胞分化和骨吸收活性的影响。对于机械勘探，RT-PCR和Western印迹用于评估NF-κB途径的激活。此外,使用悬浮阵列技术测定分泌的细胞因子和生长因子的蛋白质水平。
结果：我们的发现表明，TMAO以剂量依赖性方式增强RANKL和M-CSF诱导的破骨细胞形成和骨吸收。机械上,TMAO触发NF-κB途径和破骨细胞相关基因的上调（NFATc1，c-Fos，NF-κBp65、Traf6和组织蛋白酶K)。此外，TMAO显著升高氧化应激和炎症因子水平。
结论：结论：TMAO通过激活NF-κB信号通路增强RANKL和M-CSF诱导的RAW264.7细胞破骨细胞分化和炎症反应。这些发现为骨质疏松症治疗的进一步学术和临床研究提供了新的理论基础。
