# Urolithin B suppressed osteoclast activation and reduced bone loss of osteoporosis via inhibiting ERK/NF‐κB pathway

## TL;DR

Urolithin B reduces bone loss in osteoporosis by inhibiting osteoclast activity through a specific signaling pathway.

## Contribution

Urolithin B is shown to suppress osteoclast activation and bone loss via the ERK/NF-κB pathway in both in vitro and in vivo models.

## Key findings

- Urolithin B inhibits osteoclast differentiation and function in vitro.
- Urolithin B reduces bone resorption and osteoclast-related gene expression in ovariectomized mice.
- Urolithin B suppresses the ERK/NF-κB signaling pathway, which is key to osteoclast activation.

## Abstract

The main target of current drugs for alleviating bone loss is osteoclasts. However, the long‐term application of such drugs will also cause side effects. Therefore, it is of great need to develop new and safer therapeutics for osteoporosis. In recent years, drug development based on gut microbiota has gradually attracted attention. This manuscript investigates the inhibitory effect of urolithin B (UB) on osteoclastogenesis and differentiation in vitro and in ovariectomized (OVX) mice.

CCK‐8 was used to analyse the cytotoxicity of UB; BMMs cells were differentiated into osteoclasts by RANKL, and respectively treated with 1, 5, and 25 μmol/L UB during this process. After one week of intervention, tartrate‐resistant acid phosphatase (TRAP) staining was used to analyse the number and average area of osteoclasts. F‐actin staining and immunofluorescence staining were conducted to evaluate the morphology and function of osteoclasts. Bone resorption function of osteoclasts was detected by Pit Formation Assay. The expression of osteoclast‐related protein genes in RAW264.7 cells were investigated via western blot and RT‐PCR assays. Western blot analysis of RANKL‐mediated activation of MAPK/NF‐κB pathway after 0, 5, 15, 30, 60 min of intervention. For in vivo experiments, OVX mice received intraperitoneal injection of 10, 50 mg/kg every two days, 8 weeks later, the femurs of mice were taken for morphological analysis, and the serum content of CTX‐1, a bone metabolism index, was analysed.

UB could inhibit the osteoclast differentiation of rankl‐induced bone marrow macrophages (BMMs) and RAW264.7 cells in vitro, suppress the uptake activity of hydroxyapatite and expression of osteoclast‐related gene MMP9, CTSK, NFATc1 and c‐fos. Furthermore, UB repressed the rankl‐induced phosphorylation and degradation of IκB and the phosphorylation of P65 in the NF‐κB pathway of RAW264.7 cells, and also down‐regulated the phosphorylation level of ERK in the MAPK pathway. For in vivo studies, UB‐treated OVX mice showed more significant improved various parameters of distal femur compared with the control group, with fewer NFATc1, MMP9 and TRAP‐positive osteoclasts in bone tissues, and less serum content of CTX‐1.

Urolithin B attenuated bone loss in OVX mice by inhibiting the formation and activation of osteoclasts via down‐regulation of the ERK/NF‐κB signalling pathway.

Urolithin B can alleviate bone loss in ovariectomized mice in vivo and inhibit the formation and activation of RANKL induced osteoclasts in vitro. As shown in the figure, urolithin B inhibits the activation of downstream transcription factors NFATc1 and c‐fos by down regulating the phosphorylation levels of p65 and ERK, and reduces the expression of osteoclast related functional proteins CTSK, TRAP and MMP9 to inhibit the formation and activation of osteoclasts.