Inhibition Of iNOS Expression Research Articles

Palmitoyl Ethanol Amide in Prophylaxis and Treatment of Viral Infections

1. Abstract There is currently a pressing need to develop new therapeutic alternatives to the annual influenza vaccines and the existing antiviral agents such as oseltamivir and zanamivir. Palmitoyl Ethanol Amide (PEA), an endogenous anti-inflammatory compound and PPAR alpha and gamma agonist, available as food supplement, might be such an innovation. PEA has been tested in a variety of animal models and reduced mortality after inoculation with various microorganisms. PEA may also modulate ‘cytokine storm’, and reduces the secretion of pro-inflammatory proteins such as NGF, CXCL1, IL-1β, IL-6 and TNF-alpha. PEA inhibits iNOS expression and nuclear NF-κ B translocation. PEA further inhibits overactive mast cells, which play a role in the pathogenesis of the ‘cytokine storm’. Furthermore, PEA has been clinically evaluated in 6 randomized double blind placebo controlled trials in over 3000 patients and was found to be effective and safe in the prophylaxis and treatment of influenza and respiratory tract infections. We will review data supporting PEA’s role as an adjunct to antiviral treatment and discuss some of its supposed mechanism of action. 2. Keywords: Cytokine Storm; Flu; Infection; Inflammation; Mast Cells; PPAR

Inhibition of inducible nitric oxide synthase (iNOS) by simvastatin attenuates cardiac hypertrophy in rats.

The left ventricular hypertrophy (LVH) occurs in response to the haemodynamic overload in some physiological and pathological conditions. This study was designed to investigate the possible cardioprotective effect of simvastatin (SIM) treatment against isoproterenol (ISO)-induced LVH and the probable underlying mechanism in adult male Wistar rats. Animals were allocated into four groups. Rats of control group received normal saline orally for 30 days and intraperitoneally for the last 7 days. Rats of SIM group received SIM orally (10 mg/kg/day in saline) for 30 days. Rats of ISO group received normal saline orally for 30 days and ISO intraperitoneally (5 mg/kg) for the last 7 days to induce LVH. Rats of ISO/SIM group received SIM for 30 days and ISO intraperitoneally for the last 7 days. At the end of the experiment, all animals were sacrificed by cervical decapitation under anaesthesia. Truncal blood was collected and serum was separated and used for biochemical assay. The heart was dissected and processed for histological and immunohistochemical studies. The results of the present study confirmed the ISO-induced myocardial lesions including significant increase of heart weight (HW), heart weight/body weight (HW/BW) ratio, LVH, interstitial myocardial fibrosis (increased collagen types I and III), inflammatory cellular infiltration, necrosis of cardiomyocytes, and increased expression of inducible nitric oxide synthase (iNOS) and thioredoxin in cardiomyocytes. These changes were accompanied by significant increase in serum levels of troponin-T, creatine phosphokinase-MB (CPK-MB), tumour necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6). Co-administration of SIM to ISO-injected rats significantly reduced all these cardiac changes and serum biochemical markers in addition to marked depletion of iNOS and thioredoxin expression in cardiomyocytes. It is concluded that SIM co-administration attenuated ISO-induced cardiac lesions including LVH by inhibiting iNOS expression in cardiomyocytes.

Synthesis and biological evaluation of chalcone, dihydrochalcone, and 1,3-diarylpropane analogs as anti-inflammatory agents

Twenty-one chalcones were prepared via aldol condensation and subsequent reduction of these compound led to the corresponding dihydrochalcone and 1,3-diphenylpropane derivatives. The synthetic products were examined for their effects on NO inhibition in LPS-activated mouse peritoneal macrophages. Among the tested compounds, a 1,3-diarylpropane analog, 2-(3-(3,4-dimethoxyphenyl)propyl)-5-methoxyphenol (3p), displayed the most significant inhibitory effects against NO production. To investigate the mechanism of action, the effects of 3p on iNOS and COX-2 protein expression were studied by immunoblot. The results concluded that 3p is capable of inhibiting iNOS expression in LPS-induced RAW264.7 cells via attenuation of NF-κB signaling by ERK, p38, and JNK.

Inducible and neuronal nitric oxide synthases exert contrasting effects during rat intestinal recovery following fasting.

We investigated the effects of endogenous inducible (iNOS) and neuronal nitric oxide synthase on recovery from intestinal mucosal atrophy caused by fasting-induced apoptosis and decreased cell proliferation during refeeding in rats. Rats were divided into five groups, one of which was fed ad libitum, and four of which underwent 72 h of fasting, followed by refeeding for 0, 6, 24, and 48 h, respectively. iNOS and neuronal nitric oxide synthase mRNA and protein levels in jejunal tissues were measured, and mucosal height was histologically evaluated. Apoptotic indices, interferon-γ (IFN-γ) transcription levels, nitrite levels (as a measure of nitric oxide [NO] production),8-hydroxydeoxyguanosine formation (indicating reactive oxygen species [ROS] levels), crypt cell proliferation, and the motility indices (MI) were also estimated. Associations between mucosal height and NOS protein levels were determined using Spearman's rank correlation test. Notably, we observed significant increases in mucosal height and in neuronal nitric oxide synthase mRNA and protein expression as refeeding time increased. Indeed, there was a significant positive correlation between neuronal nitric oxide synthase protein level and mucosal height during the 48-h refeeding period ( r = 0.725, P < 0.01). Conversely, iNOS mRNA and protein expression decreased according to refeeding time, with a significant negative correlation between iNOS protein level and mucosal height being recorded during the 48-h refeeding period ( r = -0.898, P < 0.01). We also noted a significant negative correlation between jejunal neuronal nitric oxide synthase and iNOS protein concentrations over this same period ( r = -0.734, P < 0.01). Refeeding also restored the decreased jejunal MI caused by fasting. Our finding suggests that refeeding likely repairs fasting-induced jejunal atrophy by suppressing iNOS expression and subsequently inhibiting NO, ROS, and IFN-γ as apoptosis mediators, and by promoting neuronal nitric oxide synthase production and inducing crypt cell proliferation via mechanical stimulation. Impact statement Besides providing new data confirming the involvement of iNOS and nNOS in intestinal mucosal atrophy caused by fasting, this study details their expression and function during recovery from this condition following refeeding. We demonstrate a significant negative correlation between iNOS and nNOS levels during refeeding, and associate this with cell proliferation and apoptosis in crypts and villi. These novel findings elucidate the relationship between these NOS isoforms and its impact on recovery from intestinal injury. A mechanism is proposed comprising the up-regulation of nNOS activity by mechanical stimulation due to the presence of food in the intestine, restricting iNOS-associated apoptosis and promoting cell proliferation and gut motility. Our investigation sheds light on the molecular basis behind the repercussions of total parenteral nutrition on intestinal mucosal integrity, and more importantly, the beneficial effects of early enteral feeding.

Therapeutic Benefits of Mesenchymal Stromal Cells in a Rat Model of Hemoglobin-Induced Hypertensive Intracerebral Hemorrhage.

Previous studies have shown that bone marrow mesenchymal stromal cell (MSC) transplantation significantly improves the recovery of neurological function in a rat model of intracerebral hemorrhage. Potential repair mechanisms involve anti-inflammation, anti-apoptosis and angiogenesis. However, few studies have focused on the effects of MSCs on inducible nitric oxide synthase (iNOS) expression and subsequent peroxynitrite formation after hypertensive intracerebral hemorrhage (HICH). In this study, MSCs were transplanted intracerebrally into rats 6 hours after HICH. The modified neurological severity score and the modified limb placing test were used to measure behavioral outcomes. Blood–brain barrier disruption and neuronal loss were measured by zonula occludens-1 (ZO-1) and neuronal nucleus (NeuN) expression, respectively. Concomitant edema formation was evaluated by H&E staining and brain water content. The effect of MSCs treatment on neuroinflammation was analyzed by immunohistochemical analysis or polymerase chain reaction of CD68, Iba1, iNOS expression and subsequent peroxynitrite formation, and by an enzyme-linked immunosorbent assay of pro-inflammatory factors (IL-1β and TNF-α). The MSCs-treated HICH group showed better performance on behavioral scores and lower brain water content compared to controls. Moreover, the MSC injection increased NeuN and ZO-1 expression measured by immunochemistry/immunofluorescence. Furthermore, MSCs reduced not only levels of CD68, Iba1 and pro-inflammatory factors, but it also inhibited iNOS expression and peroxynitrite formation in perihematomal regions. The results suggest that intracerebral administration of MSCs accelerates neurological function recovery in HICH rats. This may result from the ability of MSCs to suppress inflammation, at least in part, by inhibiting iNOS expression and subsequent peroxynitrite formation.

Evaluation of Antiinflammatory, Antioxidant and Antiproliferative Activities of Quassia borneensis Noot. (Simaroubaceae) Extracts

Quassia borneensis has been traditionally used as antihypertensive agent without any scientific literature on its mechanism of action. The objective of this study was to evaluate the antiinflammatory, antioxidant and antiproliferation properties of Q. borneensis extracts. The hexane, chloroform and aqueous extracts of root and bark of Q. borneensis were subjected to nitric oxide (NO) inhibition assay in LPS-stimulated RAW 264.7 cells. Expression of inducible NO synthase (iNOS) protein level was analyzed by Western blot. The antioxidant and antiproliferative activities of the extracts on HL-60 cells were determined using Ferric Reducing Antioxidant Power (FRAP) and MTT assays, respectively. The chloroform extract of Q. borneensis root obtained by soxhlet method (CSR) significantly inhibited 97.64 ± 0.96% of NO production (p < 0.001) and suppressed iNOS expression (p < 0.05) at the highest concentration of 1.0 μg/ml. The chloroform extract of bark obtained by maceration (CMB) exhibited the highest antioxidant capacity in the absence and presence of HL-60 cells, where the FRAP value were 125.45 ± 9.10 μM FeSO4.7H2O and 181.55 ± 3.45 μM FeSO4.7H2O, respectively. The greatest inhibition of HL-60 cell proliferation was exhibited by the chloroform extract of bark obtained by soxhlet method (CSB) with the IC50 of 5.0 μg/ml. The findings suggested that the chloroform extracts of Q. borneensis possess antiinflammatory, antioxidant and antiproliferative activities. DOI : http://dx.doi.org/10.17576/JSKM-2017-1501-05

Anti-inflammatory Effect of Etlingera pavieana (Pierre ex Gagnep.) R.M.Sm. Rhizomal Extract and Its Phenolic Compounds in Lipopolysaccharide-Stimulated Macrophages.

Background:In our continuing search for anti-inflammatory agents from Thai herbs, Etlingera pavieana (Pierre ex Gagnep.) R.M.Sm. showed potent inhibition on nitric oxide (NO) production in lipopolysaccharide (LPS)-induced macrophages. However, the mechanism behind its inhibitory effect has not been yet explored, and little is known regarding its bioactive compounds responsible for the anti-inflammatory effect.Objective:In the present study, anti-inflammatory effect of hexane, ethyl acetate, and water fractions of rhizomal ethanol extracts of E. pavieana was evaluated for their inhibition on NO production and mechanism in LPS-stimulated macrophages. Active compounds responsible for such anti-inflammatory activity were identified.Materials and Methods:Inhibitory activities on NO production were performed in LPS-stimulated RAW264.7 macrophage. Cytotoxicity of plant extracts was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, mRNA and protein expressions by reverse transcription-polymerase chain reaction and Western blotting analysis, respectively. Anti-inflammatory compounds were isolated by activity-guided isolation technique using column chromatography.Results:Ethyl acetate fraction of E. pavieana (EPE) showed the most potent inhibitory effect on NO production in macrophages. EPE significantly decreased NO production and inhibited inducible nitric oxide synthase (iNOS) protein and mRNA expression in a dose-dependent manner. Furthermore, the level of nuclear factor-kappa B p65 subunit was markedly reduced in activated cells treated with EPE. Four phenolic compounds, 4-methoxycinnamyl alcohol (1), trans-4-methoxycinnamaldehyde (2), 4-methoxycinnamyl p-coumarate (3), and p-coumaric acid (4), were obtained from bioactivity-guided isolation technique.Conclusions:The anti-inflammatory property contained in E. pavieana rhizome extract and conferred through inhibition of iNOS expression, and NO formation provides scientific evidence and support for the development of new anti-inflammatory agents based on extracts from this plant.SUMMARY Ethyl acetate fraction (EPE) of Etlingera pavieana showed the most potent inhibitory effect on NO production in LPS-induced macrophagesFour phenolic compounds, 4-methoxycinnamyl alcohol (1), trans-4-methoxycinnamaldehyde (2), 4-methoxycinnamyl p-coumarate (3) and p-coumaric acid (4), responsible for the anti-inflammatory effect of EPE were isolated. Abbreviations used: EPE: Ethyl acetate fraction of Etlingera pavieana; EPH: Hexane fraction of Etlingera pavieana; EPW: Water fraction of Etlingera pavieana; NO: Nitric oxide (NO); LPS: Lipopolysaccharide; iNOS: Inducible nitric oxide synthase (iNOS); MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NF-κB: Nuclear factor-kappa B; DMSO: Dimethyl sulfoxide; EtOAc: Ethylacetate; MeOH: Methanol; AG: Aminoguanidine; DCM: Dichloromethane; MCA: 4-methoxycinnamyl alcohol; MCD: trans-4-methoxycinnamaldehyde; MCC: 4-methoxycinnamyl p-coumarate; CM: p-coumaric acid.

Synthesis of Clovamide Analogues That Inhibit NO Production in Activated BV-2 Microglial Cells.

A series of methyl ester of clovamide analogues, where the hydroxyl group of catechol moiety in caffeic acid and L-3,4-dihydroxyphenylalanine (L-dopa) was replaced with various functional groups, were synthesized and their inhibitory effects on nitric oxide (NO) production and inducible NO synthase (iNOS) expression in lipopolysaccharide (LPS)-induced BV2 cells were tested. Among the synthesized compounds, 3,5-ditrifluoromethyl analogue 9l (IC50=2.8 µM) exhibited a potency about 26.3 times greater than that of the parent compound 9a (IC50=73.6 µM) and suppressed NO production dose-dependently without cytotoxicity. Compound 9l also inhibited iNOS expression in LPS-induced BV2 cells at 2.5, 5 and 10 µM concentrations. These results suggested that the dihydroxyl group of catechol moiety in caffeic acid unit is not essential for the suppression of NO production and that 9l has potential as a potent inhibitor of NO production.

Anti-inflammatory and burn injury wound healing properties of the shell of Haliotis diversicolor.

BackgroundThe shell of Haliotis diversicolor, or shijueming (SJM), is a type of traditional Chinese medicine. The SJM has appeared in historical records as early as the third and fourth centuries. Historical records have revealed that SJM had mainly been used to treat eye diseases. After the Qing Dynasty (1757), records had emerged, detailing the use of SJM for treating skin injuries, particularly for treating poorly managed ulcers or traumatic wounds. Furthermore, in our anti-inflammation-screening system, SJM significantly inhibited the expression of pro-inflammatory proteins. Previous studies have yet to adopt an animal model to verify the phenomenon and described in the historical records regarding the efficacy of SJM in promoting wound healing. Besides, the mechanism of wound healing effect of SJM is also not clear.MethodsThis study applied in vitro and in vivo models, tissue section analysis, and western blotting to evaluate the effect of SJM on wound healing. The RAW 264.7 cells were used in anti-inflammatory activity assay and phagocytic assay. Male Wistar rats were used to evaluate the effect of SJM on burn injury healing. A copper block (2 × 2 cm, 150 g) preheated to 165 °C in a dry bath was used to contact the skin area for 10 s, thus creating a full-thickness burn injury. The results were analyzed by hematoxylin and eosin staining, picrosirius red staining and Western blotting.ResultsThe results revealed that in the in vitro model, the presence of SJM decreased the inducible nitric oxide synthase (iNOS) expression and enhanced the functions of macrophages. The results of the rat burn injury model revealed that SJM decreased neutrophil infiltration, promoted wound healing, thus increasing the collagen I content and promoting the expression of transforming growth factor-beta 1 (TGF-β1) protein. We speculate that the effect and mechanism of SJM on promoting wound healing is related to macrophage activation. In the inflammation phase, SJM alleviates inflammation by inhibiting iNOS expression and removing neutrophils through phagocytosis. Furthermore, SJM induces the secretion of TGF-β1, converting collagen during the tissue remodeling phase.ConclusionsAccording to our review of relevant literature, this is the first study that applied an evidence-based method to verify that SJM alleviates inflammation, enhances phagocytosis, and triggers wound healing after burn injury. The study findings reveal that SJM provides a promising therapeutic option for treating burn injury.

1400 W ameliorates acute hypobaric hypoxia/reoxygenation-induced cognitive deficits by suppressing the induction of inducible nitric oxide synthase in rat cerebral cortex microglia

Nitric oxide (NO) is involved in neuronal modifications, and overproduction of NO contributes to memory deficits after acute hypobaric hypoxia-reoxygenation. This study investigated the ability of the iNOS inhibitor 1400W to counteract spatial memory deficits following acute hypobaric hypoxia-reoxygenation, and to affect expression of NOS, NO, 3-NT and MDA production, and apoptosis in rat cerebral cortex. We also used primary rat microglia to investigate the effect of 1400W on expression of NOS, NO, 3-NT and MDA production, and apoptosis. Acute hypobaric hypoxia-reoxygenation impaired spatial memory, and was accompanied by activated microglia, increased iNOS expression, NO, 3-NT and MDA production, and neuronal cell apoptosis in rat cerebral cortex one day post-reoxygenation. 1400W treatment inhibited iNOS expression without affecting nNOS or eNOS. 1400W also reduced NO, 3-NT and MDA production, and prevented neuronal cell apoptosis in cerebral cortex, in addition to reversing spatial memory impairment after acute hypobaric hypoxia-reoxygenation. Hypoxia-reoxygenation activated primary microglia, and increased iNOS and nNOS expression, NO, 3-NT, and MDA production, and apoptosis. Treatment with 1400W inhibited iNOS expression without affecting nNOS, reduced NO, 3-NT and MDA production, and prevented apoptosis in primary microglia. Based on the above findings, we concluded that the highly selective iNOS inhibitor 1400W inhibited iNOS induction in microglial cells, and reduced generation of NO, thereby mitigating oxidative stress and neuronal cell apoptosis in the rat cerebral cortex, and improving the spatial memory dysfunction caused by acute hypobaric hypoxia-reoxygenation.

Flavonoids and biphenylneolignans with anti-inflammatory activity from the stems of Millettia griffithii

Five new flavonoids, griffinones A–E (1–5), a new biphenylneolignan, griffilignan A (6) and ten known compounds were isolated from the n-hexane and EtOAc extracts of Millettia griffithii. The structures of the new compounds were determined by 1D and 2D NMR, and by HRMS. The anti-inflammatory activity of the isolated compounds was evaluated on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW 264.7 cells. Among the isolated compounds, compounds 1, 2 and 14 showed significant anti-inflammatory activity with IC50 values of 20.4, 2.1 and 35.7μM, respectively and no obvious toxicities were observed at 100μM. Western blot and PCR assay further showed that inhibition of nitric oxide production by compound 2 was associated with suppression of iNOS expression. Modeling studies suggested that the amino group, phenyl ring as well as the isopentenyl tails of compound 2 could help bind to iNOs.

Anti-Inflammatory Effects of Melandrii Herba Ethanol Extract via Inhibition of NF-κB and MAPK Signaling Pathways and Induction of HO-1 in RAW 264.7 Cells and Mouse Primary Macrophages.

Melandrii Herba (MH) is a traditional Asian medicinal herb used to treat breast cancer, anuria, and diseases of lactation. However, its biological properties and molecular mechanisms have not been fully elucidated. The purpose of this study was to investigate the anti-inflammatory activity and underlying molecular mechanism of MH ethanol extract (MHE) on the lipopolysaccharide (LPS)-mediated inflammatory response in macrophages. MHE cytotoxicity was determined using a cell counting kit (CCK) assay. The effects of MHE on the production of NO, inflammatory cytokines, and related proteins and mRNAs were determined using the Griess test, ELISA, Western blotting, and real-time RT-PCR, respectively. In addition, intracellular signaling pathways, such as NF-κB, MAPK, and HO-1, were analyzed using Western blotting. Our results revealed that MHE treatment significantly inhibited the secretion of NO and inflammatory cytokines, including TNF-α, IL-6, and IL-1β in macrophages, at sub-cytotoxic concentrations. Furthermore, MHE treatment inhibited iNOS expression and induced HO-1 expression. Finally, the transcriptional activities of NF-κB and MAPK activation were significantly suppressed by MHE in LPS-stimulated macrophages. The results indicate that MHE exerts anti-inflammatory effects by suppressing inflammatory mediator production via NF-κB and MAPK signaling pathways inhibition and induction of HO-1 expression in macrophages. Therefore, our results suggest the potential value of MHE as an inflammatory therapeutic agent developed from a natural substance.

Gastroprotective effect of bezafibrate, a peroxisome proliferator activated receptor α agonist and its mechanism in a rat model of aspirin-induced gastric ulcer

SummaryBackgroundThe aim of the present study was to demonstrate the antiulcer activity and mechanism of bezafibrate in a rat model of aspirin‐induced gastric ulcer.MethodsWe used an aspirin‐induced gastric ulcer model. Bezafibrate was administered orally in graded doses (10 mg/kg, 25 mg/kg, 50 mg/kg, 100 mg/kg, and 200 mg/kg) to detect the best effective antiulcer dose of bezafibrate. The parameters measured were: ulcer index, histopathological scoring of gastric ulcer, gastric juice analysis, gastric mucosal lipid peroxidation parameters, estimation of NO metabolite in blood, mRNA expression of inducible NO synthase iNOS and constitutive NO synthase (cNOS) in gastric mucosa, and gastric mucosal DNA fragmentation.ResultsThe dose‐dependent antiulcer activity of bezafibrate was shown by the ulcer index and histopathological score. Bezafibrate (100 mg/kg) significantly reduced total acidity, free acidity, and pepsin activity, and increased total hexoses and total proteins. Bezafibrate (100 mg/kg) also significantly reduced lipid peroxidation, inhibited iNOS expression, preserved cNOS expression, and inhibited DNA fragmentation.ConclusionBezafibrate can decrease aspirin‐induced gastric mucosal injury via reducing lipid peroxidation, inhibiting iNOS expression, preserving cNOS expression, and decreasing DNA fragmentation.

Inducible Nitric Oxide Synthase Expression in Liver Injury: Liver Protective Effects on Primary Rat Hepatocytes.

Following injury to the liver, liver cells, including Kupffer cells and hepatocytes express inducible nitric oxide synthase (iNOS), followed by the production of excess levels of nitric oxide (NO). NO produced by iNOS has been found to contribute to liver injury. Treatment of primary cultures of rat hepatocytes with the proinflammatory cytokine interleukin (IL)-1β stimulated iNOS expression and NO production. Experiments with this in vitro hepatocyte model of liver injury and with in vivo animal models of liver injury have demonstrated that drugs showing a liver-protective effect in vivo also inhibited the induction of iNOS expression and NO production both in vivo and in vitro. Thus, in this in vitro hepatocyte model, the prevention of iNOS expression and NO production are considered indicators of liver protection. This review describes a simple in vitro liver injury model, consisting of IL-1β-stimulated cultured hepatocytes, and methods used to analyze the mechanisms of action of drugs that inhibit iNOS expression. This in vitro hepatocyte model may be used to assess the liver-protective effects of pharmaceutical agents, herbal medicines, and certain types of foods.

Steroidal glycosides from the roots of Cynanchum stauntonii and their effects on the expression of iNOS and COX-2

Six new steroidal glycosides, named stauntosides O-T (1–6), along with eight known compounds (7–14), were obtained from the 95% aqueous ethanol extract of the roots of Cynanchum stauntonii. Their chemical structures were elucidated by IR, HR ESI-MS/MS, 1H- and 13C NMR, 1H-1H COSY, HSQC, HSQC-TOCSY, and HMBC spectroscopic analyses, which showed interesting 13,14:14,15-disecopregnane-type or 14,15-secopregnane-type C21 steroidal glycosides. The glycosides’ anti-inflammatory effects were investigated by detecting the inhibitory effects of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) on RAW246.7 murine macrophage cells stimulated by lipopolysaccharide (LPS). Our results showed that compounds 1, 5, 8, 9, 11, and 13 could significantly inhibit iNOS expression, and compounds 5 and 7 could clearly reduce COX-2 expression in RAW246.7 cells stimulated by LPS compared to cells stimulated with LPS and not treated with other compounds. Thus, compounds 1, 5, 7–9, 11, and 13 have the potential to mediate anti-inflammatory effects, with compound 5 having a greater anti-inflammatory effect than the other compounds.

P746. Anti-inflammatory properties of Faecalibacterium prausnitzii in the absence and presence of cigarette smoke using a novel epithelium-gut microbe coculture system

Background: Inflammatory bowel disease (IBD) is characterised bya recurrent inflammation of the gut epithelium. Although the pathogenesis is still largely unknown, different factors contribute to the disease, including genetic and environmental factors. Cigarette smoking is the most-studied environmental factor associated with IBD. Moreover, gut microbiota play a central role in the pathogenes is ofIBD. In this study we aimed to investigate the effects of host-microbe interactions on the gut epithelium with a potentially beneficial bacterium,Faecalibacterium prausnitzii, in a recently established coculture system for anaerobic bacteria and oxygen-requiring humanintestinal epithelial (Caco-2) cells. Hence, we analysed the effect of an inflammatory cytokine (TNF-α) and cigarette smoke extract(CSE) on the growth of F. prausnitzii and expression of inflammatory markers in Caco-2 cells. Methods: F. prausnitzii and human Caco-2 cells were cocultured in the ‘Human oxygen Bacteria anaerobic’ (HoxBan) system and exposed to TNF-α and/or CSE. Bacterial growth was monitored visually, and expression of the inflammatory marker iNOS and oxidative stress marker HO-1 were determined by quantitative PCR. Results: F. prausnitzii grew well in the HoxBan coculturing system in the presence of TNF-α or with added CSE, with no observable difference to control conditions. F. prausnitzii strongly suppressed iNOS expression, both in TNF-α treated and control Caco-2 cells.CSE did not induce iNOS or HO-1 expression in Caco-2 cells,but F. prausnitzii strongly suppressed iNOS expression both in the absence and presence of CSE, with similar trends observed for HO-1. Conclusions: We conclude that F. prausnitzii produces potent antiinflammatoryfactors, which is independent of cigarette smoking.F. prausnitzii and its secreted products are therefore interesting targetsfor the treatment of intestinal inflammation, in particular forIBD

Antileishmanial Activity and Inducible Nitric Oxide Synthase Activation by RuNO Complex.

Parasites of the genus Leishmania are capable of inhibiting effector functions of macrophages. These parasites have developed the adaptive ability to escape host defenses; for example, they inactivate the NF-κB complex and suppress iNOS expression in infected macrophages, which are responsible for the production of the major antileishmanial substance nitric oxide (NO), favoring then its replication and successful infection. Metal complexes with NO have been studied as potential compounds for the treatment of certain tropical diseases, such as ruthenium compounds, known to be exogenous NO donors. In the present work, the compound cis-[Ru(bpy)2SO3(NO)]PF6, or RuNO, showed leishmanicidal activity directly and indirectly on promastigote forms of Leishmania (Leishmania) amazonensis. In addition, treatment with RuNO increased NO production by reversing the depletion of NO caused by Leishmania. We also found increased expression of Akt, iNOS, and NF-κB in infected and treated macrophages. These results demonstrated that RuNO was able to kill the parasite by NO release and modulate the transcriptional capacity of the cell.

Levosimendan inhibits interleukin-1β-induced apoptosis through activation of Akt and inhibition of inducible nitric oxide synthase in rat cardiac fibroblasts

Levosimendan, a calcium sensitizer, known as an inotropic agent has a cytoprotective effect against apoptosis in cardiomyocytes. However, the cytoprotective effect of levosimendan on cardiac fibroblasts has not been clarified. The aim of this study was to examine whether levosimendan modulates interleukin (IL)-1β-induced apoptosis in adult rat cardiac fibroblast. Cardiac fibroblasts were isolated from adult male Wistar rats. Apoptosis of cardiac fibroblasts was evaluated by 4′,6-diamidino-2-phenylindole staining and TdT-mediated d-UTP nick end labeling (TUNEL)-based staining. Expression of inducible nitric oxide (NO) synthase (iNOS) and phosphorylation of Akt (Ser473) were determined by Western blotting. Assessment of NO production in the culture medium was performed by using 4,5-diaminofluorescein as a fluorescent probe. Immunofluorescence staining was performed to examine cytochrome-c translocation. Levosimendan (3–100μM) concentration-dependently inhibited IL-1β (4ng/ml, 24h)-induced apoptotic changes in cardiac fibroblasts, such as cellular shrinkage, nuclear condensation and the increase of TUNEL-positive cells. Levosimendan inhibited IL-1β-induced iNOS expression and subsequent NO production. 1400W, an iNOS inhibitor, suppressed the IL-1β-induced apoptosis. Levosimendan alone-treatment concentration-dependently increased phosphorylation of Akt (Ser473). LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor, reversed the protective effect of levosimendan on IL-1β-induced apoptosis in TUNEL assay. In addition, levosimendan and 1400W inhibited the IL-1β-induced cytosolic translocation of cytochrome-c. LY294002 reversed the suppressive effects of levosimendan. The present study for the first time demonstrated that levosimendan inhibits IL-1β-induced apoptosis via the activation of PI3K/Akt pathway and the inhibition of iNOS expression in adult rat cardiac fibroblasts.

Soluble DPP-4 up-regulates toll-like receptors and augments inflammatory reactions, which are ameliorated by vildagliptin or mannose-6-phosphate

ObjectiveStudies have shown that dipeptidyl peptidase-4 (DPP-4) inhibitors have anti-inflammatory effects. Soluble DPP-4 (sDPP-4) has been considered as an adipokine of which actions need to be further characterized. MethodsWe investigated the pro-inflammatory actions of sDPP-4 and the anti-inflammatory effects of DPP-4 inhibition, using vildagliptin, as an enzymatic inhibitor, and mannose-6-phosphate (M6P) as a competitive binding inhibitor. ResultsIn lipopolysaccharide (LPS)-stimulated RAW264.7 cells, vildagliptin suppressed the increased expression of inducible nitric oxide synthase (iNOS) and phosphorylated JNK (pJNK), activation of the NF-κB pathway, and the resultant NO and proinflammatory cytokine production. Although sDPP-4 alone did not affect the protein level of iNOS or pJNK or the production of NO in RAW264.7 cells, it did amplify iNOS expression, NO responses, and proinflammatory cytokine production in LPS-stimulated RAW264 cells. As a probable mechanism, we found that sDPP-4 caused dose-dependent increases in the expression levels of toll-like receptor 4 (TLR4) and TLR2 in RAW264.7 cells, and that these alterations were inhibited by vildagliptin, M6P, or bisindolylmaleimide II, a protein kinase C inhibitor. Either vildagliptin or M6P suppressed iNOS expression and NO and cytokine production in LPS+DPP-4-co-stimulated macrophages, while combined treatment of the co-stimulated cells with both agents had increased anti-inflammatory effects compared with either treatment alone. Intravenous injection of sDPP-4 to C57BL/6J mice increased the expression of both TLRs in kidney and white adipose tissues. ConclusionOur findings suggest that sDPP-4 enhances inflammatory actions via TLR pathway, while DPP-4 inhibition with either an enzymatic or binding inhibitor has anti-inflammatory effects.

Inhibition of iNOS activity enhances the anti-tumor effects of alpha-galactosylceramide in established murine cancer model.

Alpha-garactosylceramide (GalCer) has been shown to have anti-tumor effect in the basic research and clinical studies. However, anti-tumor effect of GalCer is limited. The administration of GalCer increases the production of IFN-γ which is involved in the suppression of tumor growth. On the other hand, the enhancement of IFN-γ production increases immunosuppressive factors such as nitric oxide. This suppressive action might impair the anti-tumor effect of GalCer. In the present study, we evaluated the anti-tumor effect of GalCer in the absence of inducible nitric oxide synthase (iNOS). In lung metastatic model, the number of tumor nodules in the lung of iNOS-KO mice treated with GalCer was significantly reduced compared with that of WT mice treated with GalCer. Moreover, L-NAME, which is the inhibitor for iNOS, enhanced the anti-tumor effect of GalCer in lung metastatic model. The frequency of CD8+ cells in bronchoalveolar lavage fluid increased in iNOS-KO mice treated with GalCer. The administration of GalCer increased the frequency of myeloid-derived suppressor cells (MDSCs) in the lung from tumor-bearing WT mice, but the increase of MDSCs in the lung was not induced in iNOS-KO mice. The subcutaneous tumor experiments revealed that the administration of GalCer in the absence of iNOS expression significantly enhanced the induction of tumor antigen-specific response. Finally, our results indicated that the inhibition of iNOS expression could enhance the therapeutic efficacy of GalCer via the increase of tumor antigen-specific immune response and the suppression of MDSCs.