Endogenous Metabolite; NF-κB; JNK; ERK; Akt

Urolithin B inhibited the production of NO and pro-inflammatory cytokines, while increased anti-inflammatory cytokine IL-10 in LPS-stimulated BV2 microglial cells. In addition, urolithin B inhibited NO, TNF-α, and IL-6 production in lipoteichoic acid (LTA) or polyinosinic-polycytidylic acid (poly(I:C))-stimulated BV2 cells, suggesting that the anti-inflammatory effect of urolithin B is not confined to LPS stimulation. Urolithin B also showed an antioxidant effect by reducing intracellular reactive oxygen species (ROS) production and NADPH oxidase subunit expression, and by upregulating the antioxidant hemeoxygenase-1 expression via Nrf2/ARE signaling. More detailed mechanistic studies showed that urolithin B inhibited NF-κB activity by reducing the phosphorylation and degradation of IκBα. In addition, urolithin B suppressed the phosphorylation of JNK, ERK, and Akt, and enhanced the phosphorylation of AMPK, which is associated with anti-inflammatory and antioxidant processes [1].
Urolithin B enhances the growth and differentiation of C2C12 myotubes by increasing protein synthesis and repressing the ubiquitin-proteasome pathway. Genetic and pharmacological arguments support an implication of the androgen receptor. Signalling analyses suggest a crosstalk between the androgen receptor and the mTORC1 pathway, possibly via AMPK.[2].

Urolithin B inhibits microglial activation in LPS-injected mouse brains [1]
To verify its anti-inflammatory effects in vivo, we examined the effect of urolithin B on microglial activation in LPS-injected mouse brains. Microglial activation was investigated by quantifying the immunoreactivity for Iba1. Systemic LPS administration increased the number of Iba1-positive cells with densely stained amoeboid cell bodies in comparison with the number observed in the control group. However, urolithin B significantly reduced the number of Iba1-positive cells in the cortex, hippocampus, and substantia nigra, indicating that urolithin B efficiently suppressed microglial activation under neuroinflammatory conditions in vivo.
In vivo experiments confirm that urolithin B induces muscle hypertrophy in mice and reduces muscle atrophy after the sciatic nerve section [2].

The effects of urolithin B on the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and cytokines were examined in BV2 microglial cells using enzyme-linked immunosorbent assay (ELISA), reverse transcription polymerase chain reaction (RT-PCR), and Western blot analysis. Microglial activation in the lipopolysaccharide (LPS)-injected mouse brain was assessed using immunohistochemistry. The detailed molecular mechanisms underlying the anti-inflammatory and antioxidant effects of urolithin B were analyzed using an electrophoretic mobility shift assay, reporter gene assay, Western blot, and RT-PCR [1].

BV2 cells (1 × 105 cells/well in a 24-well plate) were pretreated with urolithin B for 1 h and stimulated by LPS (100 ng/ml), poly(I: C) (25 µg/ml), or LTA (10 µg/ml) for 16 h. The supernatants were then collected, and the accumulated nitric oxide level was determined using Griess reagent (Promega, Madison, WI). The concentrations of TNF-α, IL-6, IL-1β, and IL-10 in conditioned media were measured using enzyme-linked immunosorbent assay (ELISA) according to the supplier's instructions (BD Biosciences, San Jose, CA). The intracellular accumulation of ROS was measured using 2′,7′-dichlorofluorescein (H2DCF-DA). BV2 cells were stimulated with LPS for 16 h and stained with 20 µM H2DCF-DA for 1 h at 37°C. DCF fluorescence intensity was measured at an excitation wavelength of 485 nm and an emission wavelength of 535 nm on a fluorescence plate reader [1].

LPS-induced inflammation and the administration of urolithin B [1]
ICR mice (male, 32–37 g, 7 weeks old) were purchased from Orient Bio (Seoul, Korea), and housed under controlled conditions (21°C, 12 h dark/light cycle) with free access to food and water during the experimental session. All experiments were approved by the Institutional Animal Care and Use Committee of Ewha Womans University (IACUC #2014-0274) and were carried out in accordance with the guidelines of the National Institute of Health's Guide for the Care and Use of Laboratory Animals. Systemic inflammation was induced by the administration of LPS (5 mg/kg, intraperitoneal; i.p.). Urolithin B (50 mg/kg, i.p.) was dissolved in vehicle (1% DMSO in normal saline) and administered to the animals daily for 4 days prior to LPS injections.

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Immunohistochemistry [1]
After 6 h of LPS treatment, mice were anesthetized with pentobarbital and transcardially perfused with saline and 4% paraformaldehyde. Brains were post-fixed in 4% paraformaldehyde and then washed with phosphate buffered saline (PBS) 3 times for 10 min each. The tissues were then left to soak in 30% sucrose solution, following which 30 µm-thick sections were cut using a cryostat. Floating sections were treated with 0.3% hydrogen peroxide in PBS for 30 min to neutralize endogenous peroxidase activity and then rinsed in PBS containing 10% Triton X-100 (PBST). The sections were incubated in PBS containing 4% bovine serum albumin for 1 h, and then incubated overnight with the primary antibody against Iba1 (1:500). The sections were incubated with biotinylated secondary antibody (1:1000) for 1 h, incubated with avidin-biotin-horseradish peroxidase (HRP) complex (Vector Laboratories, Burlingame, CA), and then washed with PBST. The reaction was visualized using 3,3′-diaminobenzidine tetrahydrochloride (DAB). The sections were dehydrated and cover slipped for viewing under a light microscope.

[1]. Anti-inflammatory and antioxidant mechanisms of urolithin B in activated microglia. Phytomedicine. 2019 Mar 1;55:50-57.

[2]. Urolithin B, a newly identified regulator of skeletal muscle mass. J Cachexia Sarcopenia Muscle. 2017 Aug;8(4):583-597.

[3]. Urolithin B suppresses tumor growth in hepatocellular carcinoma through inducing the inactivation of Wnt/β-catenin signaling. J Cell Biochem. 2019 Oct;120(10):17273-17282.

Urolithin B is a coumarin compound. It has been reported to be found in pomegranate (Punica granatum) and yellow-footed bat (Trogopterus xanthipes), with relevant data available. Background: Urolithin B is one of the gut microbial metabolites of ellagitannins and is present in various plant-based foods, including pomegranates, berries, walnuts, tropical fruits, and medicinal herbs. Although various biological activities of urolithin B have been reported, its anti-inflammatory and antioxidant effects in neuroinflammation have not been clearly confirmed. Objective: This study aimed to investigate the anti-inflammatory and antioxidant effects of urolithin B in activated microglia and elucidate its potential molecular mechanisms. Study Design: Enzyme-linked immunosorbent assay (ELISA), reverse transcription polymerase chain reaction (RT-PCR), and Western blotting were used to detect the effects of urolithin B on the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and cytokines in BV2 microglia. Immunohistochemistry was used to assess the activation of microglia in the brains of mice injected with lipopolysaccharide (LPS). This study employed electrophoretic mobility shift analysis, reporter gene detection, Western blotting, and RT-PCR to analyze the molecular mechanisms of the anti-inflammatory and antioxidant effects of urolithin B. The results showed that urolithin B inhibited the production of nitric oxide (NO) and pro-inflammatory cytokines in LPS-stimulated BV2 microglia, while increasing the expression of the anti-inflammatory cytokine IL-10. Furthermore, urolithin B inhibited the production of NO, tumor necrosis factor-α (TNF-α), and IL-6 in BV2 cells stimulated by lipoteichoic acid (LTA) or poly(I:C), suggesting that the anti-inflammatory effect of urolithin B is not limited to LPS stimulation. Urolithin B also exerts its antioxidant effect by reducing the production of intracellular reactive oxygen species (ROS) and the expression of NADPH oxidase subunits, as well as by upregulating the expression of the antioxidant enzyme heme oxygenase-1 through the Nrf2/ARE signaling pathway. More detailed mechanistic studies showed that urolithin B inhibited NF-κB activity by reducing the phosphorylation and degradation of IκBα. In addition, urolithin B inhibited the phosphorylation of JNK, ERK and Akt and enhanced the phosphorylation of AMPK, which is associated with anti-inflammatory and antioxidant processes. Finally, we confirmed that urolithin B inhibited microglial activation in the brains of LPS-injected mice. Conclusion: The potent anti-inflammatory and antioxidant effects of urolithin B may provide therapeutic potential for neuroinflammatory diseases associated with oxidative stress and microglial activation. [1]

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Background: Muscle size control is an important part of health. In fact, skeletal muscle atrophy can lead to decreased strength, reduced quality of life and metabolic disorders. Therefore, to address this widespread health problem, strategies aimed at mitigating muscle atrophy and promoting muscle growth are needed to address various (patho)physiological conditions such as sarcopenia, immobilization, malnutrition, or cachexia. In this study, we tested the effects of urolithin B (an ellagitannin-derived metabolite) on skeletal muscle growth. Methods: C2C12 myotubes were treated with 15 μM urolithin B for 24 hours. In in vivo experiments, mice were implanted with a micro-osmotic pump and infused with 10 μg urolithin B daily for 28 days. We also investigated muscle atrophy in sciatic nerve-denervated mice that received urolithin B in the same manner. Results: Our experiments showed that urolithin B promotes the growth and differentiation of C2C12 myotubes by increasing protein synthesis and inhibiting the ubiquitin-proteasome pathway. Genetic and pharmacological evidence supports the involvement of the androgen receptor. Signaling pathway analysis indicated crosstalk between the androgen receptor and the mTORC1 pathway, possibly mediated by AMPK. In vivo experiments confirmed that urolithin B could induce muscle hypertrophy in mice and reduce muscle atrophy after sciatic nerve transection. Conclusion: This study highlights the potential application value of urolithin B in the treatment of muscle atrophy associated with various (patho)physiological conditions. [2] Dietary ellagitannins (ETs) intake has been shown to be beneficial for a variety of chronic diseases, including cancer and cardiovascular diseases. Urolithin, a gut microbiota metabolite derived from ETs, is considered to be the molecule responsible for these health benefits. Previous studies have shown that urolithin has antiproliferative effects on prostate cancer, breast cancer and colon cancer. However, its mechanism of action on hepatocellular carcinoma (HCC) is unclear. This article aims to explore the role of urolithin B (UB), a member of the urolithin family, in hepatocellular carcinoma (HCC). We evaluated the effects of UB on HCC cell viability, cell cycle and apoptosis and found that UB could inhibit HCC cell proliferation by inducing cell cycle arrest and apoptosis. In addition, UB could increase the expression of phosphorylated β-catenin and block its translocation from the nucleus to the cytoplasm, thereby inhibiting the Wnt/β-catenin signaling pathway. Using a xenograft mouse model, we found that UB can inhibit tumor growth in vivo. In summary, our data show that UB can inhibit HCC cell proliferation in vitro and in vivo by inhibiting the Wnt/β-catenin signaling pathway, suggesting that UB may be a potential candidate drug for developing anticancer drugs targeting HCC. [3]

C13H8O3

212.2008

212.047

C, 73.58; H, 3.80; O, 22.62

1139-83-9

5380406

Light yellow to yellow solid powder

1.395g/cm3

432.6ºC at 760 mmHg

196.6ºC

4.34E-08mmHg at 25°C

1.679

Endogenous Metabolite; Monophenols

2.651

1

3

0

16

289

0

O1C(C2=C([H])C([H])=C([H])C([H])=C2C2C([H])=C([H])C(=C([H])C1=2)O[H])=O

WXUQMTRHPNOXBV-UHFFFAOYSA-N

InChI=1S/C13H8O3/c14-8-5-6-10-9-3-1-2-4-11(9)13(15)16-12(10)7-8/h1-7,14H

3-hydroxy-6H-benzo[c]chromen-6-one

Urolithin B; NSC94726; 3 hydroxy Urolithin; NSC-94726; Urolithin-B; NSC 94726; 3-hydroxy Urolithin;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~250 mg/mL (~1178.13 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (9.80 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)