Increased Glycolytic Activity Is Part of Impeded M1(LPS) Macrophage Polarization in the Presence of Urolithin A.

Urolithin A is a gut metabolite of ellagitannins and reported to confer health benefits, e.g., by increased clearance of damaged mitochondria by macroautophagy or curbed inflammation. One targeted cell type are macrophages, which are plastic and able to adopt pro- or anti-inflammatory polarization states, usually assigned as M1 and M2 macrophages, respectively. This flexibility is tightly coupled to characteristic shifts in metabolism, such as increased glycolysis in M1 macrophages, and protein expression upon appropriate stimulation. This study aimed at investigating whether the anti-inflammatory properties of U: rolithin A may be driven by metabolic alterations in cultivated murine M1(lipopolysaccharide) macrophages. Expression and extracellular flux analyses showed that urolithin A led to reduced il1ß, il6, and nos2 expression and boosted glycolytic activity in M1(lipopolysaccharide) macrophages. The pro-glycolytic feature of UROLITHIN A: occurred in order to causally contribute to its anti-inflammatory potential, based on experiments in cells with impeded glycolysis. Mdivi, an inhibitor of mitochondrial fission, blunted increased glycolytic activity and reduced M1 marker expression in M1(lipopolysaccharide/UROLITHIN A: ), indicating that segregation of mitochondria was a prerequisite for both actions of UROLITHIN A: . Overall, we uncovered a so far unappreciated metabolic facet within the anti-inflammatory activity of UROLITHIN A: and call for caution about the simplified notion of increased aerobic glycolysis as an inevitably proinflammatory feature in macrophages upon exposure to natural products.

Elevated PINK1/Parkin-Dependent Mitophagy and Boosted Mitochondrial Function Mediate Protection of HepG2 Cells from Excess Palmitic Acid by Hesperetin.

Deregulation of mitochondrial functions in hepatocytes contributes to many liver diseases, such as nonalcoholic fatty liver disease (NAFLD). Lately, it was referred to as MAFLD (metabolism-associated fatty liver disease). Hesperetin (Hst), a bioactive flavonoid constituent of citrus fruit, has been proven to attenuate NAFLD. However, a potential connection between its preventive activities and the modulation of mitochondrial functions remains unclear. Here, our results showed that Hst alleviates palmitic acid (PA)-triggered NLRP3 inflammasome activation and cell death by inhibition of mitochondrial impairment in HepG2 cells. Hst reinstates fatty acid oxidation (FAO) rates measured by seahorse extracellular flux analyzer and intracellular acetyl-CoA levels as well as intracellular tricarboxylic acid cycle metabolites levels including NADH and FADH2 reduced by PA exposure. In addition, Hst protects HepG2 cells against PA-induced abnormal energetic profile, ATP generation reduction, overproduction of mitochondrial reactive oxygen species, and collapsed mitochondrial membrane potential. Furthermore, Hst improves the protein expression involved in PINK1/Parkin-mediated mitophagy. Our results demonstrate that it restores PA-impaired mitochondrial function and sustains cellular homeostasis due to the elevation of PINK1/Parkin-mediated mitophagy and the subsequent disposal of dysfunctional mitochondria. These results provide therapeutic potential for Hst utilization as an effective intervention against fatty liver disease.

Sulforaphane impedes mitochondrial reprogramming and histone acetylation in polarizing M1 (LPS) macrophages.

M1 (LPS) macrophages are characterized by a high expression of pro-inflammatory mediators, and distinct metabolic features that comprise increased glycolysis, a broken TCA cycle, or impaired OXPHOS with augmented mitochondrial ROS production. This study investigated whether the phytochemical sulforaphane (Sfn) influences mitochondrial reprogramming during M1 polarization, as well as to what extent this can contribute to Sfn-mediated inhibition of M1 marker expression in murine macrophages. The use of extracellular flux-, metabolite-, and immunoblot analyses as well as fluorescent dyes indicative for mitochondrial morphology, membrane potential or superoxide production, demonstrated that M1 (LPS/Sfn) macrophages maintain an unbroken TCA cycle, higher OXPHOS rate, boosted fusion dynamics, lower membrane potential, and less superoxide production in their mitochondria when compared to control M1 (LPS) cells. Sustained OXPHOS and TCA activity but not the concomitantly observed high dependency on fatty acids as fuel appeared necessary for M1 (LPS/Sfn) macrophages to reduce expression of nos2, il1ß, il6 and tnfα. M1 (LPS/Sfn) macrophages also displayed lower nucleo/cytosolic acetyl-CoA levels in association with lower global and site-specific histone acetylation at selected pro-inflammatory gene promoters than M1 (LPS), evident in colorimetric coupled enzyme assays, immunoblot and ChIP-qPCR analyses, respectively. Supplementation with acetate or citrate was able to rescue both histone acetylation and mRNA expression of the investigated M1 marker genes in Sfn-treated cells. Overall, Sfn preserves mitochondrial functionality and restricts indispensable nuclear acetyl-CoA for histone acetylation and M1 marker expression in LPS-stimulated macrophages.

Targeting PHGDH reverses the immunosuppressive phenotype of tumor-associated macrophages through α-ketoglutarate and mTORC1 signaling.

Phosphoglycerate dehydrogenase (PHGDH) has emerged as a crucial factor in macromolecule synthesis, neutralizing oxidative stress, and regulating methylation reactions in cancer cells, lymphocytes, and endothelial cells. However, the role of PHGDH in tumor-associated macrophages (TAMs) is poorly understood. Here, we found that the T helper 2 (Th2) cytokine interleukin-4 and tumor-conditioned media upregulate the expression of PHGDH in macrophages and promote immunosuppressive M2 macrophage activation and proliferation. Loss of PHGDH disrupts cellular metabolism and mitochondrial respiration, which are essential for immunosuppressive macrophages. Mechanistically, PHGDH-mediated serine biosynthesis promotes α-ketoglutarate production, which activates mTORC1 signaling and contributes to the maintenance of an M2-like macrophage phenotype in the tumor microenvironment. Genetic ablation of PHGDH in macrophages from tumor-bearing mice results in attenuated tumor growth, reduced TAM infiltration, a phenotypic shift of M2-like TAMs toward an M1-like phenotype, downregulated PD-L1 expression and enhanced antitumor T-cell immunity. Our study provides a strong basis for further exploration of PHGDH as a potential target to counteract TAM-mediated immunosuppression and hinder tumor progression.

LC-MS- and 1H NMR-Based Metabolomics to Highlight the Impact of Extraction Solvents on Chemical Profile and Antioxidant Activity of Daikon Sprouts (Raphanus sativus L.).

Currently, the interest of consumers towards functional foods as source of bioactive compounds is increasing. The sprouts of Raphanus sativus var longipinnatus (Brassicaceae) are "microgreens" popular, especially in gourmet cuisine, for their appealing aspect and piquant flavour. They represent a functional food due to their high nutritional value and health-promoting effects. Herein, the sprouts of daikon were extracted by different solvent mixtures to highlight how this process can affect the chemical profile and the antioxidant activity. An in-depth investigation based on a preliminary LC-ESI/LTQOrbitrap/MS profiling was carried out, leading to the identification of nineteen compounds, including glucosinolates and hydroxycinnamic acid derivatives. An undescribed compound, 1-O-feruloyl-2-O-sinapoyl-ß-D-glucopyranoside, was isolated, and its structure was elucidated by NMR spectroscopy. The phenolic content and radical scavenging activity (DPPH and TEAC assays), along with the ability to activate Nrf2 (Nrf2-mediated luciferase reporter gene assay) of polar extracts, were evaluated. The results showed the highest antioxidant activity for the 70% EtOH/H2O extract with a TEAC value of 1.95 mM and IC50 = 93.97 µg/mL in the DPPH assay. Some 50% and 70% EtOH/H2O extracts showed a pronounced concentration-dependent induction of Nrf2 activity. The extracts of daikon sprouts were submitted to 1H NMR experiments and then analyzed by untargeted and targeted approaches of multivariate data analysis to highlight differences related to extraction solvents.

Norbergenin prevents LPS-induced inflammatory responses in macrophages through inhibiting NFκB, MAPK and STAT3 activation and blocking metabolic reprogramming.

Inflammation is thought to be a key cause of many chronic diseases and cancer. However, current therapeutic agents to control inflammation have limited long-term use potential due to various side-effects. This study aimed to examine the preventive effects of norbergenin, a constituent of traditional anti-inflammatory recipes, on LPS-induced proinflammatory signaling in macrophages and elucidate the underlying mechanisms by integrative metabolomics and shotgun label-free quantitative proteomics platforms. Using high-resolution mass spectrometry, we identified and quantified nearly 3000 proteins across all samples in each dataset. To interpret these datasets, we exploited the differentially expressed proteins and conducted statistical analyses. Accordingly, we found that LPS-induced production of NO, IL1ß, TNFα, IL6 and iNOS in macrophages was alleviated by norbergenin via suppressed activation of TLR2 mediated NFκB, MAPKs and STAT3 signaling pathways. In addition, norbergenin was capable of overcoming LPS-triggered metabolic reprogramming in macrophages and restrained the facilitated glycolysis, promoted OXPHOS, and restored the aberrant metabolites within the TCA cycle. This is linked to its modulation of metabolic enzymes to support its anti-inflammatory activity. Thus, our results uncover that norbergenin regulates inflammatory signaling cascades and metabolic reprogramming in LPS stimulated macrophages to exert its anti-inflammatory potential.

Cultured rat aortic vascular smooth muscle cells do not express a functional TRPV1.

We showed previously that capsaicin, an active compound of chili peppers, can inhibit platelet-derived growth factor-induced proliferation in primary rat vascular smooth muscle cells (VSMCs). The inhibition of BrdU incorporation by capsaicin in these cells was revoked by BCTC, which might be explained by a role of TRPV1 in VSMCs proliferation. To further pursue the hypothesis of a TRPV1-dependent effect of capsaicin, we investigated TRPV1 expression and function. Commercially available antibodies against two different TRPV1 epitopes (N-terminus and C-terminus) were rendered invalid in detecting TRPV1, as shown: i) in western blot experiments using control lysates of TRPV1-expressing (PC-12 and hTRPV1 transfected HEK293T) and TRPV1-downregulated (CRISPR/Cas gene edited A10) cells, and ii) by substantial differences in staining patterns between the applied antibodies using fluorescence confocal microscopy. The TRPV1 agonists capsaicin, resiniferatoxin, piperine and evodiamine did not increase intracellular calcium levels in primary VSMCs and in A10 cells. Using RT qPCR, we could detect a rather low TRPV1 expression in VSMCs at the mRNA level (Cp value around 30), after validating the primer pair in NGF-stimulated PC-12 cells. We conclude that rat vascular smooth muscle cells do not possess canonical TRPV1 channel activity, which could explain the observed antiproliferative effect of capsaicin.

Branched-chain ketoacids derived from cancer cells modulate macrophage polarization and metabolic reprogramming.

Macrophages are prominent immune cells in the tumor microenvironment that can be educated into pro-tumoral phenotype by tumor cells to favor tumor growth and metastasis. The mechanisms that mediate a mutualistic relationship between tumor cells and macrophages remain poorly characterized. Here, we have shown in vitro that different human and murine cancer cell lines release branched-chain α-ketoacids (BCKAs) into the extracellular milieu, which influence macrophage polarization in an monocarboxylate transporter 1 (MCT1)-dependent manner. We found that α-ketoisocaproate (KIC) and α-keto-ß-methylvalerate (KMV) induced a pro-tumoral macrophage state, whereas α-ketoisovalerate (KIV) exerted a pro-inflammatory effect on macrophages. This process was further investigated by a combined metabolomics/proteomics platform. Uptake of KMV and KIC fueled macrophage tricarboxylic acid (TCA) cycle intermediates and increased polyamine metabolism. Proteomic and pathway analyses revealed that the three BCKAs, especially KMV, exhibited divergent effects on the inflammatory signal pathways, phagocytosis, apoptosis and redox balance. These findings uncover cancer-derived BCKAs as novel determinants for macrophage polarization with potential to be selectively exploited for optimizing antitumor immune responses.

Sulforaphane diminishes moonlighting of pyruvate kinase M2 and interleukin 1ß expression in M1 (LPS) macrophages.

Murine macrophages activated by the Toll-like receptor 4 agonist lipopolysaccharide (LPS) polarize to the M1 type by inducing proinflammatory marker proteins and changing their energy metabolism to increased aerobic glycolysis and reduced respiration. We here show that the aliphatic isothiocyanate sulforaphane (Sfn) diminishes M1 marker expression (IL-1ß, IL-6, TNF-α, iNOS, NO, and ROS) and leads to highly energetic cells characterized by both high glycolytic and high respiratory activity as assessed by extracellular flux analysis. Focusing on a potential connection between high glycolytic activity and low IL-1ß expression in M1 (LPS/Sfn) macrophages, we reveal that Sfn impedes the moonlighting function of pyruvate kinase M2 (PKM2) in M1 macrophages. Sfn limits mono/dimerization and nuclear residence of PKM2 accompanied by reduced HIF-1α levels, Stat3 phosphorylation at tyrosine 705, and IL-1ß expression while preserving high levels of cytosolic PKM2 tetramer with high glycolytic enzyme activity. Sfn prevents glutathionylation of PKM2 in LPS-stimulated macrophages which may account for the reduced loss of PKM2 tetramer. Overall, we uncover PKM2 as a novel affected hub within the anti-inflammatory activity profile of Sfn.

In Silico and In Vitro Approach to Assess Direct Allosteric AMPK Activators from Nature.

The 5'-adenosine monophosphate-activated protein kinase (AMPK) is an important metabolic regulator. Its allosteric drug and metabolite binding (ADaM) site was identified as an attractive target for direct AMPK activation and holds promise as a novel mechanism for the treatment of metabolic diseases. With the exception of lusianthridin and salicylic acid, no natural product (NP) is reported so far to directly target the ADaM site. For the streamlined assessment of direct AMPK activators from the pool of NPs, an integrated workflow using in silico and in vitro methods was applied. Virtual screening combining a 3D shape-based approach and docking identified 21 NPs and NP-like molecules that could potentially activate AMPK. The compounds were purchased and tested in an in vitro AMPK α 1 ß 1 γ 1 kinase assay. Two NP-like virtual hits were identified, which, at 30 µM concentration, caused a 1.65-fold (± 0.24) and a 1.58-fold (± 0.17) activation of AMPK, respectively. Intriguingly, using two different evaluation methods, we could not confirm the bioactivity of the supposed AMPK activator lusianthridin, which rebuts earlier reports.

Natural Products, the Continuous Source of Therapeutic Molecules for Various Diseases: Literature Landscape Analysis.

BACKGROUND: Substances present in nature have been a continuous source for the development of drugs for cardiovascular and infectious diseases, cancer, and many other diseases. As the literature concerning these natural products grows, it becomes more difficult for a reader to quickly grasp the essential facts and develop a well-informed impression of this field of research. Until now, it has also been difficult to determine which natural products and research objectives were gaining the most attention as measured by a number of citations. OBJECTIVE: The current study of all published articles concerned with natural products sought to identify which natural products and which research objectives are connected with the major contributors to scientific journals based on the number of relevant publications and the number of times each publication was cited elsewhere. METHODS: Bibliometric data, including citation data, were extracted from the Web of Science database using the search string TS=("natural product*)" and analyzed by the VOSviewer software. RESULTS: The search yielded 63,194 articles, with more than half of the manuscripts published since 2012. The ratio of original articles to reviews was 5.8:1. The major contributing countries were the United States, China, Germany, Japan, and India. Articles were published mainly in journals focused on chemistry, pharmacology or biochemistry. Curcumin, resveratrol, and terpenoids were the most frequently cited natural products. CONCLUSION: The results of the current study provide researchers from different backgrounds and healthcare professionals with a brief overview of the major trends in natural-product research in the form of a citation-based summary of the relevant literature.

Fast and Highly Efficient Affinity Enrichment of Azide-A-DSBSO Cross-Linked Peptides.

Cross-linking mass spectrometry is an increasingly used, powerful technique to study protein-protein interactions or to provide structural information. Due to substochiometric reaction efficiencies, cross-linked peptides are usually low abundance. This results in challenging data evaluation and the need for an effective enrichment. Here we describe an improved, easy to implement, one-step method to enrich azide-tagged, acid-cleavable disuccinimidyl bis-sulfoxide (DSBSO) cross-linked peptides using dibenzocyclooctyne (DBCO) coupled Sepharose beads. We probed this method using recombinant Cas9 and E. coli ribosome. For Cas9, the number of detectable cross-links was increased from ∼100 before enrichment to 580 cross-links after enrichment. To mimic a cellular lysate, E. coli ribosome was spiked into a tryptic HEK background at a ratio of 1:2-1:100. The number of detectable unique cross-links was maintained high at ∼100. The estimated enrichment efficiency was improved by a factor of 4-5 (based on XL numbers) compared to enrichment via biotin and streptavidin. We were still able to detect cross-links from 0.25 µg cross-linked E. coli ribosomes in a background of 100 µg tryptic HEK peptides, indicating a high enrichment sensitivity. In contrast to conventional enrichment techniques, like SEC, the time needed for preparation and MS measurement is significantly reduced. This robust, fast, and selective enrichment method for azide-tagged linkers will contribute to mapping protein-protein interactions, investigating protein architectures in more depth, and helping to understand complex biological processes.

Short Chain (≤C4) Esterification Increases Bioavailability of Rosmarinic Acid and Its Potency to Inhibit Vascular Smooth Muscle Cell Proliferation.

Rosmarinic acid is a natural phenolic acid and active compound found in many culinary plants, such as rosemary, mint, basil and perilla. Aiming to improve the pharmacokinetic profile of rosmarinic acid and its activity on vascular smooth muscle cell proliferation, we generated a series of rosmarinic acid esters with increasing alkyl chain length ranging from C1 to C12. UHPLC-MS/MS analysis of rat blood samples revealed the highest increase in bioavailability of rosmarinic acid, up to 10.52%, after oral administration of its butyl ester, compared to only 1.57% after rosmarinic acid had been administered in its original form. When added to vascular smooth muscle cells in vitro, all rosmarinic acid esters were taken up, remained esterified and inhibited vascular smooth muscle cell proliferation with IC50 values declining as the length of alkyl chains increased up to C4, with an IC50 of 2.84 µM for rosmarinic acid butyl ester, as evident in a resazurin assay. Vascular smooth muscle cells were arrested in the G0/G1 phase of the cell cycle and the retinoblastoma protein phosphorylation was blocked. Esterification with longer alkyl chains did not improve absorption and resulted in cytotoxicity in in vitro settings. In this study, we proved that esterification with proper length of alkyl chains (C1-C4) is a promising way to improve in vivo bioavailability of rosmarinic acid in rats and in vitro biological activity in rat vascular smooth muscle cells.

Tylophorine reduces protein biosynthesis and rapidly decreases cyclin D1, inhibiting vascular smooth muscle cell proliferation in vitro and in organ culture.

BACKGROUND: Tylophorine (TYL) is an alkaloid with antiproliferative action in cancer cells. Vascular smooth muscle cell (VSMC) proliferation and neointima formation contribute to restenosis after percutaneous coronary interventions. HYPOTHESIS/PURPOSE: Our goal was to examine the potential of TYL to inhibit VSMC proliferation and migration, and to dissect underlying signaling pathways. STUDY DESIGN AND METHODS: TYL was administered to platelet-derived growth factor (PDGF-BB)-stimulated, serum-stimulated, quiescent and unsynchronized VSMC of rat and human origin. BrdU incorporation and resazurin conversion were used to assess cell proliferation. Cell cycle progression was analyzed by flow cytometry of propidium iodide-stained nuclei. Expression profiles of proteins and mRNAs were determined using western blot analysis and RT-qPCR. The Click-iT OPP Alexa Fluor 488 assay was used to monitor protein biosynthesis. RESULTS: TYL inhibited PDGF-BB-induced proliferation of rat aortic VSMCs by arresting cells in G1 phase of the cell cycle with an IC50 of 0.13 µmol/l. The lack of retinoblastoma protein phosphorylation and cyclin D1 downregulation corroborated a G1 arrest. Inhibition of proliferation and cyclin D1 downregulation were species- and stimulus-independent. TYL also decreased levels of p21 and p27 proteins, although at later time points than observed for cyclin D1. Co-treatment of VSMC with TYL and MG132 or cycloheximide (CHX) excluded proteasome activation by TYL as the mechanism of action. Comparable time-dependent downregulation of cyclin D1, p21 and p27 in TYL- or CHX-treated cells, together with decreased protein synthesis observed in the Click-iT assay, suggests that TYL is a protein synthesis inhibitor. Besides proliferation, TYL also suppressed migration of PDGF-activated VSMC. In a human saphenous vein organ culture model for graft disease, TYL potently inhibited intimal hyperplasia. CONCLUSION: This unique activity profile renders TYL an interesting lead for the treatment of vasculo-proliferative disorders, such as restenosis.

Urolithin A gains in antiproliferative capacity by reducing the glycolytic potential via the p53/TIGAR axis in colon cancer cells.

Polyphenols have shown promising bioactivity in experimental in vitro and in vivo models for cancer chemoprevention. However, consumed orally, they are often transformed by gut microbes into new active principles with so far incompletely deciphered molecular mechanisms. Here, enterolacton, S-equol and urolithin A as representatives of metabolites of lignans, isoflavones and ellagitannins, respectively, were examined for their impact on HCT116 colon cancer cell growth, cooperativity with oxaliplatin and p53 dependency in vitro. Whereas enterolacton and S-equol (≤60 µM) did not elicit growth inhibition or positive cooperativity with oxaliplatin, urolithin A showed an IC50 value of 19 µM (72 h) and synergism with oxaliplatin. Urolithin A induced p53 stabilization and p53 target gene expression, and absence of p53 significantly dampened the antiproliferative effect of urolithin A (IC50(p53-/-) = 38 µM). P53 was dispensable for the G2/M arrest in HCT116 cells but required for induction of a senescence-like phenotype upon long-term exposure and for the observed synergism with oxaliplatin. Moreover, extracellular flux analyses and knockdown approaches uncovered a reduced glycolytic potential via the p53/TIGAR axis which was linked to the higher susceptibility of wildtype cells to urolithin A. Overall, the p53 status turned out to be an important determinant for the potential benefit of dietary ellagitannins in cancer chemoprevention or use in adjuvant therapy.

Impact of glutathione modulation on the toxicity of the Fusarium mycotoxins deoxynivalenol (DON), NX-3 and butenolide in human liver cells.

DON, NX-3 and butenolide (BUT) are secondary metabolites formed by Fusarium graminearum. Evidence for formation of DON-glutathione adducts exists in plants, and also in human liver (HepG2) cells mass spectrometric evidence for GSH-adduct formation was reported. NX-3 is a DON derivative lacking structural features for Thiol-Michael addition, while BUT has the structural requirements (conjugated double bond and keto group). In the present study, we addressed whether these structural differences affect levels of intracellular reactive oxygen species in HepG2 cells, and if intracellular GSH levels influence toxic effects induced by DON, NX-3 and BUT. Pre-treatment with an inhibitor of GSH bio-synthesis, L-buthionine-[S,R]-sulfoximine, aggravated substantially BUT-induced cytotoxicity (≥50 µM, 24 h), but only marginally affected the cytotoxicity of DON and NX-3 indicating that GSH-mediated detoxification is of minor importance in HepG2 cells. We further investigated whether BUT, a compound inducing alone low oral toxicity, might affect the toxicity of DON. Under different experimental designs with respect to pre- and/or co-incubations, BUT was found to contribute to the combinatorial cytotoxicity, exceeding the toxic effect of DON alone. The observed combinatorial effects underline the potential contribution of secondary metabolites like BUT, considered to be alone of low toxicological relevance, to the toxicity of DON or structurally related trichothecenes, arguing for further studies on the toxicological relevance of naturally occurring mixtures.

Dihydrochalcone Glucosides from the Subaerial Parts of Thonningia sanguinea and Their in Vitro PTP1B Inhibitory Activities.

Six new and four known dihydrochalcone glucoside derivatives (1-10), the phenylpropanoid coniferin (11), and the lignans (+)-pinoresinol (12) and lariciresinol (13) were isolated from the subaerial plant parts of Thonningia sanguinea in the course of a screening campaign for new antidiabetic lead compounds. The structures of the new substances were elucidated by HRESIMS, NMR, GC-MS, and ECD data evaluation. 2'- O-(3-Galloyl-4,6- O- Sa-hexahydroxydiphenoyl-ß-d-glucopyranosyl)-3-hydroxyphloretin (4), 2'- O-(4,6- O- Sa-hexahydroxydiphenoyl-ß-d-glucopyranosyl)phloretin (5), 2'- O-(3- O-galloyl-4,6- O- Sa-hexahydroxydiphenoyl-ß-d-glucopyranosyl)phloretin (6), and thonningianin B (9) showed moderate protein tyrosine phosphatase-1B inhibition in an enzyme assay (IC50 values ranging from 19 to 25 µM), whereas thonningianin A (10) was identified as a more potent inhibitor (IC50 = 4.4 µM). The observed activity differences could be explained by molecular docking experiments. The activity of 10 could further be confirmed in HEPG2 liver carcinoma cells, where the compound was able to increase the level of phosphorylated insulin receptors in a concentration-dependent manner.

Novel interactomics approach identifies ABCA1 as direct target of evodiamine, which increases macrophage cholesterol efflux.

Evodiamine, a bioactive alkaloid from the fruits of the traditional Chinese medicine Evodia rutaecarpa (Juss.) Benth. (Fructus Evodiae, Wuzhuyu), recently gained attention as a dietary supplement for weight loss and optimization of lipid metabolism. In light of its use by patients and consumers, there is an urgent need to elucidate the molecular targets affected by this natural product. Using a novel interactomics approach, the Nematic Protein Organisation Technique (NPOT), we report the identification of ATP-binding cassette transporter A1 (ABCA1), a key membrane transporter contributing to cholesterol efflux (ChE), as a direct binding target of evodiamine. The binding of evodiamine to ABCA1 is confirmed by surface plasmon resonance (SPR) experiments. Examining the functional consequences of ABCA1 binding reveals that evodiamine treatment results in increased ABCA1 stability, elevated cellular ABCA1 protein levels, and ultimately increased ChE from THP-1-derived human macrophages. The protein levels of other relevant cholesterol transporters, ABCG1 and SR-B1, remain unaffected in the presence of evodiamine, and the ABCA1 mRNA level is also not altered.

Constituents of Mediterranean Spices Counteracting Vascular Smooth Muscle Cell Proliferation: Identification and Characterization of Rosmarinic Acid Methyl Ester as a Novel Inhibitor.

SCOPE: Aberrant vascular smooth muscle cell (VSMC) proliferation is involved in atherosclerotic plaque formation and restenosis. Mediterranean spices have been reported to confer cardioprotection, but their direct influence on VSMCs has largely not been investigated. This study aims at examining rosmarinic acid (RA) and 11 related constituents for inhibition of VSMC proliferation in vitro, and at characterizing the most promising compound for their mode of action and influence on neointima formation in vivo. METHODS AND RESULTS: RA, rosmarinic acid methyl ester (RAME), and caffeic acid methyl ester inhibit VSMC proliferation in a resazurin conversion assay with IC50 s of 5.79, 3.12, and 6.78 µm, respectively. RAME significantly reduced neointima formation in vivo in a mouse femoral artery cuff model. Accordingly, RAME leads to an accumulation of VSMCs in the G0 /G1 cell-cycle phase, as indicated by blunted retinoblastoma protein phosphorylation upon mitogen stimulation and inhibition of cyclin-dependent kinase 2 in vitro. CONCLUSION: RAME represses PDGF-induced VSMC proliferation in vitro and reduces neointima formation in vivo. These results recommend RAME as an interesting compound with VSMC-inhibiting potential. Future metabolism and pharmacokinetics studies might help to further evaluate the potential relevance of RAME and other spice-derived polyphenolics for vasoprotection.

An increased autophagic flux contributes to the anti-inflammatory potential of urolithin A in macrophages.

BACKGROUND: An extract of Phyllanthus muellerianus and its constituent geraniin have been reported to exert anti-inflammatory activity in vivo. However, orally consumed geraniin, an ellagitannin, shows low bioavailability and undergoes metabolization to urolithins by gut microbiota. This study aimed at comparing geraniin and urolithin A with respect to inhibition of M1 (LPS) polarization of murine J774.1 macrophages and shedding more light on possible underlying mechanisms. METHODS: Photometric, fluorimetric as well as luminescence-based assays monitored production of reactive oxygen species (ROS) and nitric oxide (NO), cell viability or reporter gene expression. Western blot analyses and confocal microscopy showed abundance and localization of target proteins, respectively. RESULTS: Urolithin A is a stronger inhibitor of M1 (LPS) macrophage polarization (production of NO, ROS and pro-inflammatory proteins) than geraniin. Urolithin A leads to an elevated autophagic flux in macrophages. Inhibition of autophagy in M1 (LPS) macrophages overcomes the suppressed nuclear translocation of p65 (NF-kB; nuclear factor kB), the reduced expression of pro-inflammatory genes as well as the diminished NO production brought about by urolithin A. The increased autophagic flux is furthermore associated with impaired Akt/mTOR (mammalian target of rapamycin) signaling in urolithin A-treated macrophages. CONCLUSIONS AND GENERAL SIGNIFICANCE: Intestinal metabolization may boost the potential health benefit of widely consumed dietary ellagitannins, as suggested by side by side comparison of geraniin and urolithin A in M1(LPS) macrophages. Increased activity of the autophagic cellular recycling machinery aids the anti-inflammatory bioactivity of urolithin A.