Trimethylamine-N-Oxide Promotes Osteoclast Differentiation and Bone Loss via Activating ROS-Dependent NF-κB Signaling Pathway

Abstract

Trimethylamine-N-oxide (TMAO), an important gut microbiota (GM)-derived metabolite, has been shown to be abnormally increased in osteoporosis. However, the role and underlying mechanism of TMAO in regulating bone loss during osteoporosis have not been fully investigated. In the current study, we found that 100–400 μM TMAO dose-dependently enhanced TRAP-positive osteoclasts, F-actin ring formation, and resorption area on bovine bone slices and up-regulated osteoclast-related gene expression (Calcr, Traf6, Dcstamp, Acp5, C-Fos, and NFATc1). Western blotting validated that TMAO not only activated NF-κB signaling pathway but also stimulated c-Fos and NFATc1 protein expression in a dose-dependent manner. Furthermore, BAY 11-7082, an NF-κB inhibitor, pretreatment markedly suppressed TRAP-positive osteoclast formation and osteoclast-related genes under TMAO treatment. BAY 11-7082 also inhibited p-p65/p65, c-Fos, and NFATc1 protein expression promoted by TMAO. Moreover, TMAO significantly increased ROS production, which was inhibited by N-acetylcysteine (NAC), an ROS antagonist. In addition, we proved that NAC pretreatment could inhibit TMAO-promoted NF-κB activation. NAC also suppressed TRAP-positive osteoclast formation, osteoclast-related gene expression, and protein expression of c-Fos and NFATc1 under TMAO treatment. In vivo studies showed significantly decreased bone mass and increased TRAP-positive osteoclasts in TMAO-treated C57BL/6 mice. Moreover, western-blotting and immunohistochemical staining showed that TMAO administration markedly stimulated NF-κB p65 expression. Additionally, TMAO administration significantly promoted the gene and protein expression of C-Fos and NFATc1. In conclusion, TMAO could promote osteoclast differentiation and induce bone loss in mice by activating the ROS-dependent NF-κB signaling pathway.

Gut microbially produced tryptophan metabolite melatonin ameliorates osteoporosis via modulating SCFA and TMAO metabolism

“…89,90 TMAO is abnormally increased during OP progression and contributes to OC differentiation by activating the ROS-dependent NF-κB signaling pathway, thereby resulting in bone loss. 26 In our study, we observed that MLT treatment markedly increased SCFA abundances and partially reduced the production of TMAO-related metabolites, which were consistent with the alteration of gut microbiota. Additionally, a striking finding is that Allobaculum and nearly all kinds of SCFAs exhibited a markedly positive correlation.…”

“…21 In addition, TMAO, the gut microbial metabolite of choline, promotes OC differentiation by activating the ROSdependent NF-κB signaling pathway. 26 Notably, common studies have long held that products of microbial aminoacid metabolism possess a deleterious effect on the host, which results in the lack of attention paid to their roles in OP. Emerging evidence suggests that tryptophan metabolism plays a central role in microbiota-host crosstalk.…”

“…found that SCFAs, the gut microbial metabolites of dietary fibers, mediate metabolic reprogramming of OCs and downregulate the expression of key osteoclast genes including TRAF6 and NFATc1 21 . In addition, TMAO, the gut microbial metabolite of choline, promotes OC differentiation by activating the ROS‐dependent NF‐κB signaling pathway 26 . Notably, common studies have long held that products of microbial amino‐acid metabolism possess a deleterious effect on the host, which results in the lack of attention paid to their roles in OP.…”

Gut microbiota-dependent trimethylamine n-oxide pathway contributes to the bidirectional relationship between intestinal inflammation and periodontitis

“…Changes in the intestinal microflora are related to many diseases that lead to bone loss, including malnutrition, IBD, obesity, and metabolic disease (Guss et al, 2017). Animal experiments demonstrated that TMAO activates the ROS-dependent NF-kB signaling pathway to enhance osteoclast polarization and cause bone loss in mice (Li et al, 2019;Wang et al, 2022a). Additionally, TMAO can induce impaired intestinal barrier function, resulting in elevated levels of IL-1b and TGF-b (Nanto-Hara et al, 2020;Li et al, 2021b;Xie et al, 2022;Zhang et al, 2022).…”

Dexamethasone Induced Osteocyte Apoptosis in Steroid‐Induced Femoral Head Osteonecrosis through ROS‐Mediated Oxidative Stress

“…ROS is the main type of free radical involved in the destruction of bone remodeling 5 . ROS inhibits the expression of Runt‐related transcription factor‐2 (Runx2) and osterix, reducing osteogenic activity, while promoting the expression of osteoclast markers such as C‐Fos, Nuclear Factor of Activated T cells‐1 (NFATc1), and Tartrate Resistant Acid Phosphatase (TRAP) 6,7 . ROS may enhance the response of osteoclast precursors to Receptor Activator of NF‐κB Ligand (RANKL), and induce the production of additional osteoclastic factors, such as IL‐1, IL‐6, and IL‐7 8,9 .…”