Inhibitory action of daunorubicin on lipopolysaccharide-stimulated inducible-type nitric oxide synthase induction in alveolar macrophages

The purpose of this study was to investigate the effects of daunorubicin on lipopolysaccharide (LPS)-stimulated inducible-type nitric oxide synthase (iNOS) expression in macrophages. LPS-stimulated iNOS expression and NO production were significantly inhibited in alveolar macrophages from rats administrated daunorubicin (4 mg/kg body weight per day) for 5 consecutive days. Incubation of macrophages with daunorubicin at 1 muM but not at 0.1 and 0.5 muM significantly inhibited LPS-stimulated NO production and iNOS induction. Activation of extracellular regulated kinase (ERK) by LPS was markedly attenuated in both macrophages isolated from in vivo daunorubicin-treated rats and those incubated in vitro with daunorubicin at 1 microM. ERK activation, iNOS induction, and NO production following LPS stimulation were all markedly inhibited in the presence of U0126, an ERK inhibitor. The viability of macrophages was decreased by incubation with daunorubicin at 0.5 and 1 microM, while treatment of rats with daunorubicin did not affect viability of macrophages isolated from the rats. These results suggest that in vivo treatment of rats with daunorubicin attenuates LPS-induced iNOS expression of macrophages through inhibition of ERK activation, while inhibition of iNOS induction by in vitro incubation with daunorubicin may be mainly due to its cytotoxicity.     

NF-kappaB signaling pathway, not IFN-beta/STAT1, is responsible for the selenium suppression of LPS-induced nitric oxide production

Upon stimulation of macrophages with lipopolysaccharide (LPS), Toll-like receptor 4 recognizes LPS, leading to expression of inducible nitric oxide synthase (iNOS), via MyD88/NF-kappaB and TRIF/IFN-beta/STAT pathways. Although selenium (Se) was reported to inhibit nitric oxide (NO) production, it is unclear which signaling pathway is inhibited by Se. Here, we investigated how Se inhibits NO production in LPS-stimulated RAW 264.7 cells. When the cells were pretreated with Se for 1 h followed by LPS treatment, iNOS mRNA expression and subsequent NO production declined significantly in a dose-dependent manner. Se inhibited IkappaBalpha degradation in the cytosol and NF-kappaB binding to its recognition site in the nucleus of the LPS-stimulated cells. Meanwhile, Se did not inhibit IFN-beta mRNA induction or STAT1 phosphorylation in the LPS-stimulated cells. These results suggest that Se down-regulates iNOS gene expression and NO production in the LPS-stimulated macrophages through inhibition of the NF-kappaB activation pathway but not the IFN-beta/STAT1 signaling pathway.     

Reduction of lipopolysaccharide-induced cyclooxygenase-2 expression in diabetic arteries

In order to clarify the effects of diabetes mellitus on induction of cyclooxygenase (COX)-2 and subsequent prostaglandin production in blood vessels, we investigated lipopolysaccharide (LPS)-induced COX-2 induction and prostacyclin production in aortic strips isolated from streptozotocin-induced diabetic rats and vehicle-injected control rats. The rats at 10 weeks after injection of streptozotocin had significantly lower body weights and higher serum glucose levels compared with those in the control rats. LPS stimulation resulted in a marked increase in the release of prostacyclin from the aortic strips. This increase was abolished in the presence of indomethacin or cycloheximide but was not affected by removal of the endothelium. In diabetic aortae, both LPS-induced prostacyclin production and COX-2 induction were diminished compared with the control aortae. No significant difference in COX-1 expression was observed between diabetic and control aortae. The diminution of LPS-induced COX-2 expression was also observed in alveolar macrophages isolated from diabetic rats. These results suggest that COX-2 expression and subsequent prostacyclin production in response to LPS are selectively attenuated in diabetic blood vessels.     

Sauchinone, a lignan from Saururus chinensis, suppresses iNOS expression through the inhibition of transactivation activity of RelA of NF-kappaB

Sauchinone, a known lignan, was isolated from the root of Saururus chinensis as an active principle responsible for inhibiting the production of NO in LPS-stimulated RAW264.7 cells by activity-guided fractionation. Sauchinone dose-dependently inhibited not only the production of NO, but also the expression of iNOS mRNA and protein in LPS-stimulated RAW 264.7 cells. Furthermore, sauchinone prevented LPS-induced NF-kappaB activation, which is known to play a critical role in iNOS expression, assessed by a reporter assay under the control of NF-kappaB. However, an electrophoretic mobility shift assay (EMSA) demonstrated that sauchinone did not suppress the DNA-binding activity of NF-kappaB or the degradation of IkappaB-alpha induced by LPS. Further analysis revealed that transactivation activity of RelA subunit of NF-kappaB was dose-dependently suppressed in the presence of sauchinone. Taken together, our results suggested that sauchinone could inhibit production of NO in LPS-stimulated RAW264.7 cells through the suppression of NF-kappaB by inhibiting transactivation activity of RelA subunit.     

Decreased modulation by lipopolysaccharide of aortic smooth muscle contractility in streptozotocin-induced hyperglycemic rats

Infection is a major complication of patients with diabetes, and endotoxemic shock is a serious complication during sepsis. The purpose of this study was to determine whether the action of bacterial lipopolysaccharide (LPS) on vasocontractility is altered in diabetic vessels. Diabetes was induced in 10-week-old Wistar rats by an intraperitoneal injection of streptozotocin. LPS-induced increase in cGMP (cyclic guanosine 3',5'-monophosphate) level was lower in aortae from streptozotocin-induced hyperglycemic (diabetic) rats than in those from vehicle-injected control rats, while LPS-induced nitric oxide production was not different in the diabetic and control aortae. Phenylephrine-induced contraction of diabetic aortae was lower than that of the control aortae. LPS treatment resulted in depression of contractile response to phenylephrine in both diabetic and control aortae, and the degree of depression was much lower in diabetic aortae. Treatment with N monomethyl l-arginine (l-NMMA) prevented diminution of phenylephrine-induced contraction of the aortae after LPS stimulation, and the degree of the preventive effect by l-NMMA was significantly lower in diabetic aortae than in the control aortae. Protein expression of inducible nitric oxide synthase detected by Western blot analysis was not different in the diabetic and control aortae. The decrease in cGMP production after LPS stimulation in diabetic aortae was not prevented by treatment of the aortae with superoxide dismutase but was partially prevented by that with Tiron (4,5-dihydroxy-1,3-benzene disulfonic acid), a cell-permeable scavenger of reactive oxygen species. These results suggest that LPS-induced depression of vasocontractility is attenuated in diabetic aortae due to a decrease in nitric oxide-stimulated cGMP production, probably resulting from increased inactivation of inducible nitric oxide by excessive intracellular oxidative stress. It is concluded that contractility of aortae from streptozotocin-induced hyperglycemic rats may be less affected by LPS during endotoxemia.     

Reduced bactericidal activity and nitric oxide production in metallothionein-deficient macrophages in response to lipopolysaccharide stimulation

This study was designed to investigate bactericidal activity of and nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated peritoneal exudate macrophages (Mvarphi) from metallothionein (MT)-null mice. Control Mvarphi had a bactericidal effect on Staphylococcus aureus, but MT-null Mvarphi had significantly lower activity. NO is an important factor in the bactericidal function of Mvarphi. LPS-stimulated MT-null Mvarphi produced less NO than those of control mice. LPS-stimulated Mvarphi produce cytokines such as tumor necrosis factor (TNF)-alpha. TNF-alpha activate Mvarphi and stimulates NO production. We evaluated NO production by TNF-alpha-stimulated Mvarphi. MT-null Mvarphi produced less NO in response to TNF-alpha stimulation. Levels of expression of inducible NO synthase (iNOS) mRNA and production of iNOS protein in response to LPS stimulation were similar in MT-null and control cells, as were levels of expression of arginase, which competes in arginine metabolism with iNOS. No notable changes were found in arginine uptake or in expression of cationic amino acid transporter 2 (a major arginine transporter in Mvarphi) between control and MT-null Mvarphi. The rate of conversion of [(14)C]-l-arginine to citrulline, which is formed with NO by the action of iNOS, was much lower in MT-null Mvarphi than in control cells. These results indicate that the reduced production of NO in MT-deficient Mvarphi is due mainly to reduced activity of iNOS. Thus, MT plays important roles in bactericidal activity, NO production, and arginine metabolism in activated Mvarphi.     

Gender-specific vascular effects elicited by chronic ethanol consumption in rats: a role for inducible nitric oxide synthase

BACKGROUND AND PURPOSE: Epidemiological data suggest that the risk of ethanol-associated cardiovascular disease is greater in men than in women. This study investigates the mechanisms underlying gender-specific vascular effects elicited by chronic ethanol consumption in rats. EXPERIMENTAL APPROACH: Vascular reactivity experiments using standard muscle bath procedures were performed on isolated thoracic aortae from rats. mRNA and protein for inducible NO synthase (iNOS) and for endothelial NOS (eNOS) was assessed by RT-PCR or western blotting, respectively. KEY RESULTS: In male rats, chronic ethanol consumption enhanced phenylephrine-induced contraction in both endothelium-intact and denuded aortic rings. However, in female rats, chronic ethanol consumption enhanced phenylephrine-induced contraction only in endothelium denuded aortic rings. After pre-incubation of endothelium-intact rings with L-NAME, both male and female ethanol-treated rats showed larger phenylephrine-induced contractions in aortic rings, compared to the control group. Acetylcholine-induced relaxation was not affected by ethanol consumption. The effects of ethanol on responses to phenylephrine were similar in ovariectomized (OVX) and intact (non-OVX) female rats. In the presence of aminoguanidine, but not 7-nitroindazole, the contractions to phenylephrine in rings from ethanol-treated female rats were greater than that found in control tissues in the presence of the inhibitors. mRNA levels for eNOS and iNOS were not altered by ethanol consumption. Ethanol intake reduced eNOS protein levels and increased iNOS protein levels in aorta from female rats. CONCLUSIONS AND IMPLICATIONS: Gender differences in the vascular effects elicited by chronic ethanol consumption were not related to ovarian hormones but seemed to involve the upregulation of iNOS.     

Enhancement of nitric oxide synthase induction in alveolar macrophages by in vivo administration of docetaxel

Effects of docetaxel, a taxane-derivative anti-cancer drug, on lipopolysaccharide (LPS)-induced nitric oxide (NO) synthesis were investigated in alveolar macrophages isolated from rats. LPS-induced NO production and inducible NO synthase (iNOS) expression were significantly enhanced in the macrophages isolated from rats injected intraperitoneally with docetaxel (4 mg/kg body weight per day for 5 consecutive days) compared with those in macrophages from control rats administrated a vehicle. In vivo administration of docetaxel augmented LPS-induced p38 activation but not extracellular signal-related kinase (ERK) activation in isolated macrophages. On the other hand, in vitro treatment of macrophages with docetaxel (5 and 10 μg/ml) inhibited LPS-induced NO production and iNOS expression. Levels of lactate dehydrogenase released from macrophages were neither affected by in vitro treatment with docetaxel (up to 10 μg/ml) nor by its in vivo administration. These results suggest that docetaxel has an enhancing action in vivo on LPS-induced iNOS expression and subsequent NO production through stimulation of p38 activity in alveolar macrophages.     

Inducible nitric oxide synthase inhibitors of Chinese herbs III. Rheum palmatum

In this paper, the effects of bioactive compounds of Rheum palmatum L. on the inhibition of NO production from RAW 264.7 cells were explored. Seven main anthraquinone derivatives were isolated from the root of R. palmatum, and of these, emodin and rhein significantly inhibited nitrite production from lipopolysaccharide (LPS)-activated RAW 264.7 cells. The IC(50) values for inhibition of nitrite production by emodin and rhein were 60.7 and 67.3 microM, respectively. After iNOS enzyme activity was stimulated by LPS for 12 h, treatment with emodin or rhein at 20 microg/ml for 18 h did not significantly inhibit NO production. The data show that the inhibitory activity of emodin and rhein is not due to direct inhibition of iNOS enzyme activity. However, expression of iNOS and the COX-2 protein was inhibited by emodin in LPS-activated RAW 264.7 cells, and PGE(2) production was reduced. Rhein also inhibited LPS-induced iNOS protein expression, but not COX-2 or PGE(2) production. On the other hand, inhibition effects on NO production from RAW 264.7 cells were enhanced and cytotoxic effects decreased by co-treatment with emodin and rhein. In conclusion, emodin and rhein are major iNOS inhibitors of R. palmatum and may possibly serve as bioactive substances for anti-inflammation effects.     

Hydrangenol inhibits lipopolysaccharide-induced nitric oxide production in BV2 microglial cells by suppressing the NF-κB pathway and activating the Nrf2-mediated HO-1 pathway

We previously demonstrated the anti-inflammatory effect of water extract of Hydrangea macrophylla in lipopolysaccharide (LPS)-stimulated macrophage cells. Here, we investigated whether hydrangenol, a bioactive component of H. macrophylla, attenuates the expression of nitric oxide (NO) and its associated gene, inducible NO synthase (iNOS), in LPS-stimulated BV2 microglial cells. Our data showed that low dosages of hydrangenol inhibited LPS-stimulated NO release and iNOS expression without any accompanying cytotoxicity. Hydrangenol also suppressed LPS-induced nuclear translocation of nuclear factor-κB (NF-κB) subunits, consequently inhibiting DNA-binding activity of NF-κB. Additionally, the NF-κB inhibitors, pyrrolidine dithiocarbamate (PDTC) and PS-1145, significantly attenuated LPS-induced iNOS expression, indicating that hydrangenol-induced NF-κB inhibition might be a key regulator of iNOS expression. Furthermore, our data showed that hydrangenol suppresses NO production by inducing heme oxygenase-1 (HO-1). The presence of cobalt protoporphyrin, a specific HO-1 inducer, potently suppressed LPS-induced NO production. Hydrangenol also promoted nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) and subsequently increased its binding activity at the specific antioxidant response element sites. Additionally, transient knockdown of Nrf2 significantly downregulated hydrangenol-induced HO-1 expression, indicating that hydrangenol-induced Nrf2 is an upstream regulator of HO-1. Taken together, these data suggest that hydrangenol attenuates NO production and iNOS expression in LPS-stimulated BV2 microglial cells by inhibiting NF-κB activation and by stimulating the Nrf2-mediated HO-1 signaling pathway. Therefore, hydrangenol is a promising therapeutic agent for treatment of LPS-mediated inflammatory diseases.     

Inhibition of inducible nitric oxide synthase expression by novel nonsteroidal anti-inflammatory derivatives with gastrointestinal-sparing properties.

1. The effects of novel nitric oxide-releasing nonsteroidal anti-inflammatory compounds (NO-NSAIDs) on induction of nitric oxide (NO) synthase by bacterial lipopolysaccharide (LPS) were examined in a murine cultured macrophage cell line, J774. 2. LPS-induced nitrite production was markedly attenuated by the nitroxybutylester derivatives of flurbiprofen (FNBE), aspirin, ketoprofen, naproxen, diclofenac and ketorolac, with each compound reducing accumulated nitrite levels by > 40% at the maximum concentrations (100 micrograms ml-1) used. 3. Further examination revealed that nitrite production was inhibited in a concentration-dependent (1-100 micrograms ml-1) manner by FNBE which at 100 micrograms ml-1 decreased LPS-stimulated levels by 63.3 +/- 8.6% (n = 7). The parent compound flurbiprofen was relatively ineffective over the same concentration-range, inhibiting nitrite accumulation by 24 +/- 0.9% (n = 3) at the maximum concentration used (100 micrograms ml-1). 4. FNBE reduced LPS-induced nitrite production when added to cells up to 4 h after LPS. Thereafter, FNBE caused very little or no reduction in nitrite levels. Furthermore NO-NSAIDs (100 micrograms ml-1) did not inhibit the metabolism of L-[3H]-arginine to citrulline by NO synthase isolated from LPS-activated macrophages. 5. Western blot analysis demonstrated that NO synthase expression was markedly attenuated following co-incubation of J774 cell with LPS (1 microgram ml-1; 24 h) and FNBE (100 micrograms ml-1; 24 h). Thus taken together, these findings indicate that NO-NSAIDs inhibit induction of NO synthase without directly affecting enzyme activity. 6. In conclusion our results indicate that NO-NSAIDs can inhibit the inducible L-arginine-NO pathway, and are capable of suppressing NO synthesis by inhibiting expression of NO synthase. The clinical implications of these findings remain to be established.     

Nitric oxide is not involved in the endotoxemia-induced alterations in Ca2+ and ryanodine responses in mouse diaphragms

Lipopolysaccharide (LPS, endotoxin)-induced diaphragmatic contractile dysfunction and sarcolemmal injury in animals has been identified. However, the precise nature of sepsis-related alterations in diaphragm myofiber function and the activity of Ca(2+) release from sarcoplasmic reticulum of skeletal muscle remain unclear. The present study investigated the in vivo effects of LPS on the Ca(2+)-dependent mechanical activity and ryanodine response in mouse diaphragm and Ca(2+) release from isolated sarcoplasmic reticulum membrane vesicles, and aimed to examine the role of nitric oxide (NO) in these responses. When diaphragms were bathed in a solution that was Cl(-)-free, Na(+)-free, but contained high K(+), a Ca(2+)-induced contracture was elicited. Increases in external Ca(2+) concentration produced increases in peak tension of Ca(2+)-induced contracture in control diaphragm, while a decrease was seen in endotoxemic diaphragm. Ryanodine induced a marked contracture in control diaphragms, which was diminished after endotoxemia. This finding is correlated with the decrease of ryanodine-induced Ca(2+) release and the suppression of [(3)H]ryanodine binding on the isolated SR of the skeletal muscle from LPS-treated rats. In mice treated with LPS significantly increased levels of plasma nitrite and serum TNF-alpha were observed, changes inhibited by aminoguanidine [an inhibitor of inducible NO synthase (iNOS)] and pentoxifylline (an inhibitor of tumor necrosis factor-alpha formation), respectively. Moreover, LPS treatment resulted in a significant expression of mRNA for iNOS in mouse diaphragms. The inhibitory effects on Ca(2+)- and ryanodine responses by LPS could be prevented by treatment with polymyxin B (LPS neutralizer) and pentoxifylline, but not by treatment with dexamethasone, N(G)-nitro- L-arginine or aminoguanidine (NOS inhibitors). These results imply that the NO-related pathway may not be involved in the dysfunction of the Ca(2+) release mechanism in the sarcoplasmic reticulum of mouse diaphragm during endotoxemia.     

Inhibition of nitric oxide synthase abrogates lipopolysaccharides-induced up-regulation of L-arginine uptake in rat alveolar macrophages

It was tested whether the inducible nitric oxide synthase (iNOS) pathway might be involved in lipopolysaccharides-(LPS)-induced up-regulation of L-arginine transport in rat alveolar macrophages (AM). AM were cultured in absence or presence of LPS. Nitrite accumulation was determined in culture media and cells were used to study [3H]-L-arginine uptake or to isolate RNA for RT - PCR. Culture in presence of LPS (1 microg ml(-1), 20 h) caused 11 fold increase of nitrite accumulation and 2.5 fold increase of [3H]-L-arginine uptake. The inducible NO synthase (iNOS) inhibitor 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT) present alone during culture had only marginal effects on [3H]-L-arginine uptake. However, AMT present during culture additionally to LPS, suppressed LPS-induced nitrite accumulation and LPS-stimulated [3H]-L-arginine uptake in the same concentration-dependent manner. AMT present only for the last 30 min of the culture period had similar effects on [3H]-L-arginine uptake. AMT present only during the uptake period also inhibited LPS-stimulated [3H]-L-arginine uptake, but with lower potency. The inhibitory effect of AMT could not be opposed by the NO releasing compound DETA NONOate. LPS caused an up-regulation of the mRNA for the cationic amino acid transporter CAT-2B, and this effect was not affected by AMT. AMT (100 microM) did not affect L-arginine transport studied by electrophysiological techniques in Xenopus laevis oocytes expressing either the human cationic amino acid transporter hCAT-1 or hCAT-2B. In conclusion, iNOS inhibition in rat AM abolished LPS-activated L-arginine uptake. This effect appears to be caused by reduced flow of L-arginine through the iNOS pathway.     

Inhibition of inducible nitric oxide synthesis by catalposide from Catalpa ovata

Catalposide (1) and two related iridoids were isolated from the stem of Catalpa ovata (Bignoniaceae) by bioassay guided fractionation. Catalposide (1) significantly inhibited the production of nitric oxide (NO) in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages in a dose-dependent manner. RT-PCR and Western blot analyses demonstrated that catalposide (1) suppressed the expression of inducible nitric oxide synthase (iNOS) gene and iNOS protein. Catalposide (1) also inhibited the activation of LPS-induced NF-kappaB as analyzed by electrophoretic mobility shift assay (EMSA). In addition to the inhibitory effect on NO production in LPS-stimulated RAW 264.7 cells, catalposide (1) significantly inhibited the NO production in cytokine-stimulated human DLD-1 and rat vascular smooth muscle (VSM) cells in a dose-dependent manner.     

YL-I-108, a synthetic chalcone derivative,inhibits lipopolysaccharide-stimulated nitric oxide production in RAW 264.7 murine macrophages: Involvement of heme oxygenase-1 induction and blockade of activator protein-1

Chalcones, a group of phenolic compounds, exhibit potent anti-inflammatory properties. In the present study, we synthesized chalcone derivative, YL-I-108 ((E)-1-(2-methoxy-4,6-bis(methoxymethoxy)phenyl)-3-(3-nitrophenyl)prop-2-en-1-one), and examined its effect on the production of pro-inflammatory mediators. Treatment of RAW 264.7 macrophages with YL-I-108 potently inhibited nitrite production stimulated by LPS. YL-I-108 treatment also markedly inhibited expressions of inducible nitric oxide synthase (iNOS) and tumor necrosis factor-α (TNF-α). Treatment of cells with YL-I-108 significantly inhibited LPS-stimulated activator protein-1 (AP-1)-dependent reporter gene expression, whereas nuclear factor-κB (NF-κB) activity was not affected, indicating that down-regulation of iNOS expression by YL-I-108 is attributed by blockade of AP-1. In addition, YL-I-108 treatment led to an increase in heme oxygenase-1 (HO-1) mRNA and protein expression, accompanied with the increased expression of nuclear factor-erythroid 2-related factor 2 (Nrf2). Treatment with SnPP, a selective HO-1 inhibitor, reversed YL-I-108-mediated suppression of nitrite production, suggesting that HO-1 induction is implicated in the suppression of NO production by YL-I-108. In contrast, SnPP treatment did not reverse YL-I-108-mediated suppression of AP-1 activation, suggesting that AP-1 inhibition by YL-I-108 is independent of HO-1 induction. Together, these results indicate that YL-I-108 suppresses NO production in LPS-stimulated macrophages via simultaneous induction of HO-1 expression and blockade of AP-1 activation.     

Inhibitory effects of nardostachin on nitric oxide, prostaglandin E2, and tumor necrosis factor-alpha production in lipopolysaccharide activated macrophages

Nardostachin, which is an iridoid isolated from Patrinia saniculaefolia, was examined by assessing its effect on the production of tumor necrosis factor-alpha (TNF-alpha) and expression of 2 enzymes, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), in lipopolysaccharide (LPS)-stimulated Raw264.7 macrophages. This compound consistently inhibited the production of nitric oxide (NO) and TNF-alpha production in a dose-dependent manner, with respective IC(50) values of 12.3 and 16.2 microM. The decrease in quantity of NO products was accompanied by a decrease in the iNOS protein level, as assessed by Western blotting probed with specific anti-iNOS antibodies. In addition, this compound also reduced the COX-2 protein expression level and the attendant PGE(2) production in LPS-stimulated macrophages. These results suggest that nardostachin may be useful for inhibiting the production of inflammatory mediators such as TNF-alpha, NO and PGE(2) in inflammatory diseases.     

A therapeutic dosage of amlodipine prevents vascular hyporeactivity induced in rats by lipopolysaccharide

The aim of this work was to investigate whether treatment with the 1,4-dihydropyridine Ca²⁺ antagonist amlodipine could affect the vascular hyporesponsiveness induced by cytokines. Endotoxemia was induced by Salmonella typhosa lipopolysaccharide (LPS) injection (4 mg kg^–1, i.p.). In endothelium-denuded rings of thoracic aorta from untreated rats, contractile response to noradrenaline was decreased after LPS injection, this effect was partially overcome by the addition of N^ω-nitro-l-arginine (l-NNA, 100 μM) into the bathing solution. In amlodipine-pretreated rats (15 mg kg^–1 day^–1, orally, for one week), the effect of LPS was lower than in untreated ones and it was completely reversed by l-NNA. The relaxation of the noradrenaline-induced tone evoked by l-arginine (10 μM) in aortae of LPS-injected rats was reduced in amlodipine-pretreated rats. Amlodipine-treatment reduced both the LPS-induced Ca²⁺-independent NOS activity in homogenates of heart and the expression of iNOS mRNA in aortae of LPS-injected rats. However, the vascular hyporeactivity induced by exposing aortae to interleukin-1β in vitro was not influenced by amlodipine (10 nM). Amlodipine (10 μM) also did not affect the production of nitrite in primary aortic smooth muscle cell culture challenged by LPS although nitrite production in macrophage culture challenged with LPS was significantly inhibited. The results show that rat pretreatment with amlodipine prevented the decrease of vascular responsiveness induced by LPS, an effect that may be at least partly related to reduction of in vivo NOS induction. The weak effect of amlodipine on the in vitro NOS induction indicates that the protective action in endotoxemia did not result from a short term interaction with L-type Ca²⁺ channels in vascular smooth muscle. Alternative mechanisms are discussed. Received: 1 April 1997 / Accepted: 6 November 1997     

Anti-Inflammatory Effect of Mangostenone F in Lipopolysaccharide-Stimulated RAW264.7 Macrophages by Suppressing NF-κB and MAPK Activation

Mangostenone F (MF) is a natural xanthone isolated from Garcinia mangostana. However, little is known about the biological activities of MF. This study was designed to investigate the anti-inflammatory effect and underlying molecular mechanisms of MF in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. MF dose-dependently inhibited the production of NO, iNOS, and pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β) in LPS-stimulated RAW264.7 macrophages. Moreover, MF decreased the NF-κB luciferase activity and NF-κB DNA binding capacity in LPS-stimulated RAW264.7 macrophages. Furthermore, MF suppressed the NF-κB activation by inhibiting the degradation of IκBα and nuclear translocation of p65 subunit of NF-κB. In addition, MF attenuated the AP-1 luciferase activity and phosphorylation of ERK, JNK, and p38 MAP kinases. Taken together, these results suggest that the anti-inflammatory effect of MF is associated with the suppression of NO production and iNOS expression through the down-regulation of NF-κB activation and MAPK signaling pathway in LPS-stimulated RAW264.7 macrophages.