Andrographolide prevents oxygen radical production by human neutrophils: possible mechanism(s) involved in its anti-inflammatory effect

We have reported that andrographolide (ANDRO), an active component of Andrographis paniculata, inhibits inflammatory responses by rat neutrophils. To further elucidate the possible mechanism(s) underlying the ANDRO's effect, N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced adhesion and transmigration of isolated peripheral human neutrophils were studied. Pretreatment with ANDRO (0.1 - 10 microM) concentration-dependently prevented fMLP-induced neutrophil adhesion and transmigration. We further examined the up-expression of surface Mac-1 (CD11b/CD18), an essential integrin mediated in neutrophil adhesion and transmigration. ANDRO pretreatment significantly decreased fMLP-induced up-expression of both CD11b and CD18. Accumulation of reactive oxygen species (ROS) as well as quick intracellular calcium ([Ca(++)](i)) mobilization induced by fMLP displays two important signalling pathways in regulating the up-expression of Mac-1 by neutrophils. That ANDRO pretreatment diminished fMLP-induced production of H(2)O(2) and O(2)*(-), but failed to block that of [Ca(++)](i) mobilization suggested that the ROS but not [Ca(++)](i) signalling could be modulated by ANDRO. To clarify whether ROS production impeded by ANDRO could be an antagonism of fMLP binding, phorbol-12-myristate-13-acetate (PMA), a direct protein kinase C (PKC) activator, was introduced to activate ROS production. PMA triggered remarkable ROS production and adhesion, and were partially reversed by ANDRO. This indicated that a PKC-dependent mechanism might be interfered by ANDRO. We conclude that the prevention of ROS production through, at least in part, modulation of PKC-dependent pathway could confer ANDRO the ability to down-regulate Mac-1 up-expression that is essential for neutrophil adhesion and transmigration.     

Prevention of macrophage adhesion molecule-1 (Mac-1)-dependent neutrophil firm adhesion by taxifolin through impairment of protein kinase-dependent NADPH oxidase activation and antagonism of G protein-mediated calcium influx

Taxifolin has been reported to down-regulate the expression of intercellular adhesion molecule-1 (ICAM-1), a receptor-mediating firm adhesion with beta2 integrin (e.g., Mac-1) expressed on leukocytes. To evaluate whether taxifolin could modulate Mac-1-dependent firm adhesion by neutrophils, and the possible mechanism(s) underlying its anti-inflammatory action, its effects on N-formyl-methionyl-leucyl-phenylalanine (fMLP) or phorbol-12-myristate-13-acetate (PMA)-activated peripheral human neutrophils were studied. Pretreatment with taxifolin (1-100 microM) concentration-dependently diminished fMLP- or (PMA)-induced Mac-1-dependent firm adhesion and upexpression of surface Mac-1. Mobilisation of intracellular calcium and production of reactive oxygen species (ROS) signal the upexpression of Mac-1 and firm adhesion by neutrophils. Taxifolin impeded the calcium influx induced by fMLP (a receptor-mediated activator) or AlF(4)(-) (a G protein-mediated activator). Taxifolin also effectively inhibited the fMLP- or PMA-induced ROS production with 50% inhibitory concentration (IC(50)) less than 10microM, possibly through impairing the activation of NADPH oxidase, a major ROS-generating enzyme in neutrophils, by restricting the activation of p38 mitogen-activated protein kinase (p38 MAPK) and protein kinase C (PKC). In conclusion, we propose that impairment of ROS production by NADPH oxidase through interfering with p38 MAPK- and/or PKC-dependent signals, and antagonism of G protein-mediated calcium influx may account for the inhibition of Mac-1-dependent neutrophil firm adhesion that confers taxifolin the anti-inflammatory activity.     

Evaluation of the anti-inflammatory activity of zhankuic acids isolated from the fruiting bodies of Antrodia camphorata

We have previously shown that a concentrated ethanol extract of the fruiting bodies of Antrodia camphorata exhibited immunomodulating effects in human leukocytes and fourteen compounds including zhankuic acids A, B, C, and antcin K were identified in the extract. In this study, an acute cellular model in isolated peripheral human neutrophils was established to elucidate the anti-inflammatory effects of these compounds. Reactive oxygen species (ROS) production and firm adhesion by neutrophils display two important responses during inflammation. To evaluate whether these compounds could prevent inflammatory responses by neutrophils, their effects on N-formyl-methionyl-leucyl-phenylalanine (fMLP) or phorbol 12-myristate-13-acetate (PMA)-activated peripheral human neutrophils were examined. Pretreatment with 1 - 25 microM of zhankuic acids A, B, C, or antcin K concentration-dependently diminished fMLP- or PMA-induced ROS production, as measured by a lucigenin-amplified chemiluminescence, with IC (50) (microM) around 5 - 20 microM. Zhankuic acids A, B, C, or antcin K also effectively inhibited the fMLP- or PMA-induced firm adhesion without interfering with the up-expression of surface Mac-1 (CD11b/CD18), a beta2 integrin mediating the firm adhesion of neutrophils to endothelium. The anti-inflammatory actions of these drugs were not due to cytotoxic effects because no significant difference in cell viability was observed compared to vehicle control. These data suggest that inhibition of both ROS production and firm adhesion by neutrophils has no significant cytotoxic effect that could give these drugs the potential to be anti-inflammatory agents for the clinical treatment.     

Anti-inflammatory effects of the partially purified extract of radix Stephaniae tetrandrae: comparative studies of its active principles tetrandrine and fangchinoline on human polymorphonuclear leukocyte functions

We hypothesized that prevention of neutrophil from activation may underlie the myocardial protective effect of the specially processed extract of radix Stephaniae tetrandrae (SPRST). Inflammatory responses in isolated peripheral human neutrophils were studied in the presence or absence of SPRST. SPRST (1-10 microg/ml) concentration-dependently prevented N-formyl-methionyl-leucyl-phenylalanine (fMLP)- or leukotriene B(4) (LTB(4))-induced neutrophil adhesion and transmigration. Comparable results were also observed in neutrophils pretreated with fangchinoline (Fan) or tetrandrine (Tet), two active components in SPRST. It has been reported that neutrophil adhesion/transmigration is mainly Mac-1 (CD11b/CD18)-dependent and could be modulated by reactive oxygen species (ROS) production. SPRST, Tet, and Fan diminished fMLP- or LTB4-induced Mac-1 up-regulation and ROS production. SPRST, Fan, Tet, and verapamil impaired fMLP-induced rapid intracellular alkalization, an essential mechanism for neutrophil ROS production, and [Ca(2+)](i) increment, suggesting that a calcium dependent pathway might be involved. Direct G protein activation by AlF(4)(-) also triggered [Ca(2+)](i) increment and adhesion that could be abolished by pertussis toxin and were partially reversed by SPRST, Fan, and Tet. These results reveal that inhibition of neutrophil adhesion and transmigration may account for SPRST's myocardial protective effect. This effect of SPRST may be mediated by component(s) in addition to Tet and Fan because combination of 0.1 microg/ml of Tet and Fan did not mimic the effect of SPRST. We conclude that SPRST exerts anti-inflammatory effects by interfering with ROS production and Ca(2+) influx through G protein modulation to prevent Mac-1 up-regulation in neutrophil activation.     

Mechanisms in mediating the anti-inflammatory effects of baicalin and baicalein in human leukocytes

To evaluate the possible mechanisms responsible for the anti-inflammatory effects of baicalin or baicalein, phorbol-12-myristate-13-acetate (PMA)- or N-formyl-methionyl-leucyl-phenylalanine (fMLP)-activated inflammatory responses of peripheral human leukocytes were studied. Both baicalin and baicalein diminished fMLP- or PMA-induced reactive oxygen intermediates production in neutrophils or monocytes. Neither baicalin nor baicalein prevented the protein kinase C (PKC)-dependent assembly of the NADPH oxidase. Conversely, myeloperoxidase (MPO) activity was inhibited by baicalin or baicalein. fMLP-induced activation of leukocytes, as reflected by increased surface expression of Mac-1 (CD11b/CD18) and Mac-1-dependent neutrophil adhesion, were also inhibited by baicalin or baicalein. Furthermore, baicalein, but not baicalin, impeded fMLP- or AlF(4)(-)-induced Ca(2+) influx. We conclude that impairment of reactive oxygen intermediates production, through scavenging reactive oxygen intermediates by baicalin, or antagonizing ligand-initiated Ca(2+) influx by baicalein, accounts for the inhibition of Mac-1-dependent leukocyte adhesion that confers the anti-inflammatory activity of baicalin or baicalein.     

Evaluation of in-vitro anti-inflammatory activity of some 2-alkyl-4,6-dimethoxy-1,3,5-triazines

The ability of some 2-alkyl(aryl)-4,6-dimethoxy-1,3,5-triazine derivatives to interfere with production of reactive oxygen species (ROS) by human phagocytes was evaluated in an in-vitro cell model. Superoxide anion (O(2)(-*)) production by human polymorphonuclear cells (PMNs), challenged by the chemotactic agent N-formylmethionyl-leucyl-phenylalanine (FMLP), was inhibited in a dose-dependent manner by all the compounds tested, compounds 3, 4 and 5 being statistically the most active. Adhesion of PMNs to vascular endothelial cells (ECs) is a critical step in recruitment and infiltration of leucocytes into tissues during inflammation, and the effects of 1,3,5-triazine derivatives on PMN adhesion to ECs from the human umbilical vein (HUVEC) were also investigated. Triazines were incubated with PMNs and HUVEC; adhesion was quantitated by computerized micro-imaging fluorescence analysis. The 1,3,5-triazines tested inhibited the adhesion evoked by pro-inflammatory stimuli, such as platelet activating factor (PAF), FMLP, phorbol myristate acetate (PMA), tumour necrosis factor-alpha (TNF-alpha) and interleukin-1beta(IL-1beta) in a dose-response manner over the concentration range 10(-9) to 10(-4)M, compounds 5 and 6 being the most active. Both of these compounds inhibited PMN adhesion to HUVEC, even when endothelial or PMN stimuli were used. Indeed, when both cell populations were activated contemporarily, the anti-adhesive effect was enhanced. The study suggests that 2-aryl-4,6-dimethoxy-1,3,5-triazines deserve further evaluation as anti-inflammatory agents.     

Tetrandrine ameliorates ischaemia-reperfusion injury of rat myocardium through inhibition of neutrophil priming and activation

1. We have previously shown that tetrandrine (TTD), a bisbenzyltetrahydroiosquinoline isolated from the Chinese herb Stephania tetrandra, inhibits neutrophil adhesion, Mac-1 expression, and reactive oxygen species (ROS) production. To examine whether inhibition of neutrophil function may confer upon TTD the ability to prevent myocardial ischaemia-reperfusion (MI/R) injury, experiments were performed on rats subjected to coronary ligation followed by reperfusion for induction of MI/R injury. 2. Intravenous administration of TTD (0.1 and 1.0 mg kg-1) 15 min prior to coronary ligation completely prevented MI/R-associated mortality. TTD pretreatment also significantly reduced MI/R-induced ventricular tachyarrhythmia, myocardial infarct size, and neutrophil infiltration. 3. However, TTD pretreatment did not influence mean arterial blood pressure, heart rate, or product of pressure-rate, indicating that TTD extenuated MI/R through mechanisms independent of modulating haemodynamics or myocardial oxygen demand. 4. Peripheral blood neutrophils were isolated for ex vivo examination of shape change and Mac-1 upregulation of neutrophils, two sensitive indicators of proinflammatory priming, as well as N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced adhesion and ROS production, parameters commonly used for the assessment of neutrophil activation. 5. Neutrophils from MI/R animals showed significant shape change and Mac-1 upregulation, both of which were prevented by TTD-pretreatments. On the other hand, fMLP-induced adhesion and ROS production of neutrophils were markedly enhanced by MI/R but diminished in TTD-pretreated animals. 6. These data suggest that the protective effect of TTD against MI/R injury can be accounted for by inhibition of neutrophil priming and activation, thereby abolishing subsequent infiltration and ROS production that cause MI/R injury.     

Effects of azelastine on neutrophil chemotaxis, phagocytosis and oxygen radical generation.

The effects of azelastine, an orally active anti-allergic drug, on several inflammatory parameters of human neutrophils, including human neutrophil chemotaxis, phagocytosis and generation of reactive oxygen species (ROS), was examined. ROS generated in a cell-free, xanthine-xanthine oxidase system was also assessed. The species investigated were superoxide radical anion (O2-), hydrogen peroxide (H2O2) and hydroxyl radical (OH.). Azelastine significantly inhibited human neutrophil phagocytosis and the generation of O2-, H2O2, OH. by human neutrophils. However, the drug did not markedly affect human neutrophil chemotaxis or the ROS levels generated in the xanthine-xanthine oxidase system. The present study indicates that azelastine may exert an anti-inflammatory action by inhibiting human neutrophil phagocytosis as well as oxygen radical generation at the sites of inflammation.     

Zymosan induces NADPH oxidase activation in human neutrophils by inducing the phosphorylation of p47phox and the activation of Rac2: Involvement of protein tyrosine kinases,PI3Kinase,PKC, ERK1/2 and p38MAPkinase

Reactive oxygen species (ROS) production by the neutrophil NADPH oxidase plays a key role in host defense against pathogens, such as bacteria and fungi. Zymosan a cell-wall preparation from Saccharomyces cerevisiae is largely used to activate neutrophils in its opsonized form. In this study, we show that non-opsonized zymosan alone induced ROS production by human neutrophils. Zymosan-induced ROS production is higher than the formyl-methionyl-leucyl-phenylalanine (fMLF)- or the phorbol myristate acetate (PMA)-induced ROS production but is lower than the one induced by opsonized zymosan. Most of the zymosan-induced ROS production is intracellular. Interestingly, zymosan induced the phosphorylation of the NADPH oxidase cytosolic component p47phox on several sites which are Ser315, Ser328 and Ser345. Zymosan induced also the activation of the small G-protein Rac2. Phosphorylation of the p47phox as well as Rac2 activation were inhibited by genistein a broad range protein tyrosine kinase inhibitor and by wortmannin a PI3Kinase inhibitor. GF109203X a PKC inhibitor inhibited phosphorylation of p47phox on Ser315 and Ser328. SB203580 and UO126, inhibitors of p38MAPK and ERK1/2-pathway, respectively, inhibited phosphorylation of p47phox on Ser345. Zymosan-induced ROS production was completely inhibited by genistein and wortmannin and partially inhibited by SB203580, UO126 and GF109203X. These results show that zymosan alone is able to activate NADPH oxidase in human neutrophils via the phosphorylation of p47phox and Rac2 activation and that a protein tyrosine kinase, PI3Kinase, p38MAPK, ERK1/2 and PKC are involved in this process. These pathways could be potential pharmacological targets to treat zymosan- and S. cerevisiae-induced inflammation.     

Effect of glycine on the release of reactive oxygen species in human neutrophils

The increase of extracellular glycine concentration prevents or mitigates a variety of pathological dysfunctional inflammatory responses. To eliminate the systemic effects of glycine as the reduction in the release of cytokines, this study was performed in isolated human neutrophils. The increase of the intracellular calcium concentration ([Ca2+](i)) and reactive oxygen species (ROS) release in cells incubated with glycine (0.1 to 10 mM) and stimulated with fMLP or PMA were compared with glycine-free controls. Glycine inhibited ROS production but increased [Ca2+](i) signal produced by fMLP. The inhibition of ROS production was observed even when glycine was added after the ROS release had reached maximal rate. The inhibitory effect was insensitive to strychnine and also obtained when PMA was used as stimulant. This study demonstrated that glycine impaired the activation of oxidative burst independently of glycine-gated chloride channel, presumably at the membrane level.     

Stereocalpin A inhibits the expression of adhesion molecules in activated vascular smooth muscle cells

Up-regulation of cell adhesion molecules on vascular smooth muscle cells (VSMCs) and leukocyte recruitment to the vascular wall contribute to vascular inflammation and atherosclerosis. Stereocalpin A, a chemical compound of the Antarctic lichen Ramalina terebarata, displays tumoricidal activity against several different tumor cell types. However, other biological activities of stereocalpin A and its molecular mechanisms remain unknown. In this study, our work is directed toward studying the in vitro effects of stereocalpin A on the ability to suppress the expression of adhesion molecules induced by TNF-α in vascular smooth muscle cells. Pretreatment of VSMCs for 2h with stereocalpin A at nontoxic concentrations of 0.1-10 μg/ml inhibited TNF-α-induced adhesion of THP-1 monocytic cells and expression of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1). Stereocalpin A reduced TNF-α-induced production of intracellular reactive oxygen species (ROS) and phosphorylation of p38, ERK, JNK and Akt. Stereocalpin A also inhibited NK-κB activation induced by TNF-α. Moreover, stereocalpin A inhibited TNF-α-induced ΙκΒ kinase activation, subsequent degradation of ΙκΒα, and nuclear translocation of NF-κB. Hence, we describe a new anti-inflammatory activity and mechanism of stereocalpin A, owing to the negative regulation of TNF-α-induced adhesion molecule and MCP-1 expression, monocyte adhesion and ROS production in vascular smooth muscle cells. These results suggest that stereocalpin A has the potential to exert a protective effect by modulating inflammation within the atherosclerotic lesion.     

Anti-inflammatory effects and mechanisms of the ethanol extract of Evodia rutaecarpa and its bioactive components on neutrophils and microglial cells

Evodia rutaecarpa is commonly used as an anti-inflammatory drug in traditional Chinese medicine. We previously identified four bioactive compounds (dehydroevodiamine (I), evodiamine (II), rutaecarpine (III), and synephrine (IV)) from the ethanol extract of E. rutaecarpa, but their effects and mechanism(s) of action remain unclear. To study the anti-inflammatory potential and the possible underlying mechanism(s), their effects on phorbol-12-myristate-13-acetate (PMA)- and N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced reactive oxygen species production in neutrophils was studied, as well as lipopolysaccharide (LPS)-induced nitric oxide (NO) production and inducible NO synthetase (iNOS) expression in microglial cells. The ethanol extract of E. rutaecarpa displayed potent antioxidative effects against both PMA- and fMLP-induced reactive oxygen species production in neutrophils (with IC50 values of around 2.7-3.3 microg/ml). Although less potent than the ethanol extract of E. rutaecarpa, compounds I-IV all concentration-dependently inhibited PMA- and fMLP-induced reactive oxygen species production, with compound IV consistently being the most potent agent among these active components. The antioxidative effects of the ethanol extract of E. rutaecarpa and these compounds were partially due to inhibition (10%-33%) of NADPH oxidase activity, a predominant reactive oxygen species-producing enzyme in neutrophils, and to a minor extent to their direct radical-scavenging properties. The ethanol extract of E. rutaecarpa also inhibited LPS-induced NO production (with an IC50 of around 0.8 microg/ml) and iNOS upregulation in microglial cells that was partially mimicked by compounds I, II, and III, but not compound IV. Our results suggest that the ethanol extract of E. rutaecarpa and its four bioactive components all exhibited anti-inflammatory activities which could be partially explained by their different potentials for inhibiting NADPH oxidase-dependent reactive oxygen species and/or iNOS-dependent NO production in activated inflammatory cells.     

Molecular mechanism of the inhibitory effect of trilinolein on endothelin-1-induced hypertrophy of cultured neonatal rat cardiomyocytes

Trilinolein, isolated from the traditional Chinese herb Sanchi ( Panax notoginseng), has been shown to have myocardial protective effects via its antioxidant ability. However, the cellular and molecular mechanisms of the protective effect of trilinolein in the heart remain to be elucidated. Oxidative mechanisms have been implicated in neonatal cardiomyocyte hypertrophy. We therefore have examined whether trilinolein attenuates reactive oxygen species (ROS) production and thus ET-1-induced hypertrophy of cardiomyocytes. Cultured neonatal rat cardiomyocytes were stimulated with ET-1 (10 nM), [3H]leucine incorporation and the beta-myosin heavy chain (beta-MyHC) promoter activity were examined. Trilinolein (1 and 10 microM) inhibited the ET-1-induced increase of [3H]-leucine incorporation in a concentration-dependent manner. Trilinolein (1 and 10 microM) also inhibited ET-1-induced beta-MyHC promoter activity in cardiomyocytes. We further examined the effects of trilinolein on ET-1-induced intracellular ROS generation by measuring a redox-sensitive fluorescent dye, 2',7'-dichlorofluorescin diacetate, fluorescence intensity. Trilinolein (1 and 10 microM) inhibited ET-1-increased intracellular ROS levels in a concentration-dependent manner. This increase of ROS by ET-1 (10 nM) or H2O2 (25 microM) was significantly inhibited by trilinolein (10 microM) and N-acetylcysteine (10 mM). Moreover, ET-1- or H2O2-induced beta-MyHC promoter activity and protein synthesis were also inhibited by trilinolein (10 microM). These data indicate that trilinolein inhibits ET-1-induced beta-MyHC promoter activity, and subsequent hypertrophy via its antioxidant ability in cardiomyocytes.     

Mechanism of anti-inflammatory action of glycyrrhizin: effect on neutrophil functions including reactive oxygen species generation

The effect of glycyrrhizin on inflammatory mediators such as neutrophil functions including reactive oxygen species (ROS) generation was examined. Glycyrrhizin significantly decreased neutrophil-generated O2-, H2O2 and OH in a dose-dependent manner. However, the drug did not reduce any of the ROS generated in a cell-free, xanthine-xanthine oxidase system. The drug did not affect neutrophil chemotaxis or phagocytosis, either. The present study indicates that glycyrrhizin is not an ROS scavenger but exerts an anti-inflammatory action by inhibiting the generation of ROS by neutrophils, the most potent inflammatory mediator at the site of inflammation.     

Sesquiterpene lactone Zaluzanin D alters MMP-9 promoter methylation in differentiated THP-1 monocytic cells and down regulates inflammatory cytokines IL-1β and TNF-α

Zaluzanin D (ZD) is a sesquiterpene lactone isolated from the leaves of Vernonia arborea. Earlier studies have highlighted the Sesquiterpene lactones (SLs) as molecules of medicinal value. The current study investigates the anti-inflammatory potential of ZD and its biotransformed derivatives in PMA differentiated human monocytic THP-1 cells. ZD and its fungal biotransformed derivatives Zaluzanin C (ZC) and 11,13- dihydrozaluzanin C (DZC) were screened for anti-inflammatory activity using their IC₅₀ concentration. ZD showed significant ability to reduce PMA mediated THP-1 activation, while both ZC and DZC did not show any anti-inflammatory activity. Further studies revealed that ZD had ability to attenuate intracellular reactive oxygen species production in THP-1 cells as confirmed with FACS and fluorescence microscope experiments. Similarly, Oil red O (ORO) assay showed ability of ZD to inhibit lipid accumulation in monocytes. ZD also significantly reduced the expression of pro-inflammatory markers, tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, MMP (Matrix metalloproteinases)-9 and MMP-2 as observed with RT-PCR and ELISA. Interestingly the molecule ZD also partially reversed DNA methylation levels in the PMA activated THP-1 cells. This indicated the ability of ZD to influence the epigenetic machinery of the cell. Overall the current study indicates that ZD has ability to attenuate inflammation in differentiated human THP-1 cells by regulating genes involved in the atherosclerosis inflammatory pathway. Thus ZD could potentially be used to modulate inflammation in atherosclerosis like disorders wherein monocytes play a key role.     

Antioxidant properties of albumin: effect on oxidative metabolism of human neutrophil granulocytes

The present study aims at investigating the effect of bovine serum albumin (BSA) on the trial of oxidative-stress. The antioxidant effects of BSA were determined by human neutrophil granulocytes oxygen free radicals and their by-products (O2-, H2O2, HOCl) productions. BSA interacts with those reactive oxygen species (ROS) in a dose-dependent manner. The 50% inhibitory concentration (IC50) of BSA estimated, after phorbol-12-myristate-13-acetate (PMA) stimulation were: 33.5 mg/ml for O2-, 6.5 mg/ml for H2O2, and 6.85 mg/ml for HOCl. When neutrophils were washed after pre-incubation with BSA, there was no significant decrease of ROS after stimulation of PMA (maximal: 15 +/- 1.2%). In the free cell experiments, IC50 for H2O2 and HOCl were 7.86 mg/ml and 0.67 mg/ml, respectively. The mechanism at which BSA acts may result from a simple chemical interaction with ROS rather than an intracellular mechanism by intervention in PMA oxidative metabolism. These antioxidant activities confer to BSA properties, which might be used to prevent damage inflicted by these ROS during inflammatory disorders.     

Inhibition of TNF-α-induced adhesion molecule expression by diosgenin in mouse vascular smooth muscle cells via downregulation of the MAPK, Akt and NF-κB signaling pathways

Atherosclerosis is a chronic inflammatory disease and the expression of adhesion molecules on vascular smooth muscle cells (VSMCs) contributes to the progress of the disease. Diosgenin, a precursor of steroid hormones, has been shown to have a variety of biological activities including anti-inflammatory activity; however, its molecular mechanisms are poorly understood. This study examined the effect of diosgenin on the expression of adhesion molecules induced by TNF-α in cultured mouse VSMC cell line, MOVAS-1. Preincubation of VSMCs for 2h with diosgenin (0.1-10 μM) dose-dependently inhibited TNF-α-induced adhesion of THP-1 monocytic cells and mRNA and protein expression of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1). Diosgenin abrogated TNF-α induced production of intracellular reactive oxygen species (ROS) and phosphorylation of p38, ERK, JNK and Akt. Diosgenin was also shown to inhibit NK-κB activation induced by TNF-α. Furthermore, diosgenin inhibited TNF-α-induced IκB kinase activation, subsequent degradation of IκBα, and nuclear translocation of NF-κB. Our results indicate that diosgenin inhibits the adhesive capacity of VSMC and the TNF-α-mediated induction of ICAM-1 and VCAM-1 in VSMC by inhibiting the MAPK/Akt/NF-κB signaling pathway and ROS production, which may explain the ability of diosgenin to suppress inflammation within the atherosclerotic lesion and modulate immune response.     

Comparison of the effects of cisplatin and a novel platinum polymer conjugate on the production of reactive oxygen species by human neutrophils in vitro

We have investigated the effects of cisplatin and 2 novel, water‐soluble platinum (Pt) polymer conjugates, Pt‐6 and Pt‐7, on the production of reactive oxygen species (ROS) by unstimulated human neutrophils, as well as by cells activated with phorbol myristate acetate (PMA). Production of ROS was measured using a lucigenin‐enhanced chemiluminescence (LECL) procedure. Addition of cisplatin to neutrophils resulted in an abrupt, dose‐related, but small increase in the spontaneous generation of ROS by the cells, whereas the PMA‐activated responses were substantially increased by pretreatment of neutrophils with the antineoplastic agent. Importantly, neither the spontaneous nor the PMA‐activated LECL responses of neutrophils were altered after treatment with either Pt‐6 or Pt‐7. Taken together with their impressive and selective anti‐proliferative effects on tumor cells, the absence of pro‐oxidative interactions of Pt‐6 and Pt‐7 with human neutrophils further underscores the clinical potential of these novel anticancer agents. Drug Dev. Res. 66:204–209, 2006. © 2006 Wiley‐Liss, Inc.     

Inhibitory effects of resveratrol analogs on unopsonized zymosan-induced oxygen radical production

Resveratrol, a natural hydroxystilbene, has been reported to have anti-inflammatory and anticarcinogenic activities. Inhibitory effects of resveratrol and its analogs on reactive oxygen species (ROS) production in unopsonized zymosan-stimulated murine macrophage Raw264.7 cells, human monocytes, and neutrophils were analyzed to investigate if the anti-inflammatory and anticarcinogenic activities of resveratrol are related to the inhibition of ROS production. Resveratrol was a potent inhibitor of ROS production in both unopsonized zymosan-stimulated Raw264.7 cells and human monocytes and neutrophils. Resveratrol exhibited 50% inhibition values (IC50) of 17 microM in activated Raw264.7 cells, 18 microM in human monocytes, and 23 microM in human neutrophils. 3,5-Dihydroxy-4'-methoxystilbene or 3,4'-dimethoxy-5-hydroxystilbene exhibited IC50 values of 63 or 73 microM in Raw264.7 cells, 51 or >100 microM in human monocytes, and 10 or 37 microM in human neutrophils, respectively. Trimethylresveratrol, piceid, and 3,5-dihydroxy-4'-methoxystilbene-3-O-beta-D-glucoside were weak inhibitors of ROS production. Thus, resveratrol was identified as a potent inhibitor of ROS production, which might be one biochemical mechanism related to its anti-inflammatory and anticarcinogenic activities. The number and position of hydroxy substituents in resveratrol analogs seem to play an important role in the inhibitory potency of ROS production.