Treatment of mice with urolithin-A (Sigma-Aldrich, Munich, Germany) in drinking water was performed with a daily dose of 0.114 mg urolithin-A per kg body weight per day. This was achieved by dissolving the compound in autoclaved tap water (ad libitum) to a final concentration of 22.8 mg/L. Mice received access to the urolithin-A solution from two days after the first C. jejuni infection. The placebo control mice received autoclaved tap water instead.
Urolithin a
Manufactured by Merck Group
Sourced in United States, Germany
Urolithin A is a metabolite produced by the human gut microbiome from dietary polyphenols. It is a natural compound found in various plant-based foods. Urolithin A has been the subject of scientific research, but its core function is not the focus of this product description.
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17 protocols using urolithin a
1
Urolithin-A Treatment in C. jejuni Mice
2
In Vitro Evaluation of Urolithin A and Ellagic Acid
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All chemicals, urolithin A (UA) and ellagic acid (EA) used in this study were from Sigma Chemical (St. Louis, MO, USA). POM extracts (Bulk Supplements; 40% EA) were obtained from Amazon. T84 human colonic cell line (CCL-248) and AML12 normal mouse liver cells (CRL-2254) were purchased from the ATCC (American Type Culture Cells, Manassas, VA). Other materials not described here were the highest grades available and/or the same, as recently described [19] , [33] (link), [34] (link).
3
Urolithin A Modulates CD8+ T Cell Gene Expression
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OVA257‐264 (SIINFEKL) peptide‐specific OT‐I CD8+ CTLs were stimulated with 10 nm OVA257‐264 peptide in RPMI 1640 medium containing 10% FBS, 50 U ml−1 penicillin‐streptomycin, 50 µm 2‐Mercaptoethanol, 100 U ml−1 human recombinant IL‐2, and 10 µm Urolithin A (UA) (Sigma‐Aldrich) for 72 h. CAR T cells were treated with 10 µm UA or DMSO for 48 h. Total RNA was extracted from CD8+ CTLs and CAR T cells that were treated by Mock or UA using TRIzol reagent according to the manufacturer's protocol (cat#15596026, Invitrogen, Thermo Fisher Scientific Inc.). Extracted RNA was quantified and reverse transcribed into cDNA with random primers using the Revert Aid First Strand cDNA Synthesis Kit (cat#K1642, Thermo Fisher Scientific Inc.) according to the manufacturer's manuals. The qPCR was performed with a SYBR Premix Ex Taq II kit (TaKaRa, Japan) using Bio‐Rad CFX Connect (Bio‐Rad Laboratories, Inc., Berkeley, CA, USA) with the following cycle conditions: an initial incubation at 95 °C/3 min and 39 cycles of 95, 60, and 72 °C/20 s. Expression values were normalized to β‐actin, and fold induction was normalized to the control using the 2−ΔΔCt Method. The primers are listed in Table S2 (Supporting Information).
4
Leukemic Cell Cytotoxicity of Urolithins
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Leukemic cells, namely, Jurkat and K562 cells, were obtained from ATCC, USA. All cell lines were maintained in RPMI (Gibco, Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco one-shot, Brazil), 50 U/mL penicillin, and 50 mg/mL streptomycin (Gibco) at 37 °C and 5% CO2. The cells were treated with either vehicle control or with different concentrations of urolithin A (Sigma SML1791, St. Louis, MO, USA) and urolithin B (Sigma SML1649) for 48 h [34 (link)].
5
Screening Natural Compounds for DNMT Inhibition
6
Profiling Compounds in CareVid™ Tea Powder
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The nine compounds (1 –9 ) were determined using HPLC-MS to be present in CareVidTM as described previously [1 (link)]. Extracts were prepared initially from 20 g each of CareVidTM Tea powder. The extracts were methylene chloride, ethanol, methanol, water, acidified water (0.1 M HCl), and steeping in hot water (>90 °C). Metabolic profiling of the extracts was performed by UHPLC-UV-HRMS/MS on a Vanquish UHPLC system (Thermo Scientific, Waltham, MA, USA) coupled to a 100 Hz photodiode array detector (PDA) and an Orbitrap Fusion Tribrid (Thermo Scientific) high-resolution tandem mass spectrometer (Thermo Scientific, Waltham, MA, USA). A detailed protocol that was followed is described in Rotich et al., 2021 (Figure 1 ). For the assay, pure standards of pellitorine (1 ), oleuropein (2 ) and ellagic acid (9 ), urolithin A (10 ), and urolithin B (11 ) were bought from Sigma Aldrich, whereas pure standards of magnoflorine (3 ) earlier isolated from the seed cake of Croton megalocarpus, crotocorylifuran (6 ) earlier isolated from Croton megalocarpoides, crotepoxide (4 ) earlier isolated from Croton alienus, ent-kaurane-16β,17-diol (5 ) earlier isolated from Croton haumanianus, and lupeol (7 ) and betulin (8 ) isolated in substantial quantities from CareVidTM were obtained in the Jodrell Laboratory, Kew, UK.
7
Myocardial Ischemia/Reperfusion Injury Protocol
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I/R injury was conducted as previously described [25 (link)]. In brief, mice were anesthetized with 1%–3% isoflurane through inhalation (Baxter, USA). The left anterior descending (LAD) coronary artery was temporarily ligated for 45 min, followed by 0–24 h reperfusion. Sham operation was performed by opening the chest and exposing the pericardium of anesthetized mice, leaving the LAD intact. For each experimental condition, a minimum of 6 mice were examined. After reperfusion, 2% Evans blue/saline were injected into mice to demarcate the ischemic area at risk (AAR). Then, the hearts were excised and cut into five 1-mm slices, which were incubated with 1% 2,3,5-triphenyltetrazolium chloride (TTC) for 10 min at 37 °C to demonstrate infarct size (IS). The percentage of IS and AAR of individual myocardial sections was multiplied by the corresponding section weight. To inhibit apoptosis or necroptosis, mice were intraperitoneally injected with Z-VAD-FMK (Selleck Chemicals, USA; No. S7023, 20 mg/kg body weight) or Necrostatin-1 (Selleck Chemicals; No. S8037, 5 mg/kg) 12 h before I/R injury. To activate or inhibit mitophagy, mice were intraperitoneally injected with a mitophagy activator (Urolithin A, Sigma, USA, No. SML1791, 10 mg/kg) or antagonist (MF-094, Sigma, No. SML2501, 20 mg/kg) 12 h before I/R injury.
8
Dissolution and Preparation of Bioactive Compounds
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The chemical substances such as ellagic acid, gallic acid, punicalagin (mixture of anomers), punicalin, umifenovir, urolithin A were obtained from Sigma Chemical Co. (St. Louis, USA). Solvents used for dissolution of the tested compounds were dimethylsulfoxide (DMSO) and phosphate-buffered saline (PBS) which were also obtained from Sigma Chemical Co. (St. Louis, USA). DMSO didn’t exceed screening kit recommended concentration.
9
Transient Transfection and Luciferase Assay
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Methodology for transient transfection of cells with DNA plasmids/luciferase-containing vectors is detailed elsewhere [13 (link)]. Twenty-two to 24 h post-transfection, the cells were treated with either an ethanol (EtOH) or dimethyl sulfoxide (DMSO) vehicle control or the ligand for the nuclear receptor (usually 1,25D), along with urolithin A (Sigma-Aldrich, St. Louis, MO) and/or resveratrol (Enzo Life Sciences, Farmingdale, NY) added as a nutraceutical adjuvant in select experiments as detailed in the figure legends. After incubation, cells were lysed and the lysates were then analyzed sequentially for Firefly and Renilla luciferase, followed by computation of transcriptional activity as described previously [13 (link)].
10
Urolithin A Modulates Inflammation
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Urolithin A (≥97% purity by HPLC, Figure 1 A) was purchased from Sigma-Aldrich (St. Louis, MO, USA), cell viability assays (MTT), and poly(I:C) were purchased from InvivoGen (San Diego, CA, USA). Enzyme-linked immunosorbent assay (ELISA) kits were purchased from R&D Systems (Minneapolis, MN, USA). DAPI solution was purchased from Sigma-Aldrich (St. Louis, MO, USA). The inhibitor PD98059 was purchased from Enzo Life Sciences (Farmingdale, NY, USA). Antibodies against β-actin, TLR3, TRIF, IRF, STAT1, NF-κB, I-κB, COX-2, iNOS, Nrf2, and phosphorylated IRF (pIRF), STAT1, NF-κB (pNF-κB), and IκB (pIκB) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against JNK, ERK, p38, and phosphorylated JNK (pJNK), ERK (pERK), and p38 (pp38) were purchased from Millipore (Billerica, MA, USA).
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