Main Research Findings

1) Treatment with Urolithin B was found to relieve osteoarthritis and decrease cartilage degeneration through the inhibition of inflammatory processes.

2) Through the inhibition of the ERK/NF-kB pathways, Urolithin B was found to suppress the activation of osteoclasts and reduce osteoporosis-related bone loss.

 

Selected Data

1) This study completed by the research team of Xue et al employed a comprehensive set of in vitro, ex vivo and in vivo approaches to evaluate the effects of urolithin B (UB) on chondrocyte inflammation, extracellular matrix (ECM) metabolism and osteoarthritis (OA) progression. UB was prepared in DMSO and stored at −20 °C; standard laboratory reagents, culture media and antibodies were used for cell-based assays, biochemical measurements and immunodetection. Primary chondrocytes were isolated from three‑day‑old C57BL/6 mouse knee cartilage by collagenase II digestion (8–12 h), filtered and cultured in DMEM with 10% fetal bovine serum until 80–90% confluence. Cells were plated at defined densities of 4 × 10^5 cells/well in 6‑well plates for many assays, and treated after 24 h with pro‑inflammatory stimuli including: 10 ng/mL IL‑1β or 50 ng/mL TNF‑α to model the OA inflammatory microenvironment [1].

ATDC5 cells were used for nuclear/cytoplasmic fractionation experiments to assess NF‑κB P65 translocation. Cell viability was assessed using the CCK‑8 assay across a UB concentration range from 0–20 μM, and multiple exposure times between 48 h, 96 h, 7 d to confirm non‑toxicity prior to functional assays. To examine matrix deposition and ECM integrity in vitro, chondrocyte micromass cultures were formed by spotting 20 μL droplets of concentrated chondrocyte suspension into 12‑well plates, permitting adherent matrix formation for 24 h then exposing cultures to IL‑1β or TNF‑α with or without UB for seven days; macroscopic matrix deposition was visualized by safranin O, Alcian blue and toluidine blue staining. For molecular readouts, qRT‑PCR and western blotting were applied: RNA was isolated following 48 h of treatment with UB at doses of 0, 5, and 10 μM. They were then reverse transcribed and quantified by SYBR Green qPCR using primer sets with β‑actin serving as the internal control [1].

Protein extracts were prepared in RIPA buffer with protease/phosphatase inhibitors, quantified by BCA, separated by SDS‑PAGE and probed with validated antibodies against iNOS, COX‑2, MMP13, ADAMTS5, COL2A1, aggrecan, NF‑κB P65 and IκB‑α/p‑IκB‑α, and visualized with HRP‑conjugated secondary antibodies. Nuclear–cytoplasmic fractionation was performed on ATDC5 cells pretreated with UB for 2 h then stimulated for short time points between 0–60 minutes, to monitor P65 nucleocytoplasmic shuttling by western blot. Immunofluorescence microscopy (DAPI counterstain) was used to visualize P65 localization and COL2A1 / MMP13 distribution in cultures, quantified by ImageJ and imaged using a Magscanner KF‑PRO‑120 system.

To probe direct molecular interactions potentially underlying P65 translocation inhibition, molecular docking was performed against an AlphaFold‑predicted full‑length NF‑κB P65, targeting the NLS motif 301KRKR304. UB and the known inhibitor JSH‑23 structures were docked to compare binding modes, hydrogen bonds and calculated binding energies. For in vivo translational relevance, a surgical anterior cruciate ligament transection (ACLT) model of OA was executed in eighty‑week male C57BL/6 mice under anesthesia. The animals were then randomized into various groups including: sham, ACLT, and ACLT + UB. One week after ACLT, the UB treatment group received intraperitoneal UB 50 mg/kg every two days for eight weeks while controls received a vehicle [1].

Endpoints in vivo included micro‑computed tomography (micro‑CT) to evaluate periarticular bone changes and osteophytes, histological assessment of cartilage by safranin O/fast green and OARSI semi‑quantitative scoring, immunofluorescence detection of COL2A1 and MMP13 in joint sections, and manual counting of osteophytes by double‑blind observers. Statistical analyses were preplanned: experiments were performed in triplicate or with group sizes reported, data was reported as mean ± SD, and comparisons used Student’s t‑test or one‑/two‑way ANOVA with Tukey’s post hoc where appropriate. Overall, the methods integrate cellular assays of inflammation and ECM metabolism, mechanistic NF‑κB pathway interrogation, computational docking, and an established surgical OA model with morphological and histological outcome measures to assess UB’s anti‑inflammatory and chondroprotective actions [1].

2) This comprehensive study completed by the research team of Li et al aimed to investigate the inhibitory effects of Urolithin B (UB) on osteoclastogenesis and bone loss, both in vitro and in vivo, particularly focusing on its impact on the ERK/NF-κB signaling pathway. The research commenced with an in vitro phase, utilizing two primary cell models: bone marrow-derived macrophages (BMMs) isolated from 6-week-old C57BL/6 mice and RAW264.7 cells, a murine macrophage cell line. All cells were maintained under standard culture conditions at 37°C with 5% CO2 in DMEM medium supplemented with 10% FBS and antibiotics [2].

Initial experiments focused on assessing the cytotoxicity of UB using the CCK-8 assay. BMMs and RAW264.7 cells were seeded in 96-well plates and exposed to various concentrations of UB, including 0, 1, 5, 25, 50, 100, and 150 µmol/L, for 1, 2, or 3 days. Cell viability was determined by measuring absorbance at 450 nm, with concentrations showing minimal toxicity selected for subsequent experiments. Osteoclast differentiation in vitro was induced by treating BMMs with 50 ng/mL M-CSF and 50 ng/mL RANKL, critical cytokines for osteoclast formation. During this differentiation process, cells were concurrently treated with different concentrations of UB including 0, 1, 5, and 25 µmol/L, for 5 to 7 days, depending on the specific assay.

To evaluate the extent of osteoclast formation and function, several detailed in vitro assays were employed. Tartrate-resistant acid phosphatase (TRAP) staining was performed on differentiated BMMs to quantify the number and average area of multi-nucleated osteoclasts, which are characteristic of mature osteoclasts. F-actin staining and immunofluorescence were used to visualize the morphology of osteoclasts, specifically the formation of actin rings essential for bone resorption, and to assess the expression of functional proteins such as MMP9. DAPI staining was used to identify cell nuclei. The bone resorption capabilities of osteoclasts were directly measured using a Pit Formation Assay, where BMMs were cultured on osteo-assay plates. After UB treatment, the bone resorption pits were imaged and quantitatively analyzed using IMAGEJ software to determine the resorbed area [2].

Molecular analyses included Western blot and quantitative RT-PCR to investigate the expression of osteoclast-related protein genes and key signaling molecules. Western blotting was performed on RAW264.7 cells treated with RANKL and various UB concentrations, allowing for the analysis of proteins like MMP9, CTSK, c-fos, and NFATc1. Furthermore, the study meticulously examined the RANKL-mediated activation of the MAPK/NF-κB pathway by Western blot, assessing the phosphorylation and degradation levels of IκB and P65, both NF-κB pathway components, as well as the phosphorylation levels of ERK, JNK, and p38, MAPK pathway components, at various time points including 0, 5, 15, 30, 60 min, following intervention. Specific pathway inhibitors, SCH772984 for ERK and SC75741 for P65, were also utilized to further dissect the involvement of these pathways. Quantitative RT-PCR was used to analyze the mRNA expression levels of osteoclast-related genes, MMP9, CTSK, TRAP, V-ATPase, c-fos, and NFATc1, in BMMs under similar treatment conditions [2].

For the in vivo component, a well-established ovariectomized (OVX) mouse model of osteoporosis was utilized. Forty 6-week-old C57BL/6 mice were randomly divided into four groups: sham-operated control, OVX vehicle control, OVX + low-dose UB of 10 mg/kg, and OVX + high-dose UB of 50 mg/kg. OVX surgery involved bilateral ovariectomy, while sham-operated mice underwent a similar procedure without ovary removal. Three weeks post-surgery, UB was administered via intraperitoneal injection every two days for 8 weeks. After the intervention period, blood samples were collected to measure serum C-telopeptide of type I collagen (CTX-1), a bone metabolism marker, using an ELISA kit. The femurs of the mice were harvested and fixed for morphological and histological analyses.

Micro-CT scanning was employed to obtain high-resolution 3D images of the left femurs. Detailed structural parameters of the bone were measured and analyzed using CTAn software, including bone mineral density (BMD), bone surface area (BS), bone volume (BV), bone volume to total volume ratio (BV/TV), bone surface to bone volume ratio (BS/BV), trabecular separation (Tb.Sp), and number of trabecular bone (Tb.N). Histological analyses involved decalcifying and embedding femurs in paraffin, followed by sectioning and staining with Hematoxylin and Eosin (H&E) to visualize overall bone morphology and TRAP staining to quantify osteoclast presence in bone tissues. Immunohistochemical staining was performed to detect and quantify MMP9 and NFATc1 positive cells in the bone sections, providing further insights into osteoclast activity in vivo. All experimental data were expressed as mean ± standard deviation, and statistical significance was determined using t-tests and one-way ANOVA [2].

 

Discussion

1) The results of the study performed by Xue et al revealed that Urolithin B (UB) demonstrated robust anti‑inflammatory, anti‑catabolic and chondroprotective effects across in vitro and in vivo models, acting predominantly through inhibition of the NF‑κB signaling cascade and consequent suppression of downstream mediators that drive matrix degradation. In cell viability experiments UB, in doses up to 20 μM, IL‑1β, and TNF‑α produced no cytotoxicity under the tested conditions, validating subsequent functional assays. Quantitative PCR and western blot analyses showed that both 10 ng/mL of IL‑1β and 50 ng/mL TNF‑α strongly induced key pro‑inflammatory mediators iNOS and COX‑2; UB co‑treatment reduced these inductions dose‑dependently, with significant downregulation at 5–10 μM in mRNA and protein measures [1].

UB also reversed the inflammatory cytokine‑driven dysregulation of ECM‑related gene expression: IL‑1β/TNF‑α markedly upregulated catabolic enzymes MMP3, MMP13 and ADAMTS5 while suppressing anabolic markers SOX9, COL2A1 and aggrecan. UB treatment, at doses ranging between 5–10 μM, significantly attenuated MMP/ADAMTS induction and restored expression of SOX9, COL2A1 and aggrecan in a concentration‑dependent manner as quantified by qPCR. Micromass cultures corroborated these molecular endpoints at the matrix level: IL‑1β/TNF‑α reduced safranin O, Alcian blue and toluidine blue staining as an indicator of diminished proteoglycan/collagen matrix, whereas UB preserved matrix deposition in a dose‑dependent fashion after seven days.

Immunofluorescence and western blotting confirmed UB’s protein‑level effects: UB decreased MMP13 and ADAMTS5 protein abundance and increased COL2A1 and aggrecan protein expression under inflammatory challenge. Mechanistically, UB impaired NF‑κB pathway activation: IL‑1β/TNF‑α promoted phosphorylation of IκB‑α and nuclear translocation of NF‑κB P65. UB significantly reduced IκB‑α phosphorylation over time and inhibited P65 nuclear accumulation as revealed by nuclear–cytoplasmic fractionation and immunofluorescence. These findings support a model in which UB limits IκB phosphorylation, prevents ubiquitin‑mediated IκB degradation, sequesters P65 in the cytoplasm and thereby reduces transcription of proinflammatory genes [1].

Molecular docking targeted the P65 nuclear localization signal (NLS) and demonstrated that UB binds the NLS region and forms hydrogen bonds with ARG302, much like the established inhibitor JSH‑23, suggesting a plausible mechanism by which UB could interfere with importin α3 binding and nuclear import of P65. Translating these in vitro effects to an animal OA model, systemic UB at a dose of 50 mg/kg every two days starting one week after ACLT, significantly mitigated OA pathology. Micro‑CT revealed reduced periarticular hyperostosis and osteophyte formation compared with ACLT controls, and manual osteophyte counts were lower in UB‑treated mice.

Histologically, safranin O/fast green staining and OARSI scoring indicated UB preserved cartilage structure and reduced cartilage erosion relative to ACLT, while immunofluorescence of joint sections showed UB decreased MMP13 and increased COL2A1 in situ. Organ histology indicated no overt UB toxicity in treated mice. Collectively, the results demonstrate that UB potently suppresses inflammatory signaling via NF‑κB inhibition, reduces expression and activity of major matrix‑degrading enzymes, promotes ECM component expression, preserves matrix deposition in chondrocyte cultures, and delays structural progression of OA in the ACLT model, supporting UB as a promising candidate for further preclinical development as an anti‑osteoarthritic agent [1].

2) The results of the study by Li et al investigating Urolithin B (UB)’s impact on osteoclast activation and bone loss yielded significant findings across both in vitro and in vivo experiments. Initially, cell viability assays demonstrated that UB exhibited minimal cytotoxicity at concentrations up to 25 µmol/L in both BMMs and RAW264.7 cells, making these concentrations suitable for subsequent in vitro studies on osteoclast differentiation and function. However, concentrations exceeding 50 µmol/L significantly affected cell viability, guiding the selection of experimental doses [2].

In the in vitro assessment of osteoclast differentiation, UB effectively inhibited RANKL-induced osteoclast formation in a concentration-dependent manner. TRAP staining, a hallmark assay for osteoclasts, revealed a marked reduction in both the number and average area of osteoclasts in BMMs treated with UB compared to the RANKL-induced control group. This inhibitory effect became increasingly pronounced with higher UB concentrations of 1, 5, and 25 µmol/L. Further visualization using immunofluorescence staining corroborated these findings, showing that UB treatment significantly suppressed the formation of F-actin rings, critical structures for osteoclast adhesion and bone resorption. Moreover, the number of nuclei per osteoclast was significantly reduced in the presence of UB, indicating impaired osteoclast maturation. The expression of functional osteoclast-related proteins, such as MMP9, was also observed to be clearly inhibited by UB treatment in vitro [2].

The study then directly assessed UB’s effect on osteoclast bone resorption function using the Pit Formation Assay. The results unequivocally demonstrated that UB significantly suppressed the formation of bone resorption pits. Quantitatively, the resorbed hydroxyapatite area was reduced in a dose-dependent fashion, with UB doses of 1, 5, and 25 µmol/L decreasing the resorbed area from 51.25% in the RANKL group to 29.44%, 18.83%, and 4.84%, respectively, highlighting its potent anti-resorptive properties.

At the molecular level, Western blot analysis revealed that UB downregulated the expression of key osteoclast-related proteins, including MMP9, CTSK, c-fos, and NFATc1, in a concentration-dependent manner in RAW264.7 cells. Time-course experiments further indicated that UB reversed the RANKL-induced increase in these proteins, showing significant inhibitory effects after 12 hours, 1 day, and 3 days of intervention. Quantitative RT-PCR experiments confirmed these protein-level observations, demonstrating a significant decrease in the mRNA expression levels of osteoclast-related genes MMP9, CTSK, TRAP, V-ATPase, c-fos, and NFATc1, in BMMs treated with UB, reinforcing its role in inhibiting osteoclast gene transcription [2].

A crucial aspect of the study was elucidating the underlying signaling pathways. Western blot analysis showed that UB repressed RANKL-induced osteoclastogenesis by modulating both the NF-κB and MAPK pathways. Specifically, UB was found to significantly decrease the RANKL-induced phosphorylation and subsequent degradation of IκBα, an inhibitory protein of NF-κB, and the phosphorylation of P65, a key component of the NF-κB pathway. The peak phosphorylation of P65, which occurred rapidly after RANKL addition, was notably attenuated and delayed in UB-treated cells.

Concurrently, UB significantly down-regulated the phosphorylation levels of ERK, a critical component of the MAPK pathway, while also showing inhibitory trends for p-JNK and p-p38. These inhibitory effects on ERK and P65 phosphorylation were observed to be concentration-dependent. To validate these pathway involvements, specific inhibitors were used: SCH772984, an ERK inhibitor, significantly inhibited ERK phosphorylation and synergistically enhanced UB’s reduction of CTSK and NFATc1 expression. Similarly, SC75741, a P65 inhibitor, decreased P65 phosphorylation and osteoclast-related protein expression, although UB alone demonstrated a more potent therapeutic effect than SC75741. When combined, UB and SC75741 showed an even greater reduction in osteoclast-related proteins [2].

The in vivo experiments confirmed UB’s therapeutic potential in an OVX-induced osteoporosis mouse model. Micro-CT analysis of the femurs revealed that UB treatment significantly improved several bone structural parameters that were compromised in OVX mice. Specifically, UB increased BMD, BV, and the ratio of bone volume to total volume (BV/TV), while decreasing Tb.Sp and increasing the number of Tb.N in a concentration-dependent manner. Histological examination with H&E staining further demonstrated that UB treatment decreased OVX-induced bone mass loss. TRAP staining of bone tissue sections showed a significant reduction in the number of TRAP-positive osteoclasts per bone surface in UB-treated groups, confirming its inhibitory effect on osteoclast activity in vivo.

Figure 1: Changes in B) BMD, C) BS, D) BV, E) BV/TV, F) BS/TV, G) Tb.Sp, and H) Tb.N in response to treatment with a vehicle, a low dose of UB, or a high dose of UB.

Immunohistochemical analysis further supported these findings, revealing fewer MMP9 and NFATc1 positive cells in the bone tissue of UB-treated mice compared to the OVX control. Finally, serum analysis showed that UB treatment significantly reduced the content of CTX-1, a key bone resorption marker, in the serum of OVX mice. Importantly, no significant signs of drug toxicity were observed in the liver and kidney sections of UB-treated mice, suggesting a favorable safety profile. Collectively, these results strongly indicate that UB attenuates bone loss in OVX mice by inhibiting osteoclast formation and activity via the downregulation of the ERK/NF-κB signaling pathway [2].

 

Disclaimer

**LAB USE ONLY**
*This information is for educational purposes only and does not constitute medical advice. THE PRODUCTS DESCRIBED HEREIN ARE FOR RESEARCH USE ONLY. All clinical research must be conducted with oversight from the appropriate Institutional Review Board (IRB). All preclinical research must be conducted with oversight from the appropriate Institutional Animal Care and Use Committee (IACUC) following the guidelines of the Animal Welfare Act (AWA).

 

Citation

[1] Xue H, Zhou H, Lou Q, et al. Urolithin B reduces cartilage degeneration and alleviates osteoarthritis by inhibiting inflammation. Food Funct. 2024;15(7):3552-3565. Published 2024 Apr 2. doi:10.1039/d3fo03793b

[2] Li Y, Zhuang Q, Tao L, et al. Urolithin B suppressed osteoclast activation and reduced bone loss of osteoporosis via inhibiting ERK/NF-κB pathway. Cell Prolif. 2022;55(10):e13291. doi:10.1111/cpr.13291

 

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