Effects of 6-formylpterin, a xanthine oxidase inhibitor and a superoxide scavenger, on production of nitric oxide in RAW 264.7 macrophages

As well as superoxide generated from neutrophils, nitric oxide (NO) produced by inducible nitric oxide synthase (iNOS) in macrophages plays an important role in inflammation. We previously showed that 6-formylpterin, a xanthine oxidase inhibitor, has a superoxide scavenging activity. In the present study, to elucidate other pharmacological activities of 6-formylpterin, we investigated the effects of 6-formylpterin on production of nitric oxide (NO) in the murine macrophage cell line RAW 264.7 stimulated by lipopolysaccharide (LPS) and interferon-gamma (INF-gamma). 6-Formylpterin suppressed the expression of iNOS, and it also inhibited the catalytic activity of iNOS, which collectively resulted in the inhibition of NO production in the stimulated macrophages. However, 6-formylpterin did not scavenge the released NO from an NO donor, S-nitroso-N-acetylpenicillamine (SNAP). These results indicate that 6-formylpterin inhibits pathological NO generation from macrophages during inflammation, but that it does not disturb the physiological action of NO released from other sources.     

Inhibition of inducible nitric oxide synthesis by azathioprine in a macrophage cell line

Azathioprine is used as an anti-inflammatory agent. Although there are numerous data demonstrating cytotoxic and immunosuppressive properties of azathioprine and its metabolite 6-mercaptopurine, the mechanism of the anti-inflammatory action of azathioprine has not yet been fully clarified. During our study, we investigated the effects of azathioprine on the inducible nitric oxide synthase (iNOS) in lipopolysaccharide stimulated murine macrophages (RAW 264.7) by measurement of iNOS protein (immunoblotting), iNOS mRNA (semiquantitative competitive RT-PCR), and NO production (nitrite levels). Azathioprine (0-210 muM) induces a concentration dependent inhibition of inducible nitric oxide synthesis (IC50: 33.5 muM). iNOS protein expression showed a concentration dependent reduction as revealed by immunoblotting when cells were incubated with increasing amounts of azathioprine. Azathioprine decreases iNOS mRNA levels as shown by semiquantitative competitive RT-PCR. In contrast, 6-mercaptopurine showed no inhibition of inducible nitric oxide synthesis. Azathioprine did not reduce iNOS mRNA stability after the addition of actinomycin D. Enzymatic activity assays with increasing concentrations of azathioprine (0-210 muM) showed no statistically significant inhibition of iNOS enzyme activity compared to cell lysates without azathioprine. Nuclear translocation of NF-kappaB p65 subunit and binding of NF-kappaB p50 subunit from nuclear extracts to a biotinylated-consensus sequence was unaffected by azathioprine treatment. iNOS inhibition by azathioprine was associated with a decreased expression of IRF-1 (interferon regulatory factor 1) and IFN-beta (beta-interferon) mRNA. Azathioprine induced iNOS inhibition seems to be associated with an action of the methylnitroimidazolyl substituent. This suggests a route to the rational design of nontoxic anti-inflammatory agents by replacing the 6-mercaptopurine component of azathioprine with other substituents. The inhibition of inducible nitric oxide synthesis might contribute to the anti-inflammatory activities of azathioprine.     

Anti-inflammatory effect of heme oxygenase 1: glycosylation and nitric oxide inhibition in macrophages

Flavonoids including the aglycones, hesperetin (HT; 5,7,3'-trihydroxy-4'-methoxy-flavanone), and naringenin (NE; 5,7,4'-trihydroxy flavanone) and glycones, hesperidin (HD; 5,7,3'-trihydroxy-4'-methoxy-flavanone 7-rhamnoglucoside) and naringin (NI; 5,7,4'-trihydroxy flavanone 7-rhamno glucoside), were used to examine the importance of rutinose at C7 on the inhibitory effects of flavonoids on lipopolysaccharide (LPS)-induced nitric oxide production in macrophages. Both HT and NE, but not their respective glycosides HD and NI, induced heme oxygenase 1 (HO-1) protein expression in the presence or absence of LPS and showed time and dose-dependent inhibition of LPS-induced nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression in RAW264.7, J774A.1, and thioglycolate-elicited peritoneal macrophages. Additive inhibitory effect of an HO-1 inducer hemin and NE or NI on LPS-induced NO production and iNOS expression was identified, and HO enzyme inhibitor tin protoporphyrin (SnPP) attenuated the inhibitory effects of HT, NE, and hemin on LPS-induced NO production. Both NE and HT showed no effect on iNOS mRNA and protein stability in RAW264.7 cells. Removal of rutinose at C7 of HD and NI by enzymatic digestion using hesperidinase (HDase) and naringinase (NIase) produce inhibitory activity on LPS-induced NO production, according to the production of the aglycones, HT and NE, by high-performance liquid chromatography (HPLC) analysis. Furthermore, the amount of NO produced by LPS or lipoteichoic acid (LTA) was significantly reduced in HO-1-overexpressing cells (HO-1/RAW264.7) compared to that in parental cells (RAW264.7). Results of the present study provide scientific evidence to suggest that rutinose at C7 is a negative moiety in flavonoid inhibition of LPS-induced NO production, and that HO-1 is involved in the inhibitory mechanism of flavonoids on LPS-induced iNOS and NO production.     

Anti-inflammatory activity of c-phycocyanin in lipopolysaccharide-stimulated RAW 264.7 macrophages

C-phycocyanin (C-PC), found in blue green algae, is often used as a dietary nutritional supplement. C-PC has been found to have an anti-inflammatory activity and exert beneficial effect in various diseases. However, little is known about its mechanism of action. Overproduction of nitric oxide (NO) derived from inducible nitric oxide synthase (iNOS) plays an important role in the pathogenesis of inflammation. The aim of this study was to determine whether C-PC inhibits production of nitrite, an index of NO, and iNOS expression in lipopolysaccharide (LPS)-treated RAW 264.7 macrophages. Our results indicated that C-PC significantly inhibited the LPS-induced nitrite production and iNOS protein expression accompanied by an attenuation of tumor necrosis factor-alpha (TNF-alpha) formation but had no effect on interleukin-10 production in macrophages. Furthermore, C-PC also suppressed the activation of nuclear factor-kappaB (NF-kappaB) through preventing degradation of cytosolic IkappaB-alpha in LPS-stimulated RAW 264.7 macrophages. Thus, the inhibitory activity of C-PC on LPS-induced NO release and iNOS expression is probably associated with suppressing TNF-alpha formation and nuclear NF-kappaB activation, which may provide an additional explanation for its anti-inflammatory activity and therapeutic effect.     

Double-stranded RNA inhibits beta-cell function and induces islet damage by stimulating beta-cell production of nitric oxide

Viral infection has been implicated as a triggering event that may initiate beta-cell damage during the development of autoimmune diabetes. In this study, the effects of the viral replicative intermediate, double-stranded RNA (dsRNA) (in the form of synthetic polyinosinic-polycytidylic acid (poly IC)) on islet expression of inducible nitric oxide synthase (iNOS), production of nitric oxide, and islet function and viability were investigated. Treatment of rat islets with poly(IC) + interferon-gamma (IFN-gamma) stimulates the time- and concentration-dependent expression of iNOS and production of nitrite by rat islets. iNOS expression and nitrite production by rat islets in response to poly(IC) + IFN-gamma correlate with an inhibition of insulin secretion and islet degeneration, effects that are prevented by the iNOS inhibitor aminoguanidine (AG). We have previously shown that poly(IC) + IFN-gamma activates resident macrophages, stimulating iNOS expression, nitric oxide production and interleukin-1 (IL-1) release. In addition, in response to tumor necrosis factor-alpha (TNF-alpha) + lipopolysaccharide, activated resident macrophages mediate beta-cell damage via intraislet IL-1 release followed by IL-1-induced iNOS expression by beta-cells. The inhibitory and destructive effects of poly(IC) + IFN-gamma, however, do not appear to require resident macrophages. Treatment of macrophage-depleted rat islets for 40 h with poly(IC) + IFN-gamma results in the expression of iNOS, production of nitrite, and inhibition of insulin secretion. The destructive effects of dsRNA + IFN-gamma on islets appear to be mediated by a direct interaction with beta-cells. Poly IC + IFN-gamma stimulates iNOS expression and inhibits insulin secretion by primary beta-cells purified by fluorescence-activated cell sorting. In addition, AG prevents the inhibitory effects of poly(IC) + IFN-gamma on glucose-stimulated insulin secretion by beta-cells. These results indicate that dsRNA + IFN-gamma interacts directly with beta-cells stimulating iNOS expression and inhibiting insulin secretion in a nitric oxide-dependent manner. These findings provide biochemical evidence for a novel mechanism by which viral infection may directly mediate the initial destruction of beta-cells during the development of autoimmune diabetes.     

Testosterone inhibits expression of inducible nitric oxide synthase in murine macrophages

We investigated the effect of testosterone, the main sexual steroid hormone in men, upon inducible nitric oxide synthesis in murine macrophages. Incubation of murine macrophages (RAW 264.7 cells) stimulated by bacterial lipopolysaccharide (2 microg/ml) with increasing amounts of testosterone (0.1-40 microM) showed a dose dependent inhibition of inducible nitric oxide synthesis. Inducible nitric oxide synthase protein expression was reduced in a dose dependent manner as revealed by immunoblotting when cells were incubated with increasing amounts of testosterone. This was associated with a decline in iNOS mRNA-levels as determined by competitive semiquantitative PCR. As nitric oxide plays an important role in immune defense and atherosclerosis prevention, testosterone-induced iNOS inhibition could lead to an elevated risk of infection as well as to the development of atherosclerotic lesions.     

Inhibition of lipopolysaccharide-induced expression of inducible nitric oxide synthase by butein in RAW 264.7 cells

Butein has been reported to exert anti-inflammatory effect but the possible mechanism involved is still unclear. Here, we report the inhibitory effect of butein on nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) gene expression. Butein also inhibited the induction of tumor necrosis factor-alpha and cyclooxygenase 2 by LPS. To further investigate the mechanism responsible for the inhibition of iNOS gene expression by butein, we examined the effect of butein on LPS-induced nuclear factor-kappaB (NF-kappaB) activation. The LPS-induced DNA binding activity of NF-kappaB was significantly inhibited by butein, and this effect was mediated through inhibition of the degradation of inhibitory factor-kappaB and phosphorylation of Erk1/2 MAP kinase. Furthermore, increased binding of the osteopontin alphavbeta3 integrin receptor by butein may explain its inhibitory effect on LPS-mediated NO production. Taken together, these results suggest that butein inhibits iNOS gene expression, providing possible mechanisms for its anti-inflammatory action.     

Oxidized low density lipoprotein inhibits inducible nitric oxide synthase, GTP cyclohydrolase I and transforming growth factor beta gene expression in rat macrophages.

Several studies have already demonstrated that oxidized- LDL decreases nitric oxide (NO) generation by cytokine-stimulated macrophages. However, the mechanisms of such an inhibition have not been yet elucidated. NO generation by inducible nitric oxide synthase (iNOS) is dependent on the presence of cofactors for NO generation, tetrathydrobiopterin (BH4) among them. The NO generation by these cells is also regulated by some endogenous inhibitors, like TGF-beta. Therefore, the aim of our recent study was to investigate the influence of ox-LDL on the expression of iNOS and GTP cyclohydrolase I (GTP-CH I), the key enzyme involved in the BH4 synthesis as well as the ox-LDL effect on TGF-beta expression in rat macrophages stimulated with IFNgamma (250 U/ml) and LPS (500 ng/ml). Macrophages, activated in this way, express iNOS, GTP-CH I, and TGF-beta mRNA. This expression was inhibited when the macrophages were preincubated for 24 hours with ox-LDL (100 microg/ml). Quantitative PCR revealed about 10-fold inhibition of iNOS gene expression by ox-LDL. As a consequence of down-regulation of iNOS and GTP-CH I genes, almost 3-fold diminished generation of NO2- by rat macrophages was observed. An inhibition of the TGFbeta mRNA expression was also found. Our studies indicate that decreased NO generation by ox-LDL treated macrophages may be the result of the diminished expression of both iNOS and GTP-CH I genes. This effect may be mediated by the activity of certain endogenous inhibitors of gene expression, however, our studies exclude the TGF-beta as a candidate for this activity.     

Inhibition of inducible nitric oxide synthesis by the herbal preparation Padma 28 in macrophage cell line

Padma 28 is a mixture of herbs used in traditional Tibetan medicine with anti-inflammatory activities. We investigated the effects of Padma 28 on nitric oxide (NO) production by the inducible nitric oxide synthase (iNOS) in lipopolysaccharide stimulated mouse macrophages (RAW 264.7). Padma 28 (0-900 microg/mL) induced a concentration dependent inhibition of inducible nitric oxide synthesis. iNOS protein expression showed a concentration dependent reduction as revealed by immunoblotting when cells were incubated with increasing amounts of Padma 28. Padma 28 decreased iNOS mRNA levels as shown by RT-PCR. Aqueous extracts from costi amari radix (costus root, the dried root of Saussurea lappa) and the outer cover of myrobalani fructus (the dried fruit of Terminalia chebula), constituents of the complex herb preparation Padma 28, were found to inhibit inducible nitric oxide synthesis by decreasing iNOS protein and iNOS mRNA levels. The inhibition of inducible nitric oxide synthesis might contribute to the anti-inflammatory activities of Padma 28.     

Role of interferon regulatory factor-1 in double-stranded RNA-induced iNOS expression by mouse islets.

Environmental factors, such as viral infection, have been implicated as potential triggering events leading to the initial destruction of pancreatic beta cells during the development of autoimmune diabetes. Double-stranded RNA (dsRNA), the active component of a viral infection that stimulates antiviral responses in infected cells, has been shown in combination with interferon-gamma (IFN-gamma) to stimulate inducible nitric oxide synthase (iNOS) expression and nitric oxide production and to inhibit beta cell function. Interferon regulatory factor-1 (IRF-1), the activation of which is induced by dsRNA, viral infection, and IFN-gamma, regulates the expression of many antiviral proteins, including PKR, type I IFN, and iNOS. In this study, we show that IRF-1 is not required for dsRNA + IFN-gamma-stimulated iNOS expression and nitric oxide production by mouse islets. In contrast to islets, dsRNA + IFN-gamma fails to induce iNOS expression or nitric oxide production by macrophages isolated from IRF-1(-/-) mice; however, dsRNA + IFN-gamma induces similar levels of IL-1 release by macrophages isolated from both IRF-1(-/-) and IRF-1(+/+) mice. Importantly, we show that dsRNA- or dsRNA + IFN-gamma-stimulated IRF-1 expression by mouse islets and peritoneal macrophages is independent of PKR. These results indicate that IRF-1 is required for dsRNA + IFN-gamma-induced iNOS expression and nitric oxide production by mouse peritoneal macrophages but not by mouse islets. These findings suggest that dsRNA + IFN-gamma stimulates iNOS expression by two distinct PKR-independent mechanisms; one that is IRF-1-dependent in macrophages and another that is IRF-1-independent in islets.     

Inhibitory effects of indole α‐lipoic acid derivatives on nitric oxide production in LPS/IFNγ activated RAW 264.7 macrophages

Alpha‐lipoic acid (α‐lipoic acid) is a potent antioxidant compound that has been shown to possess anti‐inflammatory effects. RAW 264.7 macrophages produce various inflammatory mediators such as nitric oxide, IL‐1β, IL‐6 and TNF‐alpha upon activation with LPS ( Lipopolysaccharide) and IFNγ (interferon gamma). In this study, the effect of 12 synthetic indole α‐lipoic acid derivatives on nitric oxide production and iNOS (inducible nitric oxide synthase) protein expression in LPS/IFNγ activated RAW 264.7 macrophages was determined. Cell proliferation, nitric oxide levels and iNOS protein expression were examined with thiazolyl blue tetrazolium blue test, griess assay and western blot, respectively. Our results showed that all of the indole α‐lipoic acid derivatives showed significant inhibitory effects on nitric oxide production and iNOS protein levels (p < 0.05). The most active compounds were identified as compound I‐4b, I‐4e and II‐3b. In conclusion, these indole α‐lipoic acid derivatives may have the potential for treatment of inflammatory conditions related with high nitric oxide production. Copyright © 2015 John Wiley & Sons, Ltd.     

Cell-specific inhibition of inducible nitric oxide synthase activation by leflunomide

The influence of a novel immunomodulating drug, leflunomide, on iNOS-dependent nitric oxide (NO) production in rodent macrophages and fibroblasts was investigated. Leflunomide's active metabolite A77 1726 caused a dose-dependent decrease of NO production in IFN-gamma-treated L929 fibroblasts. The observed effect was cell-specific, as well as stimulus-specific, since A77 1726 did not affect NO production in IFN-gamma-stimulated murine peritoneal macrophages or db-cAMP-treated L929 cells. A77 1726 reduced expression of IFN-gamma-induced iNOS and IRF-1 mRNA in L929 cells, while iNOS enzymatic activity remained unchanged. Specific inhibitor of MAP kinase kinase (MEK), PD98059, but not unselective protein kinase inhibitor genistein, completely mimicked cell-type-specific and stimulus-specific NO-inhibitory action of leflunomide. Therefore, the recently described inhibition of MEK/MAP pathway by leflunomide could present a possible mechanism for its suppression of iNOS activation in L929 fibroblasts. Finally, a similar inhibitory effect of A77 1726 on both NO production and iNOS mRNA expression was observed also in IFN-gamma + LPS-activated murine and rat primary fibroblasts.