Superoxide Generation In Macrophages Research Articles

Antioxidant activity of Curculigoside, a phenolic glucoside from Curculigo orchioides Gaertn

Background: Curculigoside is phenolic glucoside derived from the plant Curculigo orchioides Gaertn, a well known immune stimulatory and rejuvenating tuberous plant. A systematic analysis of the antioxidant potential of curculigoside is not reported as far. Methods: In the present study antioxidant activity of curculigoside was evaluated both in the in vivo and in vitro models. Tube test were performed to analyse 1, 1-diphenyl-2-picrylhydrazyl (DPPH), super oxide (Nitro blue tetrazolium reduction) and hydroxyl radical (Fenton reaction) scavenging effects. Besides, effect of the compound on lipid peroxidation and nitric oxide scavenging was also tested. In vivo antioxidant activity was checked by PMA (Phorbol-12-myristate-13-acetate) induced superoxide radical formation by mouse peritoneal macrophages. Result: Curculigoside was found to scavenge the hydroxyl radicals generated by Fenton reaction and also inhibited the in vitro lipid peroxidation. The compound was also found to be an effective scavenger of superoxide, DPPH and nitric oxide (NO) radicals. Curculigoside showed a significant scavenging effect on the NO release by cultured macrophages. Besides, oral administration of curculigoside inhibited superoxide generation in macrophages in vivo . Conclusion: Curculigoside was found to exhibit good free radical scavenging effects which is comparable to known standards, both in the in vitro as well as in vivo systems.

Berberine inhibits NADPH oxidase mediated superoxide anion production in macrophagesThis article is one of a selection of papers published in a Special Issue on Oxidative Stress in Health and Disease.

Oxidative stress and amplified redox signaling contribute to the pathogenesis of many human diseases including atherosclerosis. The superoxide-generating phagocytic NADPH oxidase is a key source of oxidative stress in the developing atheroma. The aim of the present study was to examine the effect of berberine, a plant-derived alkaloid, on NADPH oxidase-mediated superoxide anion production in macrophages. Lipopolysaccharide (LPS) treatment activated NADPH oxidase in THP-1 monocyte-derived macrophages and increased the intracellular level of superoxide anions. Preincubation of cells with berberine demonstrated a concentration-dependent (10-50 micromol/L) and time-dependent (6-24 h) inhibition of superoxide anion generation in LPS-stimulated macrophages. Cell viability tests confirmed that berberine, at concentrations sufficient for inhibiting NADPH oxidase-mediated superoxide anion generation in macrophages, did not affect cell viability. Real-time PCR analysis revealed that addition of berberine to the culture medium was able to reduce gp91phox mRNA expression in LPS-treated cells. Berberine also restored superoxide dismutase (SOD) activity, which was found to be inhibited by LPS treatment. In conclusion, results from the present study demonstrate that berberine can effectively reduce intracellular superoxide levels in LPS- stimulated macrophages. Such a restoration of cellular redox by berberine is mediated by its selective inhibition of gp91phox expression and enhancement of SOD activity. The therapeutic relevance of berberine in the prevention and management of atherosclerosis remains to be further investigated.

Stanniocalcin-1 suppresses superoxide generation in macrophages through induction of mitochondrial UCP2

Mammalian STC1 decreases the mobility of macrophages and diminishes their response to chemokines. In the current experiments, we sought to determine the impact of STC1 on energy metabolism and superoxide generation in mouse macrophages. STC1 decreases ATP level in macrophages but does not affect the activity of respiratory chain complexes I-IV. STC1 induces the expression of mitochondrial UCP2, diminishing mitochondrial membrane potential and superoxide generation; studies in UCP2 null and gp91phox null macrophages suggest that suppression of superoxide by STC1 is UCP2-dependent yet is gp91phox-independent. Furthermore, STC1 blunts the effects of LPS on superoxide generation in macrophages. Exogenous STC1 is internalized by macrophages within 10 min and localizes to the mitochondria, suggesting a role for circulating and/or tissue-derived STC1 in regulating macrophage function. STC1 induces arrest of the cell cycle at the G1 phase and reduces cell necrosis and apoptosis in serum-starved macrophages. Our data identify STC1 as a key regulator of superoxide generation in macrophages and suggest that STC1 may profoundly affect the immune/inflammatory response.

Anti-Inflammatory and Renal Protective Actions of Stanniocalcin-1 in a Model of Anti-Glomerular Basement Membrane Glomerulonephritis

We have previously shown that stanniocalcin-1 (STC1) inhibits the transendothelial migration of macrophages and T cells, suppresses superoxide generation in macrophages, and attenuates macrophage responses to chemoattractants. To study the effects of STC1 on inflammation, in this study we induced a macrophage- and T-cell-mediated model of anti-glomerular basement membrane disease in STC1 transgenic mice, which display elevated serum STC1 levels and preferentially express STC1 in both endothelial cells and macrophages. We examined the following parameters both at baseline and after anti-glomerular basement membrane antibody treatment: blood pressure; C(3a) levels; urine output; proteinuria; blood urea nitrogen; and kidney C(3) deposition, fibrosis, histological changes, cytokine expression, and number of T cells and macrophages. Compared with wild-type mice, after anti-glomerular basement membrane treatment STC1 transgenic mice exhibited: i) diminished infiltration of inflammatory macrophages in the glomeruli; ii) marked reduction in crescent formation and sclerotic glomeruli; iii) decreased interstitial fibrosis; iv) preservation of kidney function and lower blood pressure; v) diminished C(3) deposition in the glomeruli; and vi) reduced expression of macrophage inhibitory protein-2 and transforming growth factor-beta2 in the kidney. Compared with baseline, wild-type mice, but not STC1 transgenic mice, had higher proteinuria and a marked reduction in urine output. STC1 had minimal effects, however, on both T-cell number in the glomeruli and interstitium and on cytokine expression characteristic of either TH1 or TH2 activation. These data suggest that STC1 is a potent anti-inflammatory and renal protective protein.

Targeting NOX, INOS and COX‐2 in inflammatory cells: Chemoprevention using food phytochemicals

Biological, biochemical and physical stimuli activate inflammatory leukocytes, such as macrophages, resulting in induction and synthesis of proinflammatory proteins and enzymes, together with free radicals, as innate immune responses. On the other hand, chronic and dysregulated activation of some inducible enzymes, including NADPH oxidase (NOX), inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2, have been shown to play pivotal roles in the development of certain inflammatory diseases such as oncogenesis. While the use of synthetic agents, especially those targeting molecules, is an attractive and reasonable approach to prevent carcinogenesis, it should be noted that traditional herbs and spices also exist along with their active constituents, which have been demonstrated to disrupt inflammatory signal transduction pathways. In this mini-review, the molecular mechanisms of activation or induction of NOX, iNOS and COX-2, as well as some food phytochemicals with marked potential to regulate those key inflammatory molecules, are highlighted. For example, 1'-acetoxychavicol acetate, which occurs in the rhizomes of the subtropical Zingiberaceae plant, has been shown to attenuate NOX-derived superoxide generation in macrophages, as well as lipopolysaccharide-induced nitric oxide and prostaglandin E(2) production through the suppression of iNOS and COX-2 synthesis, respectively. Notably, this phytochemical has exhibited a wide range of cancer prevention activities in several rodent models of inflammation-associated carcinogenesis. Herein, the cancer preventive potentials of several food phytochemicals targeting the induction of NOX, iNOS and COX-2 are described.

Folic acid inhibits homocysteine-induced superoxide anion production and nuclear factor kappa B activation in macrophagesThis paper is one of a selection of papers published in this Special Issue, entitled Young Investigator's Forum.

Folic acid supplementation is a promising approach for patients with cardiovascular diseases associated with hyperhomocysteinemia. We have demonstrated that homocysteine (Hcy) activates nuclear factor-kappaB (NF-kappaB), a transcription factor that plays an important role in inflammatory responses. The aim of the present study was to investigate the effect of folic acid on Hcy-induced NF-kappaB activation in macrophages. Hcy treatment (100 micromol/L) resulted in NF-kappaB activation and increased monocyte chemoattractant protein-1 (MCP-1) expression in THP-1 derived macrophages. Hcy-induced NF-kappaB activation was associated with a significant increase in the intracellular superoxide anion levels. There was a significant increase in phosphorylation and membrane translocation of NADPH oxidase p47phox subunit in Hcy-treated cells. Addition of folic acid (200 ng/mL) to the culture medium abolished NADPH oxidase-dependent superoxide anion generation in macrophages by preventing phosphorylation of p47phox subunit. Consequently, Hcy-induced NF-kappaB activation and MCP-1 expression was inhibited. Such an inhibitory effect of folic acid was independent of its Hcy-lowering ability. Taken together, these results suggest that folic acid treatment can effectively inhibit Hcy-induced oxidative stress and inflammatory responses in macrophages. This may represent one of the mechanisms by which folic acid supplementation exerts a protective effect in cardiovascular disorders.

Antioxidant Potential of an Extract of Calendula officinalis. Flowers in Vitro. and in Vivo.

An extract of Calendula officinalis. Linn. (Compositae) was evaluated for its antioxidant potential in vitro. and in vivo.. Calendula officinalis. extract was found to scavenge superoxide radicals generated by photoreduction of riboflavin and hydroxyl radicals generated by Fenton reaction and inhibited in vitro. lipid peroxidation. Concentrations needed for 50% inhibition (IC50) were 500, 480, and 2000 µg/mL, respectively. Extract scavenged ABTS radicals and DPPH radicals and IC50 were 6.5 and 100 µg/mL, respectively. IC50 values were compared with that of ginger extract, which is a standard antioxidant extract. The drug also scavenged nitric oxide, and the IC50 was found to be 575 µg/mL. Extract also produced dose-dependent scavenging of nitric oxide in culture. The oral administration of Calendula. extract inhibited superoxide generation in macrophages in vivo. by 12.6% and 38.7% at doses of 100 and 250 mg/kg b.wt. Oral administration of Calendula officinalis. to mice for 1 month significantly increased catalase activity. The extract produced significant increase in glutathione levels in blood and liver. Glutathione reductase was found to be increased, whereas glutathione peroxidase was found to be decreased after administration of Calendula. extract. These results indicated Calendula officinalis. has significant antioxidant activity in vitro. and in vivo..

Ganoderma lucidum inhibits inducible nitric oxide synthase expression in macrophages

Nitric oxide (NO) is a principal mediator in many physiological and pathological processes. Overproduction of NO via the inducible nitric oxide synthase (iNOS) has cytotoxic effect through the formation of peroxynitrite with superoxide anion. The iNOS is mainly expressed in macrophages and is able to produce large amount of NO. The expression of iNOS is mainly regulated at the transcriptional level. The iNOS-mediated NO production plays a role in the development of atherosclerosis. Ganoderma lucidum (G. lucidum, Linzhi or Reishi) is a traditional herbal medicine which is commonly used as health supplement. Several studies have demonstrated its effectiveness against cancer, immunological disorders and cardiovascular diseases. The objective of the present study was to investigate the effect of G. lucidum on iNOS-mediated NO production in macrophages. Human monocytic cell (THP-1) derived macrophages were incubated with lipopolysaccharide (LPS) for 24 h. Such treatment significantly stimulated NO production (253% versus the control). Such a stimulatory effect was resulted from increased iNOS mRNA expression (270% versus the control) and iNOS activity (169.5% versus the control) in macrophages. The superoxide anion level was also elevated (150% versus the control) in LPS-treated macrophages. Treatment of macrophages with G. lucidum extract (100 microg/ml) completely abolished LPS-induced iNOS mRNA expression and NO production. Such an inhibitory effect of G. lucidum was mediated via its antioxidant action against LPS-induced superoxide anion generation in macrophages. These results suggest that G. lucidum may exert a therapeutic effect against atherosclerosis via ameliorating iNOS-mediated NO overproduction in macrophages.

Studies on the inactivation of superoxide dismutase activity by nitric oxide from rat peritoneal macrophages.

Rat peritoneal macrophages stimulated with lipopolysaccharide (LPS) and Phorbol myristate acetate (PMA) generated increased levels of superoxide anions (O2.-) by 122% as compared to those stimulated with PMA alone. However, Nitric oxide (NO) synthase inhibitors-n-monomethyl arginine (nMMA) or spermine-HCI lowered the enhanced levels of O2.- released by LPS treated macrophages. The Superoxide dismutase (SOD) activity in LPS treated macrophages was 51% lower than that observed in resident cells. NO synthase inhibitors prevented the loss of SOD activity in LPS treated cells. Exogenously added SOD during sensitization of cells with LPS also inactivated the enzyme. This inactivation of SOD is inhibited by Nitric oxide synthase inhibitors. PMA alone did not affect SOD activity. NO synthase inhibitors also did not affect PMA activated superoxide anion generation in macrophages. These studies indicate that nitric oxide generated by LPS treated macrophages can inactivate SOD activity.

Effect of capsaicin on phospholipase A 2 activity and superoxide generation in macrophages

The mechanism of inhibition of Ca 2+ - triggered phospholipase A 2 (PLA 2) activity and respiratory burst in macrophages by capsaicin is assessed. In the present study, it has been shown that capsaicin inhibits calcium-ionophore stimulated pro-inflammatory responses in macrophages such as generation of superoxide anion, PLA 2 activity (IC 50 = 20uM) and membrane lipid peroxidation (IC 50 = 10 uM). Both capsaicin and PLA 2 antagonists altered the kinetics of respiratory burst in a time and dose dependent manner. Arachidonic acid, linoleic acid and SDS restored capsaicin inhibited respiratory burst. Capsaicin and known PLA 2 inhibitors, dexamethasone and indomethacin, inhibited Ca 2+-dependent PLA 2 activity in vitro from macrophages. Inhibition of PLA 2 activity by capsaicin is independent of Ca 2+ and substrate concentration. Fluorescence studies suggest that capsaicin interacts directly with partially purified macrophage PLA 2. Finally, the antioxidant property of capsaicin was comparable to that of butylated hydroxy toludine (BHT). Taken together these results show that capsaicin an antiinflammatory agent with potential clinical application.

Neopterin as an Endogenous Antioxidant

The in vitro antioxidative activity of neopterin and its in vivo potency to suppress gastric ischemia were investigated. The scavenging activity of the reduced form of neopterin, 5,6,7,8-tetrahydroneopterin (NPH4), against superoxide anion radicals, showed an extremely high superoxide anion radical scavenging activity in two assay systems, i.e., hypoxanthine/xanthine oxidase (HPX/XOD) and rat macrophage/ phorbol myristate acetate (Mø/PMA) radical-generating systems. The antioxidative activities against both super oxideanion and hydroxyl radicals were confirmed by spin trapping-ESR spectrometry. In contrast, the oxidized form of neopterin (NP) did not show any activity in the HPX/XOD system, but was effective in the Mø/PMA system. These data suggest that NPH4 directly scavenges superoxide anion radicals, while NP suppresses the superoxide anion generation in macrophages through its inhibitory effect on the membrane NADPH-oxidase. Furthermore, NPH4 and NP effectively suppressed the formation of gastric injury induced by ischemia and reperfusion. These results suggest that neopterin play an important role as an endogenous antioxidant.

Inhibition of Macrophage Superoxide Generation by Dehydroepiandrosterone

To understand the anti-atherosclerotic mechanism of the steroid dehydroepiandrosterone (DHEA), the effect of DHEA on rat peritoneal macrophage superoxide generation was studied. Dehydroepiandrosterone (12.5 to 50 microM) inhibited digitonin-stimulated superoxide production in a dose-dependent manner, with 100% inhibition achieved at 50 microM. Dehydroepiandrosterone also inhibited macrophage superoxide production stimulated with phorbol myristate acetate, A23187 (calcium ionophore), sodium fluoride, and arachidonate. Dehydroepiandrosterone did not affect the activity of nicotinamide-adenine dinucleotide phosphate oxidase, which generates superoxide. Dehydroepiandrosterone inhibited superoxide formation in the presence of nicotinamide-adenine dinucleotide phosphate, potassium cyanide, and 2,4-dinitrophinal, suggesting that DHEA does not exert its effects by inhibiting glucose-6-phosphate dehydrogenase activity or mitochondrial respiration. Of the several steroids tested, epiandrosterone was as effective as DHEA in inhibiting macrophage superoxide production. Estrogen, androstenedione, and dihydroxytestosterone showed 25% inhibition, whereas pregnenolone, progesterone, testosterone, etiocholanolone, androstenediol, and DHEA-sulfate had minimal effect. The steroids cortisol and corticosterone had slight stimulatory effect. These results suggest that the anti-atherosclerotic effect of DHEA may be the result of inhibition of superoxide generation in macrophages.

Arachidonic acid release is closely related to the Fc gamma receptor-mediated superoxide generation in macrophages.

Stimulation of macrophages with IgG2 immune complexes induced dose-dependently the O2- generation and the release of arachidonic acid and its metabolites. This Fc gamma R-mediated O2- generation was inhibited by a phospholipase A2 inhibitor, 4-p-bromophenacyl bromide (4-pBPB), in parallel to the dose-dependent inhibition of arachidonic acid release. The main arachidonic acid metabolites released were shown to be prostaglandin E2 and thromboxane B2 and blocking of the production of these metabolites by indomethacin did not inhibit the O2- generation. Inhibition of the Fc gamma R-mediated O2- generation and the arachidonic acid release by the C-kinase inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), was less intense than by 4-pBPB. These results support the previously proposed hypothesis that arachidonic acid acts as an intracellular activator of the Fc gamma R-mediated O2- generation in macrophages. Although the C-kinase activation may also contribute to the activation of the O2--generating system, arachidonic acid release appears to play a major role in Fc gamma R-mediated O2- generation. In contrast, activation of C-kinase seems to be contributing mainly in the induction of both the arachidonic acid release and O2- generation by 12-o-tetradecanoylphorbol 13-acetate (TPA). Furthermore, suboptimal concentrations of TPA and arachidonate were found to act synergistically to stimulate O2- generation and the inhibition study suggested a positive synergism between C-kinase and arachidonic acid release to induce O2- generation. This synergistic action may have general importance in receptor-mediated O2- generation.

Arachidonic acid acts as an intracellular activator of NADPH-oxidase in Fc gamma receptor-mediated superoxide generation in macrophages.

A protein kinase C inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7), inhibited phorbol ester (12-o-tetradecanoylphorbol 13-acetate)-induced and Fc gamma receptor-mediated superoxide anion (O2-) generations in guinea pig macrophages, but the inhibitory effect on Fc gamma receptor-mediated O2- generation was only partial. Both O2- generations were inhibited extensively by a phospholipase A2 inhibitor, 4-p-bromophenacyl bromide (4-pBPB). It was confirmed in control experiments that H-7 and 4-pBPB had no direct inhibitory effect on NADPH-oxidase activity. Dose-dependent stimulation of O2- generation was induced by arachidonate in macrophages, and the arachidonate-induced O2- generation was not inhibited by H-7. Arachidonate could also induce NADPH-oxidase activation in a post-nuclear fraction obtained from unstimulated macrophages and this activation was not inhibited by H-7, indicating that protein kinase C activation was not involved in this cellfree system. These results support the hypothesis that the O2- generation induced by Fc gamma receptor stimulation is mainly mediated by arachidonic acid which is released by the action of phospholipase A2 activated by receptor stimulation. Arachidonic acid seems to be acting rather directly in activating the NADPH-oxidase system of macrophage membrane. Protein kinase C may have a significant role in Fc gamma receptor-mediated O2- generation but it is not obligatory, and protein kinase C seems to activate NADPH-oxidase rather indirectly, probably by inducing the arachidonic acid release.

Superoxide generation in macrophages: enzymatic basis and function

Superoxide generation in macrophages: enzymatic basis and function