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1.
Sci Rep ; 8(1): 3437, 2018 02 21.
Article in English | MEDLINE | ID: mdl-29467483

ABSTRACT

Earlier studies from our laboratory have demonstrated that clove bud oil (CBO) attenuates expression of certain virulence factors of Pseudomonas aeruginosa PAO1. Here, we probe more deeply into the effect of CBO on four pseudomonal proteases - elastase A, elastase B, protease IV and alkaline protease - each known to play key roles in disease pathogenesis. CBO inhibited the activity of these proteases present in the bacterial culture supernatant. Zymography studies indicated that these proteases can activate host matrix metalloproteases (MMPs) to establish infection, through conversion of pro-MMP-2 to active MMP-2. PAO1 is a predominant pathogen in burn wound infections and we show the modulatory effect of CBO on MMPs in an in vitro model of burn injury. Furthermore, CBO induced dose-dependent neutrophil extracellular trap formation in human neutrophils. CBO also increased the survival of C. elegans infected with PAO1, establishing an anti-infective role in a whole animal model of pathogenesis. LC-MS/MS analysis indicated that CBO treatment elicited a significant reduction of signalling molecules (Acyl-Homoserine-Lactone) involved in quorum sensing regulation. Our observations demonstrate that CBO attenuates key virulence mechanisms of this important human pathogen, while concomitantly enhancing host innate immunomodulatory functions, with potential implications for topical therapy against antibiotic-resistant infections.


Subject(s)
Anti-Bacterial Agents/pharmacology , Clove Oil/pharmacology , Pseudomonas Infections/drug therapy , Pseudomonas aeruginosa/drug effects , 3T3-L1 Cells , Animals , Anti-Bacterial Agents/therapeutic use , Bacterial Proteins/metabolism , Caenorhabditis elegans , Clove Oil/therapeutic use , Endopeptidases/metabolism , Enzyme Inhibitors/pharmacology , Extracellular Traps/drug effects , Humans , Matrix Metalloproteinase 2/metabolism , Mice , Pancreatic Elastase/metabolism , Peptide Hydrolases/metabolism , Pseudomonas Infections/metabolism , Pseudomonas Infections/microbiology , Pseudomonas aeruginosa/physiology , Quorum Sensing/drug effects , Virulence/drug effects , Virulence Factors/metabolism
2.
Exp Cell Res ; 346(2): 167-75, 2016 08 15.
Article in English | MEDLINE | ID: mdl-27448766

ABSTRACT

The complex process of wound healing is a major problem associated with diabetes, venous or arterial disease, old age and infection. A wide range of pharmacological effects including anabolic, anti-diabetic and hepato-protective activities have been attributed to Ecdysterone. In earlier studies, Ecdysterone has been shown to modulate eNOS and iNOS expression in diabetic animals and activate osteogenic differentiation through the Extracellular-signal-Regulated Kinase (ERK) pathway in periodontal ligament stem cells. However, in the wound healing process, Ecdysterone has only been shown to enhance granulation tissue formation in rabbits. There have been no studies to date, which elucidate the molecular mechanism underlying the complex cellular process involved in wound healing. The present study, demonstrates a novel interaction between the phytosteroid Ecdysterone and Nitric Oxide Synthase (NOS), in an Epidermal Growth Factor Receptor (EGFR)-dependent manner, thereby promoting cell proliferation, cell spreading and cell migration. These observations were further supported by the 4-amino-5-methylamino- 2' ,7' -difluorofluorescein diacetate (DAF FM) fluorescence assay which indicated that Ecdysterone activates NOS resulting in increased Nitric Oxide (NO) production. Additionally, studies with inhibitors of both the EGFR and ERK, demonstrated that Ecdysterone activates NOS through modulation of EGFR and ERK. These results clearly demonstrate, for the first time, that Ecdysterone enhances Nitric Oxide production and modulates complex cellular processes by activating ERK1/2 through the EGF pathway.


Subject(s)
Ecdysterone/pharmacology , Extracellular Signal-Regulated MAP Kinases/metabolism , Nitric Oxide/metabolism , 3T3-L1 Cells , Aizoaceae/chemistry , Animals , Cell Migration Assays , Cell Movement/drug effects , Cell Proliferation/drug effects , Ecdysterone/chemistry , Ecdysterone/isolation & purification , Enzyme Activation/drug effects , ErbB Receptors/metabolism , Flavonoids/pharmacology , Mice , Quinazolines/pharmacology , Signal Transduction/drug effects , Tyrphostins/pharmacology
3.
Appl Microbiol Biotechnol ; 100(8): 3681-92, 2016 Apr.
Article in English | MEDLINE | ID: mdl-26821927

ABSTRACT

Quorum sensing (QS), a communication system involved in virulence of pathogenic bacteria like Pseudomonas aeruginosa is a promising target to combat multiple drug resistance. In vitro studies using clove bud oil (CBO) in P. aeruginosa revealed a concentration dependent attenuation of a variety of virulence factors including motility, extracellular DNA, exopolysaccharides and pigment production. Furthermore, treatment with CBO demonstrated a distinct dose-dependent reduction in biofilm formation as well as promoting dispersion of already formed biofilm, observations that were also supported by porcine skin ex vivo studies. Expression studies of genes involved in signalling systems of P. aeruginosa indicated a specific decrease in transcription of pqsA, but not in the lasI or rhlI levels. Additionally, the expression of vfr and gacA genes, involved in regulation, was also not affected by CBO treatment. CBO also influenced the PQS signalling pathway by decreasing the levels of kynurenine, an effect which was reversed by the addition of exogenous kynurenine. Though the synthesis of the signalling molecules of the Las and Rhl pathways was not affected by CBO, their activity was significantly affected, as observed by decrease in levels of their various effectors. Molecular modelling studies demonstrated that eugenol, the major component of CBO, favourably binds to the QS receptor by hydrophobic interactions as well as by hydrogen bonding with Arg61 and Tyr41 which are key amino acid residues of the LasR receptor. These results thus elucidate the molecular mechanism underlying the action of CBO and provide the basis for the identification of an attractive QS inhibitor.


Subject(s)
Bacterial Proteins/genetics , Clove Oil/pharmacology , Down-Regulation/drug effects , Gene Expression Regulation, Bacterial/drug effects , Kynurenine/metabolism , Pseudomonas aeruginosa/drug effects , Bacterial Proteins/metabolism , Biofilms/drug effects , Pseudomonas aeruginosa/genetics , Pseudomonas aeruginosa/physiology , Quorum Sensing/drug effects
4.
Mol Pharmacol ; 82(4): 614-22, 2012 Oct.
Article in English | MEDLINE | ID: mdl-22745359

ABSTRACT

Cashew nut shell liquid (CNSL) has been used in traditional medicine for the treatment of a wide variety of pathophysiological conditions. To further define the mechanism of CNSL action, we investigated the effect of cashew nut shell extract (CNSE) on two matrix metalloproteinases, MMP-2/gelatinase A and MMP-9/gelatinase B, which are known to have critical roles in several disease states. We observed that the major constituent of CNSE, anacardic acid, markedly inhibited the gelatinase activity of 3T3-L1 cells. Our gelatin zymography studies on these two secreted gelatinases, present in the conditioned media from 3T3-L1 cells, established that anacardic acid directly inhibited the catalytic activities of both MMP-2 and MMP-9. Our docking studies suggested that anacardic acid binds into the MMP-2/9 active site, with the carboxylate group of anacardic acid chelating the catalytic zinc ion and forming a hydrogen bond to a key catalytic glutamate side chain and the C15 aliphatic group being accommodated within the relatively large S1' pocket of these gelatinases. In agreement with the docking results, our fluorescence-based studies on the recombinant MMP-2 catalytic core domain demonstrated that anacardic acid directly inhibits substrate peptide cleavage in a dose-dependent manner, with an IC50 of 11.11 µM. In addition, our gelatinase zymography and fluorescence data confirmed that the cardol-cardanol mixture, salicylic acid, and aspirin, all of which lack key functional groups present in anacardic acid, are much weaker MMP-2/MMP-9 inhibitors. Our results provide the first evidence for inhibition of gelatinase catalytic activity by anacardic acid, providing a novel template for drug discovery and a molecular mechanism potentially involved in CNSL therapeutic action.


Subject(s)
Anacardic Acids/pharmacology , Matrix Metalloproteinase 2/metabolism , Matrix Metalloproteinase 9/metabolism , Matrix Metalloproteinase Inhibitors/pharmacology , 3T3-L1 Cells , Anacardic Acids/isolation & purification , Anacardium/chemistry , Animals , Catalysis , Matrix Metalloproteinase Inhibitors/isolation & purification , Mice , Molecular Docking Simulation , Plant Extracts/chemistry , Plant Extracts/pharmacology
5.
Biochem Cell Biol ; 85(6): 709-20, 2007 Dec.
Article in English | MEDLINE | ID: mdl-18059529

ABSTRACT

Hepatic stellate cells are liver-specific pericytes and exist in close proximity with endothelial cells. The activation of liver pericytes is intrinsic to liver pathogenesis, and leads to endothelial dysfunction, including the low bioavailability of nitric oxide (NO). However, the role of nitric oxide in pericyte-endothelium cross-talk has not yet been elucidated. This work examines the cellular mechanism of action of NO in pericyte-mediated endothelial dysfunction. We used in vitro coculture and conditioned medium systems to study the effects of activated liver pericytes on endothelial function, and an egg yolk vascular bed model was used to study the effects of activated pericytes on angiogenesis. This study also demonstrates that activated pericytes attenuate the migration, proliferation, permeability, and NO production of endothelial cells. Our results demonstrate that activated pericytes restrict angiogenesis in egg yolk vascular bed models, and NO supplementation recovers 70% of the inhibition. Our results also demonstrate that supplementation with NO, sildenafil citrate (phosphodiesterase inhibitor), and 8-bromo-cGMP (cGMP analog) partially recovers activated-pericyte-mediated endothelium dysfunction. We conclude that NO-cGMP alleviates activated-pericyte-associated endothelial dysfunction, including angiogenesis, in a cGMP-dependent manner.


Subject(s)
Cyclic GMP/metabolism , Endothelial Cells/metabolism , Endothelial Cells/pathology , Nitric Oxide/metabolism , Pericytes/metabolism , Animals , Cell Line , Cell Membrane Permeability/drug effects , Cell Movement/drug effects , Cell Proliferation/drug effects , Cell Surface Extensions/drug effects , Chickens , Culture Media, Conditioned/pharmacology , Dose-Response Relationship, Drug , Endothelial Cells/cytology , Endothelial Cells/ultrastructure , Enzyme Inhibitors/pharmacology , Guanylate Cyclase/antagonists & inhibitors , Hepatocytes/cytology , Hepatocytes/drug effects , Hepatocytes/enzymology , Hepatocytes/ultrastructure , Humans , Hydrolysis/drug effects , Neovascularization, Physiologic/drug effects , Nitric Oxide/biosynthesis , Pericytes/cytology , Pericytes/drug effects , Pericytes/ultrastructure , Solubility/drug effects , Wound Healing/drug effects
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