rs822336 binding to C/EBPß and NFIC modulates induction of PD-L1 expression and predicts anti-PD-1/PD-L1 therapy in advanced NSCLC.

Efficient predictive biomarkers are needed for immune checkpoint inhibitor (ICI)-based immunotherapy in non-small cell lung cancer (NSCLC). Testing the predictive value of single nucleotide polymorphisms (SNPs) in programmed cell death 1 (PD-1) or its ligand 1 (PD-L1) has shown contrasting results. Here, we aim to validate the predictive value of PD-L1 SNPs in advanced NSCLC patients treated with ICIs as well as to define the molecular mechanisms underlying the role of the identified SNP candidate. rs822336 efficiently predicted response to anti-PD-1/PD-L1 immunotherapy in advanced non-oncogene addicted NSCLC patients as compared to rs2282055 and rs4143815. rs822336 mapped to the promoter/enhancer region of PD-L1, differentially affecting the induction of PD-L1 expression in human NSCLC cell lines as well as their susceptibility to HLA class I antigen matched PBMCs incubated with anti-PD-1 monoclonal antibody nivolumab. The induction of PD-L1 expression by rs822336 was mediated by a competitive allele-specificity binding of two identified transcription factors: C/EBPß and NFIC. As a result, silencing of C/EBPß and NFIC differentially regulated the induction of PD-L1 expression in human NSCLC cell lines carrying different rs822336 genotypes. Analysis by binding microarray further validated the competitive allele-specificity binding of C/EBPß and NFIC to PD-L1 promoter/enhancer region based on rs822336 genotype in human NSCLC cell lines. These findings have high clinical relevance since identify rs822336 and induction of PD-L1 expression as novel biomarkers for predicting anti-PD-1/PD-L1-based immunotherapy in advanced NSCLC patients.

Constituents from the Polar Extract of Pisolithus arhizus and Their Anti-inflammatory Activity.

The phytochemical study of the Pisolithus arhizus fruiting body methanol extract led to the isolation of six new triterpenoids (1-6) and one new naphthalenoid pulvinic acid derivative (7), together with five known compounds, including norbadione A (8). Their structure was established from 1D and 2D NMR spectroscopy and HRESIMS analyses. The absolute configuration of the triterpenoids was determined by circular dichroism. The two pulvinic acid derivatives 7 and 8, showing the highest activity in modulating IL-6 secretion, were tested for their effect on COX-2, STAT3, and p-STAT3 proteins; both compounds were able to downregulate p-STAT3.

The Bacterial Spore as a Mucosal Vaccine Delivery System.

The development of efficient mucosal vaccines is strongly dependent on the use of appropriate vectors. Various biological systems or synthetic nanoparticles have been proposed to display and deliver antigens to mucosal surfaces. The Bacillus spore, a metabolically quiescent and extremely resistant cell, has also been proposed as a mucosal vaccine delivery system and shown able to conjugate the advantages of live and synthetic systems. Several antigens have been displayed on the spore by either recombinant or non-recombinant approaches, and antigen-specific immune responses have been observed in animals immunized by the oral or nasal route. Here we review the use of the bacterial spore as a mucosal vaccine vehicle focusing on the advantages and drawbacks of using the spore and of the recombinant vs. non-recombinant approach to display antigens on the spore surface. An overview of the immune responses induced by antigen-displaying spores so far tested in animals is presented and discussed.

Diterpenoid Constituents of Psiadia punctulata and Evaluation of Their Antimicrobial Activity.

Sixteen diterpenes (1-16), along with 10 previously described compounds, including four flavonoids and six diterpenes, were isolated from the aerial parts of Psiadia punctulata growing in Saudi Arabia. The diterpene structures were elucidated using NMR spectroscopy and mass spectrometry data. Furthermore, a DFT/NMR procedure was used to suggest the relative configuration of several compounds. The labdane-derived skeletons, namely, ent-atisane, ent-beyerene, ent-trachylobane, and ent-kaurene, were identified. The extracts, fractions, and pure compounds were then tested against Staphylococcus aureus, Streptococcus mutans, Treponema denticola, and Lactobacillus plantarum. One diterpenoid, namely, psiadin, showed an additive effect with the antiseptic chlorhexidine, with a fractional inhibitory concentration index of less than 1. Additionally, psiadin showed a prospective inhibition activity for bacterial efflux pumps.

Plant Growth Promotion Function of Bacillus sp. Strains Isolated from Salt-Pan Rhizosphere and Their Biocontrol Potential against Macrophomina phaseolina.

In recent decades, intensive crop management has involved excessive use of pesticides or fertilizers, compromising environmental integrity and public health. Accordingly, there has been worldwide pressure to find an eco-friendly and safe strategy to ensure agricultural productivity. Among alternative approaches, Plant Growth-Promoting (PGP) rhizobacteria are receiving increasing attention as suitable biocontrol agents against agricultural pests. In the present study, 22 spore-forming bacteria were selected among a salt-pan rhizobacteria collection for their PGP traits and their antagonistic activity against the plant pathogen fungus Macrophomina phaseolina. Based on the higher antifungal activity, strain RHFS10, identified as Bacillus vallismortis, was further examined and cell-free supernatant assays, column purification, and tandem mass spectrometry were employed to purify and preliminarily identify the antifungal metabolites. Interestingly, the minimum inhibitory concentration assessed for the fractions active against M. phaseolina was 10 times lower and more stable than the one estimated for the commercial fungicide pentachloronitrobenzene. These results suggest the use of B. vallismortis strain RHFS10 as a potential plant growth-promoting rhizobacteria as an alternative to chemical pesticides to efficiently control the phytopathogenic fungus M. phaseolina.

Outer Membrane Vesicles Derived from Klebsiella pneumoniae Are a Driving Force for Horizontal Gene Transfer.

Gram-negative bacteria release Outer Membrane Vesicles (OMVs) into the extracellular environment. Recent studies recognized these vesicles as vectors to horizontal gene transfer; however, the parameters that mediate OMVs transfer within bacterial communities remain unclear. The present study highlights for the first time the transfer of plasmids containing resistance genes via OMVs derived from Klebsiella pneumoniae (K. pneumoniae). This mechanism confers DNA protection, it is plasmid copy number dependent with a ratio of 3.6 times among high copy number plasmid (pGR) versus low copy number plasmid (PRM), and the transformation efficiency was 3.6 times greater. Therefore, the DNA amount in the vesicular lumen and the efficacy of horizontal gene transfer was strictly dependent on the identity of the plasmid. Moreover, the role of K. pneumoniae-OMVs in interspecies transfer was described. The transfer ability was not related to the phylogenetic characteristics between the donor and the recipient species. K. pneumoniae-OMVs transferred plasmid to Escherichia coli, Salmonella enterica, Pseudomonas aeruginosa and Burkholderia cepacia. These findings address the pivotal role of K. pneumoniae-OMVs as vectors for antimicrobial resistance genes spread, contributing to the development of antibiotic resistance in the microbial communities.

Abietane Diterpenoids from the Hairy Roots of Salvia corrugata.

Salvia corrugata Vahl. is an interesting source of abietane and abeo-abietane compounds that showed antibacterial, antitumor, and cytotoxic activities. The aim of the study was to obtain transformed roots of S. corrugata and to evaluate the production of terpenoids in comparison with in vivo root production. Hairy roots were initiated from leaf explants by infection with ATCC 15834 Agrobacterium rhizogenes onto hormone-free Murashige and Skoog (MS) solid medium. Transformation was confirmed by polymerase chain reaction analysis of rolC and virC1 genes. The biomass production was obtained in hormone-free liquid MS medium using Temporary Immersion System bioreactor RITA®. The chromatographic separation of the methanolic extract of the untransformed roots afforded horminone, ferruginol, 7-O-acetylhorminone and 7-O-methylhorminone. Agastol and ferruginol were isolated and quantified from the hairy roots. The amount of these metabolites indicated that the hairy roots of S. corrugata can be considered a source of these compounds.

Labdane Diterpenoids from Salvia tingitana Etl. Synergize with Clindamycin against Methicillin-Resistant Staphylococcus aureus.

Quorum-sensing (QS) is a regulatory mechanism in bacterial communication, important for pathogenesis control. The search for small molecules active as quorum-sensing inhibitors (QSI) that can synergize with antibiotics is considered a good strategy to counteract the problem of antibiotic resistance. Here the antimicrobial labdane diterpenoids sclareol (1) and manool (2) extracted from Salvia tingitana were considered as potential QSI against methicillin-resistant Staphylococcus aureus. Only sclareol showed synergistic activity with clindamycin. The quantification of these compounds by LC-MS analysis in the organs and in the calli of S. tingitana showed that sclareol is most abundant in the flower spikes and is produced by calli, while manool is the major labdane of the roots, and is abundant also in the leaves. Other metabolites of the roots were abietane diterpenoids, common in Salvia species, and pentacyclic triterpenoids, bearing a γ-lactone moiety, previously undescribed in Salvia. Docking simulations suggested that 1 and 2 bind to key residues, involved in direct interactions with DNA. They may prevent accessory gene regulator A (AgrA) binding to DNA or AgrA activation upon phosphorylation, to suppress virulence factor expression. The antimicrobial activity of these two compounds probably achieves preventing upregulation of the accessory gene regulator (agr)-regulated genes.

Bacillus subtilis builds structurally and functionally different spores in response to the temperature of growth.

Bacterial spores are commonly isolated from a variety of different environments, including extreme habitats. Although it is well established that such ubiquitous distribution reflects the spore resistance properties, it is not clear whether the growing conditions affect the spore structure and function. We used Bacillus subtilis spores of similar age but produced at 25, 37, or 42°C to compare their surface structures and functional properties. Spores produced at the 25°C were more hydrophobic while those produced at 42°C contained more dipicolinic acid, and were more resistant to heat or lysozyme treatments. Electron microscopy analysis showed that while 25°C spores had a coat with a compact outer coat, not tightly attached to the inner coat, 42°C spores had a granular, not compact outer coat, reminiscent of the coat produced at 37°C by mutant spores lacking the protein CotG. Indeed, CotH and a series of CotH-dependent coat proteins including CotG were more abundantly extracted from the coat of 25 or 37°C than 42°C spores. Our data indicated that CotH is a heat-labile protein with a major regulatory role on coat formation when sporulation occurs at low temperatures, suggesting that B. subtilis builds structurally and functionally different spores in response to the external conditions.

The temperature of growth and sporulation modulates the efficiency of spore-display in Bacillus subtilis.

BACKGROUND: Bacterial spores displaying heterologous antigens or enzymes have long been proposed as mucosal vaccines, functionalized probiotics or biocatalysts. Two main strategies have been developed to display heterologous molecules on the surface of Bacillus subtilis spores: (i) a recombinant approach, based on the construction of a gene fusion between a gene coding for a coat protein (carrier) and DNA coding for the protein to be displayed, and (ii) a non-recombinant approach, based on the spontaneous and stable adsorption of heterologous molecules on the spore surface. Both systems have advantages and drawbacks and the selection of one or the other depends on the protein to be displayed and on the final use of the activated spore. It has been recently shown that B. subtilis builds structurally and functionally different spores when grown at different temperatures; based on this finding B. subtilis spores prepared at 25, 37 or 42 °C were compared for their efficiency in displaying various model proteins by either the recombinant or the non-recombinant approach. RESULTS: Immune- and fluorescence-based assays were used to analyze the display of several model proteins on spores prepared at 25, 37 or 42 °C. Recombinant spores displayed different amounts of the same fusion protein in response to the temperature of spore production. In spores simultaneously displaying two fusion proteins, each of them was differentially displayed at the various temperatures. The display by the non-recombinant approach was only modestly affected by the temperature of spore production, with spores prepared at 37 or 42 °C slightly more efficient than 25 °C spores in adsorbing at least some of the model proteins tested. CONCLUSION: Our results indicate that the temperature of spore production allows control of the display of heterologous proteins on spores and, therefore, that the spore-display strategy can be optimized for the specific final use of the activated spores by selecting the display approach, the carrier protein and the temperature of spore production.

Molecular Dissection of dH3w, A Fluorescent Peptidyl Sensor for Zinc and Mercury.

Previously, we reported that fluorescent peptide dansyl-HPHGHW-NH2 (dH3w), designed on the repeats of the human histidine-rich glycoprotein, shows a turn-on response to Zn(II) and a complex response to Hg(II) characterized by a turn-off phase at low Hg(II) concentrations and a turn-on phase at high concentrations. As Hg(II) easily displaces Zn(II), dH3w is a useful probe for the environmental monitoring of Hg(II). In order to investigate the molecular basis of the metal selectivity and fluorescence response, we characterized three variants, dH3w(H1A), dH3w(H3A), and dH3w(H5A), in which each of the three histidine residues was changed to alanine, and two variants with a single fluorescent moiety, namely dH3w(W6A), in which the tryptophan residue at the C-terminus was changed to alanine, and AcH3w, in which the N-terminal dansyl moiety was substituted by an acetyl group. These variants allowed us to demonstrate that all the histidine residues are essential for a strong interaction with Zn(II), whereas two histidine residues (in particular His5) and the dansyl group are necessary to bind Hg(II). The data reported herein shed light on the molecular behavior of dH3w, thus paving the way to the rational designing of further and more efficient fluorescent peptidyl probes for Hg(II).

Bioactive Ent-Kaurane Diterpenes Oridonin and Irudonin Prevent Cancer Cells Migration by Interacting with the Actin Cytoskeleton Controller Ezrin.

The ent-kaurane diterpene oridonin was reported to inhibit cell migration and invasion in several experimental models. However, the process by which this molecule exerts its anti-metastatic action has not been yet elucidated. In this article, we have investigated the anti-metastatic activity of Oridonin and of one homolog, Irudonin, with the aim to shed light on the molecular mechanisms underlying the biological activity of these ent-kaurane diterpenes. Cell-based experiments revealed that both compounds are able to affect differentiation and cytoskeleton organization in mouse differentiating myoblasts, but also to impair migration, invasion and colony formation ability of two different metastatic cell lines. Using a compound-centric proteomic approach, we identified some potential targets of the two bioactive compounds among cytoskeletal proteins. Among them, Ezrin, a protein involved in the actin cytoskeleton organization, was further investigated. Our results confirmed the pivotal role of Ezrin in regulating cell migration and invasion, and indicate this protein as a potential target for new anti-cancer therapeutic approaches. The interesting activity profile, the good selectivity towards cancer cells, and the lower toxicity with respect to Oridonin, all suggest that Irudonin is a very promising anti-metastatic agent.

Localization of a red fluorescence protein adsorbed on wild type and mutant spores of Bacillus subtilis.

BACKGROUND: Bacterial spores have been proposed as vehicles to display heterologous proteins for the development of mucosal vaccines, biocatalysts, bioremediation and diagnostic tools. Two approaches have been developed to display proteins on the spore surface: a recombinant approach, based on the construction of gene fusions between DNA molecules coding for a spore surface protein (carrier) and for the heterologous protein to be displayed (passenger); and a non-recombinant approach based on spore adsorption, a spontaneous interaction between negatively charged, hydrophobic spores and purified proteins. The molecular details of spore adsorption have not been fully clarified yet. RESULTS: We used the monomeric Red Fluorescent Protein (mRFP) of the coral Discosoma sp. and Bacillus subtilis spores of a wild type and an isogenic mutant strain lacking the CotH protein to clarify the adsorption process. Mutant spores, characterized by a strongly altered coat, were more efficient than wild type spores in adsorbing mRFP but the interaction was less stable and mRFP could be in part released by raising the pH of the spore suspension. A collection of isogenic strains carrying GFP fused to proteins restricted in different compartments of the B. subtilis spore was used to localize adsorbed mRFP molecules. In wild type spores mRFP infiltrated through crust and outer coat, localized in the inner coat and was not surface exposed. In mutant spores mRFP was present in all surface layers, inner, outer coat and crust and was exposed on the spore surface. CONCLUSIONS: Our results indicate that different spores can be selected for different applications. Wild type spores are preferable when a very tight protein-spore interaction is needed, for example to develop reusable biocatalysts or bioremediation systems for field applications. cotH mutant spores are instead preferable when the heterologous protein has to be displayed on the spore surface or has to be released, as could be the case in mucosal delivery systems for antigens and drugs, respectively.

The oxygenating constituent of 3,6-diketocamphane monooxygenase from the CAM plasmid of Pseudomonas putida: the first crystal structure of a type II Baeyer-Villiger monooxygenase.

The three-dimensional structures of the native enzyme and the FMN complex of the overexpressed form of the oxygenating component of the type II Baeyer-Villiger 3,6-diketocamphane monooxygenase have been determined to 1.9 Å resolution. The structure of this dimeric FMN-dependent enzyme, which is encoded on the large CAM plasmid of Pseudomonas putida, has been solved by a combination of multiple anomalous dispersion from a bromine crystal soak and molecular replacement using a bacterial luciferase model. The orientation of the isoalloxazine ring of the FMN cofactor in the active site of this TIM-barrel fold enzyme differs significantly from that previously observed in enzymes of the bacterial luciferase-like superfamily. The Ala77 residue is in a cis conformation and forms a ß-bulge at the C-terminus of ß-strand 3, which is a feature observed in many proteins of this superfamily.

Chemical Profiling, Antioxidant, and Anti-Inflammatory Activities of Hyoseris radiata L., a Plant Used in the Phytoalimurgic Tradition.

Hyoseris radiata L. (Asteraceae), known as "wild chicory", is a perennial herbaceous plant native to the Mediterranean region, North Africa, and West Asia. Collected from the wild, the plant is largely used in Italy for culinary purposes and in popular medicine, so that it can be included in the list of phytoalimurgic plants. The present study aimed to investigate for the first time the plant's chemical profile, through a combined UHPLC-HR-ESI-Orbitrap/MS and NMR approach, and its potential healthy properties, focusing on antioxidant and anti-inflammatory activities. The LC-MS/MS analysis and the isolation through chromatographic techniques of the plant's hydroalcoholic extract allowed the authors to identify 48 compounds, including hydroxycinnamic acids, flavonoids, megastigmane glucosides, coumarins, and lignans, together with several unsaturated fatty acids. The quantitative analysis highlighted a relevant amount of flavonoids and hydroxycinnamic acids, with a total of 12.9 ± 0.4 mg/g DW. NMR-based chemical profiling revealed the presence of a good amount of amino acids and monosaccharides, and chicoric and chlorogenic acids as the most representative polyphenols. Finally, the antioxidant and anti-inflammatory activities of H. radiata were investigated through cell-free and cell-based assays, showing a good antioxidant potential for the plant extract and a significant reduction in COX-2 expression.

Targeted metabolite profiling of Salvia rosmarinus Italian local ecotypes and cultivars and inhibitory activity against Pectobacterium carotovorum subsp. carotovorum.

Introduction: The development of agriculture in terms of sustainability and low environmental impact is, at present, a great challenge, mainly in underdeveloped and marginal geographical areas. The Salvia rosmarinus "Eretto Liguria" ecotype is widespread in Liguria (Northwest Italy), and farmers commonly use it by for cuttings and for marketing. In the present study, this ecotype was characterized in comparison with other cultivars from the same geographical region and Campania (Southern Italy), with a view to application and registration processes for the designation of protected geographical indications. Moreover, the possibility of using the resulting biomass after removing cuttings or fronds as a source of extracts and pure compounds to be used as phytosanitary products in organic farming was evaluated. Specifically, the potential of rosemary extracts and pure compounds to prevent soft rot damage was then tested. Methods: A targeted NMR metabolomic approach was employed, followed by multivariate analysis, to characterize the rosemary accessions. Bacterial soft rot assay and disk diffusion test were carried out to evaluate the activity of extracts and isolated compounds against Pectobacterium carotovorum subsp. carotovorum. Enzymatic assay was performed to measure the in vitro inhibition of the pectinase activity produced by the selected pathogen. Molecular docking simulations were used to explore the possible interaction of the selected compounds with the pectinase enzymes. Results and Discussion: The targeted metabolomic analysis highlighted those different geographical locations can influence the composition and abundance of bioactive metabolites in rosemary extracts. At the same time, genetic factors are important when a single geographical area is considered. Self-organizing maps (SOMs) showed that the accessions of "Eretto Liguria" appeared well characterized when compared to the others and had a good content in specialized metabolites, particularly carnosic acid. Soft rotting Enterobacteriaceae belonging to the Pectobacterium genus represent a serious problem in potato culture. Even though rosemary methanolic extracts showed a low antibacterial activity against a strain of Pectobacterium carotovorum subsp. carotovorum in the disk diffusion test, they showed ability in reducing the soft rot damage induced by the bacterium on potato tissue. 7-O-methylrosmanol, carnosol and isorosmanol appeared to be the most active components. In silico studies indicated that these abietane diterpenoids may interact with P. carotovorum subsp. carotovorum pectate lyase 1 and endo-polygalacturonase, thus highlighting these rosemary components as starting points for the development of agents able to prevent soft rot progression.

Two new triterpenes from Commicarpus grandiflorus (A. Rich.) Standl. aerial parts exudate.

The exudate of Commicarpus grandiflorus (A. Rich.) Standl. flowering aerial parts was investigated for its chemical composition. Nine compounds were isolated, five triterpenes and four methylated flavones, of which two were new natural triterpenes, 2α,3ß,11α-olean-18-en-2,3,11-triol (1) and 2α,3ß-olean-12-en-2,3-diol-11-one (2) that were named commicarpotriol and commicarpodiol, respectively. Structural characterization was carried out using 1D, 2D NMR, and MS techniques and the antimicrobial activity of all isolates was evaluated.

Rhanteriol, a New Rhanterium suaveolens Desf. Lignan with Pharmacological Potential as an Inhibitor of Enzymes Involved in Neurodegeneration and Type 2 Diabetes.

Several specialized plant metabolites are reported to be enzyme inhibitors. In this investigation, the phytochemical composition and the biological activity of Rhanterium suaveolens Desf. were studied. One new lignan (rhanteriol 1) and seven known secondary metabolites were isolated from the aerial parts of R. suaveolens by using different chromatographic procedures. The biological properties of the R. suaveolens extracts and the new compound were evaluated by measuring their ability to inhibit the cholinesterase and carbohydrate-hydrolyzing enzymes, using cell-free in vitro methods. The new lignan, rhanteriol, was shown to inhibit α-amylase and α-glucosidase (IC50 = 46.42 ± 3.25 µM and 26.76 ± 3.29 µM, respectively), as well as butyrylcholinesterase (IC50 = 10.41 ± 0.03 µM), with an effect comparable to that of the respective standards, acarbose and galantamine. Furthermore, docking studies were performed suggesting the interaction mode of rhanteriol with the active sites of the investigated enzymes. The obtained data demonstrated that the aerial part of R. suaveolens could represent a source of active molecules, such as rhanteriol, usable in the development of treatments for preventing or treating type 2 diabetes mellitus and neurodegeneration.

Characterization of Health Beneficial Components in Discarded Leaves of Three Escarole (Cichorium endivia L.) Cultivar and Study of Their Antioxidant and Anti-Inflammatory Activities.

Plants of genus Cichorium (Asteraceae) can be used as vegetables with higher nutritional value and as medicinal plants. This genus has beneficial properties owing to the presence of a number of specialized metabolites such as alkaloids, sesquiterpene lactones, coumarins, unsaturated fatty acids, flavonoids, saponins, and tannins. Cichorium endivia L., known as escarole, has achieved a common food status due to its nutritionary value, bitter taste, and the presence of healthy components, and is eaten cooked or raw in salads. Presently, wastes derived from the horticultural crops supply chain are generated in very large amounts. Vegetable waste comprises the discarded leaves of food sources produced during collection, handling, transportation, and processing. The external leaves of Cichorium endivia L. are a horticultural crop that is discarded. In this work, the phytochemical profile, antioxidant, and anti-inflammatory activities of hydroalcoholic extract obtained from discarded leaves of three cultivars of escarole (C. endivia var. crispum 'Capriccio', C. endivia var. latifolium 'Performance' and 'Leonida') typical horticultural crop of the Campania region were investigated. In order to describe a metabolite profile of C. endivia cultivars, the extracts were analysed by HR/ESI/Qexactive/MS/MS and NMR. The careful analysis of the accurate masses, the ESI/MS spectra, and the 1H NMR chemical shifts allowed for the identification of small molecules belonging to phenolic, flavonoid, sesquiterpene, amino acids, and unsaturated fatty acid classes. In addition, the antioxidant potential of the extracts was evaluated using cell-free and cell-based assays, as well as their cytotoxic and anti-inflammatory activity. All the extracts showed similar radical-scavenging ability while significant differences between the three investigated cultivars emerged in the cell-based assays. The obtained data were ascribed to the content of polyphenols and sesquiterpenes in the extracts. Accordingly, C. endivia by-products can be deemed an interesting material for healthy product formulations.

Design of an Herbal Preparation Composed by a Combination of Ruscus aculeatus L. and Vitis vinifera L. Extracts, Magnolol and Diosmetin to Address Chronic Venous Diseases through an Anti-Inflammatory Effect and AP-1 Modulation.

Chronic venous disease (CVD) is an often underestimated inflammatory pathological condition that can have a serious impact on quality of life. Many therapies have been proposed to deal with CVD, but unfortunately the symptoms recur with increasing frequency and intensity as soon as treatments are stopped. Previous studies have shown that the common inflammatory transcription factor AP-1 (activator protein-1) and nuclear factor kappa-activated B-cell light chain enhancer (NF-kB) play key roles in the initiation and progression of this vascular dysfunction. The aim of this research was to develop a herbal product that acts simultaneously on different aspects of CVD-related inflammation. Based on the evidence that several natural components of plant origin are used to treat venous insufficiency and that magnolol has been suggested as a putative modulator of AP-1, two herbal preparations based on Ruscus aculeatus root extracts, and Vitis vinifera seed extracts, as well as diosmetin and magnolol, were established. A preliminary MTT-based evaluation of the possible cytotoxic effects of these preparations led to the selection of one of them, named DMRV-2, for further investigation. First, the anti-inflammatory efficacy of DMRV-2 was demonstrated by monitoring its ability to reduce cytokine secretion from endothelial cells subjected to LPS-induced inflammation. Furthermore, using a real-time PCR-based protocol, the effect of DMRV-2 on AP-1 expression and activity was also evaluated; the results obtained demonstrated that the incubation of the endothelial cells with this preparation almost completely nullified the effects exerted by the treatment with LPS on AP-1. Similar results were also obtained for NF-kB, whose activation was evaluated by monitoring its distribution between the cytosol and the nucleus of endothelial cells after the different treatments.