Suppression of tumor necrosis factor-alpha and inducible nitric oxide synthase gene expression by THI 52, a new synthetic naphthyl-benzylisoquinoline alkaloid

The effects of THI 52 (1-naphthylethyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline) on (a) inducible nitric oxide synthase (iNOS) and tumor necrosis factor-alpha (TNF-alpha) expression in RAW 264.7 cells stimulated by lipopolysaccharide (LPS)/interferon gamma (IFN-gamma), (b) plasma nitrate concentration as well as iNOS protein expression (lung) in vivo in LPS-treated rats, and (c) the restoration of vascular contractility to vasoconstrictor agents in LPS-treated vessels in vitro were investigated. THI 52 concentration-dependently reduced not only nitric oxide (NO) production (IC(50) value, 12.5 microM) but also the expression of TNF-alpha and iNOS mRNA in RAW 264.7 cells. Incubation of rat endothelium-denuded thoracic aorta with LPS (300 ng/mL) in vitro for 8 hr resulted in the suppression of vasoconstrictor effects to phenylephrine (PE), effects that were restored by co-incubation with THI 52. Administration of THI 52 (10 and 20mg/kg, i.p.) 30 min before injection of LPS (10mg/kg, i.p.) resulted in a significant reduction of the expression of iNOS protein in rat lung tissue and in the plasma nitrite/nitrate (NOx) level. Addition of THI 52-treated macrophage-conditioned medium to a TNF-sensitive L929 fibroblast cell line (CCL1) increased cell viability, depending on the concentration of THI 52. Finally, THI 52 inhibited the activation of nuclear factor kappaB (NF-kappaB) by inhibition of IkappaB degradation through the prevention of IkappaB phosphorylation. Collectively, these results strongly suggest that THI 52 suppresses both TNF-alpha and iNOS gene expression by inhibiting NF-kappaB. Thus, THI 52, a new synthetic isoquinoline alkaloid, may be beneficial in inflammatory disorders where the overproduction of NO and TNF-alpha is a matter of concern.     

Nicotine enhancement of lipopolysaccharide/interferon-gamma-induced cytotoxicity with elevating nitric oxide production

Nicotine has been shown to induce relaxation via nitric oxide (NO) production with activation of endothelium nitric oxide synthase (eNOS), however the effect of nicotine on lipopolysaccharide/interferon-gamma (LPS/IFN-gamma)-induced NO production and inducible NOS (iNOS) gene expression is still undefined. Here, nicotine alone did not affect the NO and PGE2 production in RAW264.7 and primary peritoneal macrophages. Interestingly, nicotine showed the dose-dependent stimulatory effect on LPS (20 ng/ml)/IFN-gamma (10 ng/ml)-induced NO but not PGE2 production in both cells. Although nicotine stimulates NO production in the presence of LPS/IFN-gamma, LPS at the dose of 20 ng/ml, nicotine showed no obvious inductive effect on the expression of iNOS protein by Western blotting in both cells. However, nicotine significantly stimulates LPS (2.5, 5 ng/ml)/IFN-gamma (10 ng/ml)-induced iNOS expression and NO production in RAW264.7 cells. Cytotoxicity assay showed that nicotine enhanced LPS (20 ng/ml) and IFN-gamma (10 ng/ml)-induced cytotoxicity, which was inhibited by an NOS inhibitor N-nitro-L-arginine (NLA) in RAW264.7 cells. Direct and indirect NOS activity assays indicated that nicotine did not affect NOS activity. And, iNOS protein stability was not changed by nicotine after LPS/IFN-gamma treatment. These data indicates that nicotine may potentiate LPS/IFN-gamma-induced cytotoxic effects by enhancing NO production; enhancing iNOS gene expression induced by LPS/IFN-gamma is involved. A cross-talk between inflammation and smoking was proposed in the present study.     

Inhibition by N-acetyl-5-hydroxytryptamine of nitric oxide synthase expression in cultured cells and in the anaesthetized rat.

1. Induction of the calcium-independent isoform of nitric oxide (NO) synthase (iNOS) in various cell types has been implicated in the circulatory failure in experimental models of septic shock. Tetrahydrobiopterin (BH4) appears to be an essential co-factor for NO formation and therefore an inhibition of its biosynthesis represents a feasible therapeutic target. We have investigated the effects of an inhibitor of BH4 synthesis, N-acetyl-5-hydroxytryptamine (N-acetylserotonin, NAS), on the expression of iNOS in cultured macrophages and smooth muscle cells in vitro, and on the hypotensive response to bacterial lipopolysaccharide (LPS) in the anaesthetized rat in vivo. 2. NAS (0.01-5 mM) caused a concentration-dependent inhibition of the accumulation of nitrite in the conditioned medium of LPS/interferon-gamma (IFN gamma)-stimulated RAW 264.7 macrophages and interleukin-1 beta (IL-1 beta)-activated vascular smooth muscle cells (VSMC). This effect was paralleled by a similar decrease in the iNOS protein content of these cells, as determined by immunoblot analysis. 3. Pretreatment of RAW 264.7 macrophages with the BH4 precursor, dihydrobiopterin (BH2, 0.1 mM) did not restore nitrite formation in the presence of NAS (1 mM). 4. Intravenous administration of NAS (1 mg kg-1 min-1 for 30 min) in anaesthetized rats significantly reduced the fall in mean arterial blood pressure, restored the pressor response to noradrenaline (1 micrograms kg-1), and ameliorated the increase in plasma nitrite following exposure to LPS (10 mg kg-1).(ABSTRACT TRUNCATED AT 250 WORDS)     

芦荟大黄素对LPS诱导的RAW264.7细胞NO生成及iNOS表达的影响

目的观察芦荟大黄素(aloe-emodin)对脂多糖(LPS)诱导的RAW264.7细胞一氧化氮(NO)生成及诱生型一氧化氮合酶(iNOS)mRNA表达的作用。方法采用LPS诱导的RAW264.7细胞株建立细胞炎症反应模型。采用Griess试剂法测定NO释放量;采用硝普钠释放NO法测定NO自由基含量的变化;采用反转录聚合酶链反应(RT-PCR)分析iNOS mRNA表达改变。结果芦荟大黄素在0.69～2.50mg·L-1剂量范围内可抑制LPS诱导的RAW264.7细胞NO的释放,并呈剂量和时间依赖关系;芦荟大黄素在0.63～5.00mg·L-1剂量范围内可下调LPS诱导的RAW264.7细胞iNOS mRNA含量;而此范围内芦荟大黄素无直接清除NO自由基作用,不影响iNOS活性。结论芦荟大黄素可明显降低LPS诱导的RAW264.7细胞NO释放,呈时间和剂量依赖关系,此作用并非通过捕捉NO或抑制iNOS活性来实现,而是通过抑制iNOS mRNA表达发挥作用的。     

In vitro inducible nitric oxide synthesis inhibitors from Alismatis Rhizoma.

Bioassay-guided fractionation of an aqueous extract of Alismatis Rhizoma has furnished two inducible nitric oxide synthase (iNOS) inhibitory compounds, alismol (1) and alisol B monoacetate (2), together with an inactive triterpene, alisol C monoacetate (3). Compounds 1 and 2 inhibited nitric oxide (NO) synthesis in a dose-dependent manner in murine macrophage-like RAW 264.7 cells stimulated with interferon-gamma (IFN-gamma) plus lipopolysaccharide (LPS). The inhibitory effects of 1 and 2 on NO synthesis were partly due to suppression of iNOS mRNA expression as determined by Northern blotting.     

Scoparone from artemisia capillaris inhibits the release of inflammatory mediators in RAW 264.7 cells upon stimulation cells by interferon-γ plus LPS

Scoparone is a major component of the shoot of Artemisia capillaris (Compositae), which has been used for the treatment of hepatitis and biliary tract infection in oriental countries. In the present study we observed that, scorparone exhibited no cytotoxic effect in unstimulated macrophages, but reduced the release of nitric oxide (NO) and prostaglandin E2 (PGE2) upon stimulation by IFN-gamma/LPS or LPS. The inhibitory effects were found to be in conjuction with the suppression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in IFN-gamma/LPS stimulated RAW 264.7 cells. Moreover, scoparone also attenuated the production of tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta and IL-6 in LPS-stimulated RAW264.7 cells. These results suggest that scoparone decreases the production of the inflammatory mediators such as NO and PGE2 in macrophages by inhibiting iNOS and COX-2 expression.     

Differential anti-inflammatory pathway by xanthohumol in IFN-gamma and LPS-activated macrophages

Macrophages are the main cells responsible for the innate immunity, and their activation by lipopolysaccharide (LPS) from Gram-negative bacteria or interferon (IFN)-gamma from host immune cells is important for controlling infections. However, the overwhelming activation of macrophages can cause a severe inflammatory state. This study investigated the inhibitory mechanism of xanthohumol (XN) against the inflammatory effectors (IL-1beta, TNF-alpha, and iNOS) in activated RAW264.7 macrophages by using different stimuli such as LPS, IFN-gamma, or LPS plus IFN-gamma. XN is a major prenylated chalcone found in hops, which is used to add bitterness and flavor to beer. XN reduced the expression of the LPS receptor components such as TLR4 and MD2 resulting in the suppression of NF-kappaB activation in LPS-activated RAW264.7 cells. In the IFN-gamma stimulated RAW264.7 cells, the binding activity of STAT-1alpha and IRF-1 was inhibited by XN. This suggests that differential signaling pathways are used by XN for the inhibition of excess inflammatory mediators depending on the stimuli in macrophages.     

Pharmacological profile of FR260330, a novel orally active inducible nitric oxide synthase inhibitor

In this study, we examined effects of a newly synthesized chemical compound, FR260330, (2E)-3-(4-chlorophenyl)-N-[(1S)-2-oxo-2-{[2-oxo-2-(4-{[6-(trifluoromethyl)-4-pyrimidinyl]oxy}-1-piperidinyl)ethyl]amino}-1-(2-pyridinylmethyl)ethyl]acrylamide on nitric oxide (NO) production in rat splenocytes and human colon cancer cell line, DLD-1 cells. FR260330 inhibited NOx production dose dependently in both cells. In lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) treated murine macrophage cell line, RAW264.7, Western blot analysis with gel filtration chromatography revealed FR260330 might prevent dimerization of inducible nitric oxide synthase (iNOS), but had no effect on the expression of iNOS protein. Furthermore, oral administration of FR260330 reduced NOx production dose dependently in plasma from rats exposed to LPS (IC50=1.6 mg/kg). Meanwhile, higher dose (100 mg/kg) of oral administration of FR260330 did not change mean arterial blood pressure in rats. These results suggest that FR260330 might be a useful therapeutical approach to various inflammatory diseases, in which superoxide or peroxynitrite formed from iNOS-derived NO are involved.     

Dexamethasone inhibits inducible nitric-oxide synthase expression and nitric oxide production by destabilizing mRNA in lipopolysaccharide-treated macrophages

Nitric oxide (NO) production through the inducible nitric-oxide synthase (iNOS) pathway is increased in inflammatory diseases and leads to cellular injury. Anti-inflammatory steroids inhibit the expression of various inflammatory genes, including iNOS. In the present study, we investigated the mechanism how dexamethasone decreased NO production in murine J774 macrophages. Dexamethasone (0.1-10 microM) inhibited the production of NO and iNOS protein in a dose-dependent manner in cells stimulated with lipopolysaccharides (LPS). In contrast, in cells treated with a combination of LPS and interferon-gamma (IFN-gamma), dexamethasone did not reduce iNOS expression and NO formation. Dissociated glucocorticoid RU24858 inhibited iNOS expression and NO production to levels comparable with that of dexamethasone, suggesting that the reduced iNOS expression by dexamethasone is not a GRE-mediated event. In further studies, the effect of dexamethasone on iNOS mRNA levels was tested by actinomycin assay. The half-life of iNOS mRNA after LPS treatment was 5 h 40 min, and dexamethasone reduced it to 3 h. The increased degradation of iNOS mRNA was reversed by a protein synthesis inhibitor cycloheximide. iNOS mRNA was more stabile in cells treated with a combination of LPS plus IFN-gamma (half-life = 8 h 20 min), and dexamethasone had a minor effect in these conditions. In conclusion, dexamethasone decreases iNOS-dependent NO production by destabilizing iNOS mRNA in LPS-treated cells by a mechanism that requires de novo protein synthesis. Also, decreased iNOS mRNA and protein expression and NO formation by dexamethasone was not found in cells treated with a combination of LPS plus IFN-gamma, suggesting that the effect of dexamethasone is stimulus-dependent.     

Scopoletin: an inducible nitric oxide synthesis inhibitory active constituent from Artemisia feddei.

Bioassay-guided fractionation of an H2O extract of Artemisia feddei has furnished an inducible nitric oxide synthase (iNOS) inhibitory coumarin, scopoletin (1) and one of the inactive sesquiterpenes, achillin (2). Compound 1 showed inhibition of nitric oxide (NO) synthesis in a dose-dependent manner in murine macrophage-like RAW 264.7 cells stimulated with interferon-gamma (IFN-gamma) plus lipopolysaccharide (LPS). The inhibition of NO synthesis of 1 was due to suppression of iNOS mRNA and iNOS protein, as determined by Northern and Western blotting, respectively.     

Diarylheptanoids with in vitro inducible nitric oxide synthesis inhibitory activity from Alnus hirsuta

Four diarylheptanoids were isolated from the leaf of Alnus hirsuta (Betulaceae) and have been assessed for nitric oxide (NO) production inhibitory effects in vitro. Oregonin (1) and hirsutanonol (2) were found to be potent inducible nitric oxide synthase (iNOS) inhibitors. Compounds 1 and 2 showed inhibition of NO synthesis in dose-dependent manners by murine macrophage-like RAW 264.7 cells stimulated with interferon-gamma (IFN-gamma) plus lipopolysaccharide (LPS). Their 50% inhibitory concentrations (IC50) were 3.8 and 14.3 microM, respectively. The inhibitory effects of these compounds on NO synthesis were due to suppression of iNOS mRNA expression as determined by Northern blotting.     

Enantiomers of higenamine inhibit LPS-induced iNOS in a macrophage cell line and improve the survival of mice with experimental endotoxemia

The importance of development of single enantiomers (optically pure isomers) of chiral molecules has been recognized and manifested in countless pharmaceutical and biological advancement. (RS)-(+/-)-Higenamine (racemic mixture), an active ingredient of Aconite tuber, has been shown to have antioxidant activity along with inhibitory action of iNOS expression in various cells. In the present study, the effects of each enantiomer of higenamine [(S)-(-)-higenamine and (R)-(+)-higenamine] were investigated in comparison with the effects of racemic mixture [(RS)-(+/-)-higenamine] on iNOS expression and NO production in RAW 264.7 cells activated with LPS. In addition, the effects of higenamine enantiomers on the survival rates were also investigated using mice, in which each test compound was injected (i.p.) 90 min prior to LPS. All three forms of higenamine inhibited iNOS expression and reduced NO production with IC50 of 26.2, 86.3, and 53.4 microM, for (S)-, (R)-, and (RS)-higenamine, respectively. (S)-higenamine also significantly reduced serum NOx level and increased survival rates in LPS-treated mice. In contrast, (R)-isomer only showed tendency to increase the survival rates which was not statistically significant when compared to LPS-treated controls. Taken together, it was concluded that (S)-higenamine may be more beneficial than (R)-enantiomer in diseases associated with iNOS over-expression, such as septic shock.     

STAT5通路抑制剂对脂多糖诱导RAW264.7细胞的保护作用研究

目的观察信号传导及转录激活因子(STAT)5通路抑制剂对经脂多糖(LPS)诱导的小鼠巨噬细胞RAW264.7细胞株一氧化氮(NO)和诱导型一氧化氮合成酶(iNOS)表达的影响。方法将处于对数期的RAW264.7细胞分为空白对照组、STAT5通路抑制剂对照组、LPS诱导组和STAT5通路抑制剂低、中、高浓度组,实时定量聚合酶链式反应(RT-PCR)法测定iNOS mRNA的表达量,Western blot测定iNOS以及磷酸化STAT5(p-STAT5)的蛋白表达量。结果RAW264.7细胞培养24 h后,空白对照组、STAT5通路抑制剂对照组、LPS诱导组以及STAT5通路抑制剂低、中、高浓度组细胞中NO的含量差异有统计学意义(F=25.69,P<0.05);低、中、高浓度STAT5通路抑制剂对RAW264.7细胞释放NO的抑制率差异有统计学意义(F=132.49,P<0.05)。空白对照组、STAT5通路抑制剂对照组、LPS诱导组和STAT5通路抑制剂低、中、高浓度组细胞iNOS mRNA以及蛋白质的表达量比较差异有统计学意义(F=123.59、23.37,P<0.05)。空白对照组、STAT5通路抑制剂对照组、LPS诱导组和STAT5通路抑制剂低、中、高浓度组细胞p-STAT5/STAT5表达比较差异有统计学意义(F=12.07,P<0.05)。结论STAT5通路抑制剂能够抑制RAW264.7细胞iNOS的表达以及NO的产生,其机制可能与抑制STAT5磷酸化的表达有关。     

A limited series of synthetic tetrahydroisoquinoline alkaloids reduce inflammatory gene iNOS via inhibition of p-STAT-1 and suppress HMGB1 secretion in LPS-treated mice lung tissue

Tetrahydroisoquinoline alkaloids (THIs) have shown to increase survival and beneficial effect on animal model of sepsis, partly due to heme oxygenase-1 (HO-1) induction. Here, we aimed to compare a limited series of synthesized THIs on HO-1 induction and inhibitory effect of iNOS and COX-2 expression in lipopolysaccharide (LPS)-activated RAW264.7 cells. To the end, most promising compound (THI-61) was tested whether this compound reduces iNOS protein expression and inflammatory markers (HMGB1, TNF-α) in LPS-treated mice lung tissue. The results indicated that N-carbonyl substituted THI seem to affect HO-1 induction depending on which functional group is attached to C1 position. All compounds that reduce LPS-activated NF-κB-luciferase activity showed to preferential inhibition of iNOS/NO but not COX-2/PGE₂ that was partly related to inhibition of STAT-1 phosphorylation. In particular, THI-61 induced translocation of Nrf2 from cytosol into the nucleus by an increased Nrf2-ARE binding activity, and reduced IL-1β production in LPS-activated RAW264.7 cells. The reduced expression of iNOS/NO by THI-61 was reversed by siHO-1RNA-transfection. In LPS-treated mice, THI-61 significantly reduced iNOS protein in lung tissues, and HMGB1 and TNF-α levels in the BALF. We concluded that 1) lipophilic moiety of 1C substituent is much more important in N-carbonyl substituted THI for induction of HO-1, 2) newly synthesized THI-61 may be beneficial for treatment of lung injury.     

In vitro inducible nitric oxide synthesis inhibitory active constituents from Fraxinus rhynchophylla

Bioassay-guided fractionation of an H2O extract of the barks of Fraxinus rhynchophylla has furnished two inducible nitric oxide synthase (iNOS) inhibitory compounds, ferulaldehyde (1) and scopoletin (3) together with a coumarin, fraxidin (2). Compounds 1 and 3 showed inhibition of nitric oxide (NO) synthesis in a dose-dependent manner by murine macrophage-like RAW 264.7 cells stimulated with interferon-gamma (IFN-gamma) plus lipopolysaccharide (LPS). The inhibition of NO synthesis of 1 was reflected in the decreased amount of iNOS protein, as determined by Western blotting.     

Suppression by a sesquiterpene lactone from Carpesium divaricatum of inducible nitric oxide synthase by inhibiting nuclear factor-kappaB activation

Excessive nitric oxide (NO) produced by inducible NO synthase (iNOS) acts as a causative regulator in various inflammatory disease states. Carpesium divaricatum has been used in Korean traditional herbal medicine for its antipyretic, analgesic, vermifugic, and anti-inflammatory properties. We investigated the molecular mechanism for the suppression of lipopolysaccharide/interferon-gamma (LPS/IFN-gamma)-induced NO production in RAW 264.7 macrophages by the sesquiterpene lactone 2beta,5-epoxy-5,10-dihydroxy-6alpha-angeloyloxy-9beta-isobutyloxy-germacran-8alpha,12-olide (C-1), which has been identified recently as a new compound from C. divaricatum. C-1 decreased NO production in LPS/IFN-gamma-stimulated RAW 264.7 cells in a concentration-dependent manner, with an IC50 of approximately 2.16 microM; however, it had no direct effect on the iNOS activity of fully LPS/IFN-gamma-stimulated RAW 264.7 cells. Furthermore, treatment with C-1 led to a decrease in iNOS protein and mRNA. These effects appear to be due to inhibition of nuclear factor-kappaB (NF-kappaB) activation through a mechanism involving stabilization of the NF-kappaB/inhibitor of the kappaB (I-kappaB) complex, since inhibition of NF-kappaB DNA binding activity by C-1 was accompanied by a parallel reduction of nuclear translocation of subunit p65 of NF-kappaB and I-kappaBalpha degradation. Taken together, the results suggest that the ability of C-1 to inhibit iNOS gene expression may be responsible, in part, for its anti-inflammatory effects.