iNOS Induction Research Articles

Native low density lipoprotein increases the production of both nitric oxide and reactive oxygen species in the human umbilical vein endothelial cells.

Nitric oxide synthases (NOSs) are a unique family of enzymes that catalyze the production of nitric oxide (NO) from L-arginine. Atherogenic action of oxidized low-density lipoproteins (oxLDL) may be mediated partly by the formation of NO in endothelial cells. The objective of this study was to identify sources of reactive oxygen species (ROS) causing native LDL (nLDL)-induced senescence of cultured human umbilical vein endothelial cells (HUVECs). HUVECs were treated with nLDL and NO production was assessed using Griess reagent as substrate and spectrophotometry in the absence or presence of specific inhibitors of endothelial NOS (eNOS) and inducible NOS (iNOS). In addition, expression levels of eNOS and iNOS were measured with ELISA and western blotting, and ROS was evaluated using 2',7'-dichlorofluorescin diacetate (DCF-DA) and a fluorescence microplate reader. NO formation in nLDL-treated HUVECs was significantly increased. Long-term treatment with nLDL up-regulated both eNOS and iNOS proteins. Such increase of NO production in HUVECs induced by nLDL was significantly suppressed by treatment with iNOS-selective inhibitor 1400W, but not by the eNOS-selective inhibitor L-NIO. Native LDL treatment uncoupled Hsp90, the regulatory binding protein of eNOS, from the enzyme in HUVECs. Native LDL also significantly increased ROS production in HUVECs. These findings suggest that oxidative stress originated from induction of iNOS and eNOS could be a causative factor for nLDL-induced senescence of HUVECs.

Arsenite suppresses NO production evoked by lipopolysaccharide and poly(I:C) via the suppression of interferon-β expression in RAW264.7 cells.

Immunological functions are disturbed in humans who have been chronically exposed to arsenic via contaminated groundwater. Little is known about the specific mechanisms underlying the impairment of immunological defense system caused by arsenic. The activation of macrophage cells upon infection with bacteria and viruses plays important roles in the defense against these pathogens. Here we show that exposure to arsenite (As(III)) suppresses nitric oxide (NO) production in murine RAW264.7 macrophage cells stimulated with lipopolysaccharide (LPS) and poly(I:C), the compounds mimicking bacterial and viral infection, respectively. As(III) suppressed the LPS- or poly(I:C)-evoked induction of inducible NO synthase (iNOS) without affecting the transactivation of NF-κB. As the interferon (IFN)-β/STAT1 pathway is also involved in the induction of iNOS in addition to NF-κB, we examined the effects of As(III) on the expression and secretion of IFN-β, the expression of the components of IFN-α/β receptor, the phosphorylation of STAT1, and the levels of cytokines involved in STAT1 activation. The results showed that the expression and secretion of IFN-β were specifically suppressed by As(III) treatment in RAW264.7 cells stimulated with LPS or poly(I:C). These results suggest that As(III) suppresses the expression and secretion of IFN-β, leading to the reduced STAT1 activation and consequently the reduced iNOS induction in macrophage cells. Our data suggest an important role of the arsenic-induced suppression of IFN-β on the disturbances in immunological defense against both bacteria and viruses.

Glutathione inhibits expression of the proinflammatory biomarker inducible nitric oxide synthase in hepatocytes

Background: Intracellular glutathione (GSH) plays an important regulatory role in the host response to liver injury. However, there have been few scientific reports on the anti-inflammatory effects of GSH. In the inflamed liver, proinflammatory cytokines stimulate liver cells, followed by expression of inducible nitric oxide synthase (iNOS). Excessive nitric oxide (NO) levels produced by iNOS are one of the factors involved in liver injury. Therefore, inhibiting iNOS induction is important for preventing liver injury. This study aimed to investigate the protective effects of GSH on the liver by examining interleukin (IL)-1β-stimulated hepatocytes.Methods: Primary cultured rat hepatocytes were treated with IL-1β in the presence or absence of GSH. Induction of iNOS and its signaling pathway were analyzed.Results: Addition of GSH decreased IL-1β-induced iNOS protein and mRNA expression levels, which resulted in inhibition of NO production. GSH also decreased tumor necrosis factor (TNF)-α and IL-6 mRNA expression. GSH blocked “type I IL-1 receptor upregulation”, one of the essential signaling pathways for iNOS induction, through inactivation of an upstream kinase, phosphatidylinositol 3-kinase/Akt. In contrast, GSH had no effects on degradation of IκB and activation of NF-ĸB (nuclear translocation and its DNA binding). Transfection experiments revealed that GSH reduced iNOS mRNA levels at the promoter transactivation and mRNA stabilization steps. Delayed administration of GSH after IL-1β addition also inhibited iNOS induction. Conclusions: Our study suggests that GSH affects induction of inflammatory mediators, including iNOS and TNF-α, indicating its therapeutic potential for organ injuries, especially for the liver.Keywords: glutathione, inducible nitric oxide synthase, liver injury, primary cultured hepatocytes, type I interleukin-1 receptor, tumor necrosis factor-α

Intraphagolysosomal conditions predispose to Staphylococcus epidermidis small colony variants persistence in macrophages.

Staphylococcus epidermidis small colony variants can survive inside macrophages and their survival has been proposed as a pivotal process in the pathogenesis of biomaterial associated infections. In the present study the intracellular location of clinical isolates of SCV and parental wild type strains inside macrophages was determined. Furthermore, the effect of IFN-γ and rapamycin on the level of SCV/WT as well as lysosomes colocalisation and iNOS induction in THP-activated macrophages in response to WT and SCV strains of Staphylococcus epidermidis were examined. It was demonstrated that SCV strain of S. epidermidis can survive and persist inside macrophages and its intracellular survival is supported by the induction of phagosomal acidification. The ability to reduce the high proportion of LysoTracker positive SCV containing phagosomes was exclusively found when IFN-γ was used. The findings suggest that IFN-γ mediates SCV killing via two distinct mechanisms, phagosome alkalisation and an increased iNOS synthesis, so the cytokine may control S. epidermidis WT and SCV infection in macrophages. Staphylococcus epidermidis SCV is a less potent stimulus of iNOS than the WT strain and the feature may help SCV to persist in hostile environment of macrophages. Rapamycin treatment did not influence the iNOS synthesis but reduced the percentage of both bacterial strains within acidic organelles. However, the percentage of SCV within LysoTracker positive organelles, even though reduced comparing to non-primed cells, was higher than in the WT strain indicating that Staphylococcus epidermidis possesses unique metabolic features allowing SCV to survive within macrophages.

Adjuvant role of macrophages in stem cell-induced cardiac repair in rats

Bone marrow-derived mesenchymal stem cells (BMMSCs) are used extensively for cardiac repair and interact with immune cells in the damaged heart. Macrophages are known to be modulated by stem cells, and we hypothesized that priming macrophages with BMMSCs would enhance their therapeutic efficacy. Rat bone marrow-derived macrophages (BMDMs) were stimulated by lipopolysaccharide (LPS) with or without coculture with rat BMCs. In the LPS-stimulated BMDMs, induction of the inflammatory marker iNOS was attenuated, and the anti-inflammatory marker Arg1 was markedly upregulated by coculture with BMMSCs. Myocardial infarction (MI) was induced in rats. One group was injected with BMMSCs, and a second group was injected with MIX (a mixture of BMMSCs and BMDMs after coculture). The reduction in cardiac fibrosis was greater in the MIX group than in the BMC group. Cardiac function was improved in the BMMSC group and was substantially improved in the MIX group. Angiogenesis was better in the MIX group, and anti-inflammatory macrophages were more abundant in the MIX group than in the BMMSC group. In the BMMSCs, interferon regulatory factor 5 (IRF5) was exclusively induced by coculture with macrophages. IRF5 knockdown in BMMSCs failed to suppress inflammatory marker induction in the macrophages. In this study, we demonstrated the successful application of BMDMs primed with BMMSCs as an adjuvant to cell therapy for cardiac repair.

The two phases of the clinical validation of preclinical translational mechanistic research on PDE5 inhibitors since Viagra's advent. A personal perspective.

The FDA approval of Viagra (sildenafil) for the on demand treatment of erectile dysfunction (ED) through relaxation of the corporal and cavernosal vascular smooth muscle that results in an increase in blood flow to the corporal tissues stemmed from 2 decades of research, mainly at academic centers. This culminated in the finding of the nitric oxide/cGMP pathway as the mediator of penile erection, followed by some years of basic studies and clinical validation at Pfizer. Further on, new translational laboratory and animal research from our group initiated a second phase when we proposed an alternative therapeutic schedule and mechanism of action for PDE5 inhibitors (PDE5i) in both corporal veno-occlusive dysfunction (CVOD) and Peyronie's disease (PD), specifically, continuous long-term administration (CLTA) to achieve sustained levels of cGMP within the penis. Due to the extended half-life of the long-acting PDE5i, tadalafil, this new alternative encompasses preferentially daily administration, although shorter half-life PDE5i, like sildenafil and vardenafil work too, depending on the duration, dose, and frequency of their administration This novel use was initially supported by showing the antifibrotic/antioxidant effects of nitric oxide and cGMP, produced by the induction of iNOS, as a mechanism of defense against collagen deposition in the localized fibrotic plaque of PD in an avascular tissue, the tunica albuginea. Our studies on iNOS and the progressive diffuse fibrosis occurring in the smooth muscle in CVOD, led to proposing the CLTA of PDE5i for maintaining sustained cGMP levels both in PD and in CVOD in order to halt or regress the penile fibrosis. In CVOD, we showed that PDE5i protect the corporal smooth muscle and reduce myofibroblast activation and number, counteracting the underlying corporal tissue pathology that causes CVOD, and potentially ameliorating long-term CVOD or even curing it. This review is focused on this novel PDE5i anti-fibrotic therapeutic concept.

Metformin has a protective effect through the inhibition of inducible nitric oxide synthase gene expression in hepatocytes

Rationale: Metformin is treated in patients with type II diabetes. However, there is few scientific evidence on the mechanisms involved in its anti-inflammatory effects. In inflamed liver, pro-inflammatory cytokines including tumor necrosis factor (TNF)-α stimulate liver cells (Kupffer cells and hepatocytes), leading to the induction of inducible nitric oxide synthase (iNOS) and NO production. Excessive NO produced by iNOS has been implicated as one of the factors in liver injury. It is essential to inhibit iNOS induction for the prevention of liver injury. We examined whether metformin influenced iNOS induction and, if so, the mechanisms in cultured hepatocytes (“in vitro injury model”).

Olive oil polyphenols reduce oxysterols -induced redox imbalance and pro-inflammatory response in intestinal cells

Dietary habits may strongly influence intestinal homeostasis. Oxysterols, the oxidized products of cholesterol present in cholesterol-containing foodstuffs, have been shown to exert pro-oxidant and pro-inflammatory effects, altering intestinal epithelial layer and thus contributing to the pathogenesis of human inflammatory bowel diseases and colon cancer. Extra virgin olive oil polyphenols possess antioxidant and anti-inflammatory properties, and concentrate in the intestinal lumen, where may help in preventing intestinal diseases. In the present study we evaluated the ability of an extra virgin olive oil phenolic extract to counteract the pro-oxidant and pro-inflammatory action of a representative mixture of dietary oxysterols in the human colon adenocarcinoma cell line (Caco-2) undergoing full differentiation into enterocyte-like cells. Oxysterols treatment significantly altered differentiated Caco-2 cells redox status, leading to oxidant species production and a decrease of GSH levels, after 1 h exposure, followed by an increase of cytokines production, IL-6 and IL-8, after 24 h. Oxysterol cell treatment also induced after 48 h an increase of NO release, due to the induction of iNOS. Pretreatment with the phenolic extract counteracted oxysterols effects, at least in part by modulating one of the main pathways activated in the cellular response to the action of oxysterols, the MAPK-NF-kB pathway. We demonstrated the ability of the phenolic extract to directly modulate p38 and JNK1/2 phosphorylation and activation of NF-kB, following its inhibitor IkB phosphorylation. The phenolic extract also inhibited iNOS induction, keeping NO concentration at the control level. Our results suggest a protective effect at intestinal level of extra virgin olive oil polyphenols, able to prevent or limit redox unbalance and the onset and progression of chronic intestinal inflammation.

P-365 - Beneficial effects of inhibiting iNOS activity versus iNOS transcription in anti-tumor photodynamic therapy

We have shown that various cancer cells can exploit endogenous nitric oxide (NO) to resist elimination by photodynamic therapy (PDT) or cisplatin treatment. Inducible NO synthase (iNOS) was rapidly and persistently upregulated after treatment and the resulting NO signaled not only for greater resistance, but greater growth, migration, and invasion aggressiveness of surviving cells. NO-mediated hyper-aggressiveness has been also observed in non-targeted bystander cells. In the present study involving human glioblastoma U87 cells, we compared the pro-PDT effectiveness of inhibiting iNOS activity, using 1400 W, with inhibiting NF-κB-mediated iNOS transcription, using a BET bromodomain inhibitor (JQ1). iNOS was upregulated 2–3-fold in PDT-treated U87 cells and Bay11–7082 inhibition of NF-κB-activating kinase IKK prevented this response. JQ1 strongly suppressed basal as well as post-PDT iNOS induction. JQ1 synergized with PDT in enhancing either apoptotic cell killing or suppressing hyper-invasiveness of PDT-surviving cells. This is the first evidence for JQ1's superior effectiveness in suppressing iNOS antagonism to an anticancer therapy. Since several BET bromodomain inhibitors are now in clinical trials for various cancers, the translational potential of our findings are clear.

Effects of oral cimetidine on the reproductive system of male rats.

Cimetidine is widely used for the treatment of digestive tract ulcers, but it induces testis injury. To explore the mechanisms underlying cimetidine-induced toxicity towards the testis, the effects of oral cimetidine on the reproductive system of male rats were assessed. Cimetidine was orally administered to male rats at 20, 40 or 120 mg/kg/day for 9 weeks. The rats were then euthanized, and serum, testis, epididymis, prostate gland, seminal vesicle, preputial gland, levator ani muscle and sphincter ani samples were collected. Sperm parameters were obtained by computer-assisted sperm analysis. Serum hormone levels were measured by ELISA. Protein expression levels were detected by immunohistochemistry. Apoptosis was assessed with the DeadEnd™ Colorimetric Apoptosis Detection System. The results indicated that the sperm average path velocity, straight line velocity and curvilinear velocity were significantly decreased in the 120 mg/kg cimetidine group compared with the control group, while luteinizing hormone and testosterone levels were significantly higher compared with the control group. Testicular lesions were observed by histopathology in the 120 mg/kg cimetidine group. The amounts of cells positive for cyclooxygenase-2 (COX-2) and nuclear factor κB (NF-κB) were increased in the 120 mg/kg cimetidine group compared with the control group. The amounts of cells positive for iNOS were increased in all cimetidine treatment groups. In addition, apoptotic cells were significantly more abundant in the 120 mg/kg cimetidine group compared with the control group, as indicated by terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling. Overall, 9 weeks of oral cimetidine induced pathological changes in the testicles and hormone secretion disorder in rats. COX-2, iNOS and NF-κB upregulation and induction of apoptosis may be associated with the reproductive toxicity caused by cimetidine.

Discovery of N-{3-[(ethanimidoylamino)methyl]benzyl}-l-prolinamide dihydrochloride: A new potent and selective inhibitor of the inducible nitric oxide synthase as a promising agent for the therapy of malignant glioma

In mammalian cells, aberrant iNOS induction may have detrimental consequences, and seems to be involved in the proliferation and progression of different tumors, such as malignant gliomas. Therefore, selective inhibition of iNOS could represent a feasible therapeutic strategy to treat these conditions. In this context, we have previously disclosed new acetamidines able to inhibit iNOS with a very high selectivity profile over eNOS or nNOS. Here we report the synthesis of a new series of compounds structurally related to the leading scaffold of N-[(3-aminomethyl)benzyl] acetamidine (1400 W), together with their in vitro activity and selectivity. Compound 39 emerged as the most promising molecule of this series, and it was ex vivo evaluated on isolated and perfused resistance arteries, confirming a high selectivity toward iNOS inhibition. Moreover, C6 rat glioma cell lines biological response to 39 was investigated, and preliminary MTT assay showed a significant decrease in cell metabolic activity of C6 rat glioma cells. Finally, results of a docking study shed light on the binding mode of 39 into NOS catalytic site.

L-Carnitine has a liver-protective effect through inhibition of inducible nitric oxide synthase induction in primary cultured rat hepatocytes

Background: L-Carnitine has protective effects on various injured organs. However, it has not been reported whether L-carnitine influences the induction of inducible nitric oxide synthase (iNOS) expression during inflammation. Nitric oxide (NO) produced by iNOS is an inflammatory indicator in organs which become inflamed, including the liver.Objective: This study aimed to examine whether L-carnitine influences the induction of iNOS gene expression in inflammatory cytokine-stimulated hepatocytes and the mechanisms involved in the action. Methods: L-Carnitine was added into the primary cultures of rat hepatocytes stimulated by interleukin-1β (an in vitro liver injury model). The production of NO and induction of iNOS and its signaling pathway were analyzed.Results: Transfection experiments with iNOS promoter-luciferase constructs revealed how L-carnitine inhibited iNOS mRNA synthesis activity and reduced its stability. In support of this observation, L-carnitine reduced iNOS mRNA and iNOS protein expression levels, resulting in reduced NO production. L-Carnitine blocked two essential pathways for iNOS induction: IκB kinase (IκB degradation/NF-κB activation) and phosphatidylinositol 3-kinase/Akt (type I IL-1 receptor upregulation).Conclusions: L-Carnitine inhibited the induction of inflammatory mediator iNOS, partially through inhibition of NF-κB activation, which demonstrated L-carnitine has protective effects in an in vitro liver injury model. L-Carnitine may have therapeutic potential for organ injuries, including the liver.Keywords: L-carnitine, hepatic encephalopathy, inducible nitric oxide synthase, liver injury, primary cultured hepatocytes, nuclear factor-κB, type I interleukin-1 receptor

Metformin inhibits expression of the proinflammatory biomarker inducible nitric oxide synthase in hepatocytes

Background: Metformin is used to treat patients with type II diabetes. However, there are few scientific reports on its anti-inflammatory effects. In the inflamed liver, proinflammatory cytokines stimulate liver cells, followed by inducible nitric oxide synthase (iNOS) expression. Excessive NO levels produced by iNOS have been implicated as a factor in liver injury. As a result, it is essential to inhibit iNOS induction to prevent liver injury.Objective: This study aimed to investigate liver protective effects of metformin by examining interleukin (IL)-1β-stimulated hepatocytes. Methods: Primary cultured rat hepatocytes were treated with interleukin (IL)-1β in the presence or absence of metformin. iNOS induction and its signaling pathway were analyzed.Results: Metformin decreased iNOS protein and mRNA expression, resulting in the inhibition of hepatic NO production. Metformin also reduced tumor necrosis factor (TNF)-α and IL-6 mRNA expression. Metformin inhibited an essential signaling pathway for iNOS induction, type I IL-1 receptor upregulation. Transfection experiments revealed that metformin reduced iNOS mRNA levels through both promoter transactivation and mRNA stabilization. Delayed metformin administration after IL-1β addition also inhibited iNOS induction. Conclusions: Metformin affects the induction of inflammatory mediators including iNOS and TNF-α, demonstrating its therapeutic potential for organ injuries, including the liver.Keywords: metformin, inducible nitric oxide synthase, liver injury, primary cultured hepatocytes, type I interleukin-1 receptor, tumor necrosis factor-α

Protection by different classes of dietary polyphenols against palmitic acid-induced steatosis, nitro-oxidative stress and endoplasmic reticulum stress in HepG2 hepatocytes

Polyphenol-rich functional foods have shown promise in ameliorating NAFLD but the relative effectiveness and mechanisms of different polyphenols are largely unknown. In a model of steatosis using HepG2 hepatocytes exposed to palmitic acid, we investigated the effect of selected polyphenols (resveratrol, quercetin, catechin, cyanidin, kuromanin) and berberine. Exposure to palmitic acid produced steatosis and intracellular ROS production, which were prevented by all of the polyphenols and berberine, at 10 µM. Palmitic acid produced a 245% increase in mRNA for inducible nitric oxide synthase (iNOS) that was almost completely prevented by all of the polyphenols (but not by berberine). Most of the polyphenols also partially inhibited palmitic acid-induced increases in markers of endoplasmic reticulum stress, and decreases in mitochondrial content and mitochondrial membrane potential. In conclusion, the polyphenols all similarly prevented steatosis, ROS generation and iNOS induction, and protected against ER stress and mitochondrial impairment.

Immunomodulatory Activity of Sulfonamide Chalcone Compounds in Mice Infected with Filarial Parasite, Brugia malayi.

Diethyl carbamazine (DEC) is being used as a sole drug to treat the lymphatic filariasis, although encountered with many limitations. Importantly, DEC works with putative host immunomodulating activities without any direct antifilarial effect. This study aimed to assess the possible modulatory effect on host immune system by sulfonamide chalcone compound, having direct antifilarial activity. The immunomodulatory activity of DEC and/or chalcone compound, 4t on mice peritoneal exudate cells (PECs) was analyzed initially in vitro. This was followed by the study of in vivo effect of these test agents in the parasitaemic BALB/c mice induced by Brugia malayi microfilariae. Cytokine profile and iNOS induction were measured from PECs of mice. 4t compound showed anti-inflammatory activity in vivo in contrast to DEC. Further 4t was found to increase anti-inflammatory and regulatory cytokines, IL-10 and TGF-β gene expression with down regulation of pro-inflammatory cytokines TNF-α and IFN-γ and iNOS in mice PECs in in vitro. In conclusion, chalcones having direct antifilarial effect also upsurges anti-inflammatory host immune response. Therefore, the results might be envisaged as 4t to be a better alternative to DEC in the chronic case of lymphatic filariasis.

Benefits of VCE-003.2, a cannabigerol quinone derivative, against inflammation-driven neuronal deterioration in experimental Parkinson\u2019s disease: possible involvement of different binding sites at the PPAR\u03b3 receptor

BackgroundNeuroprotection with cannabinoids in Parkinson’s disease (PD) has been afforded predominantly with antioxidant or anti-inflammatory cannabinoids. In the present study, we investigated the anti-inflammatory and neuroprotective properties of VCE-003.2, a quinone derivative of the non-psychotrophic phytocannabinoid cannabigerol (CBG), which may derive its activity at the peroxisome proliferator-activated receptor-γ (PPARγ). The compound is also an antioxidant.MethodsWe evaluated VCE-003.2 in an in vivo [mice subjected to unilateral intrastriatal injections of lipopolysaccharide (LPS)] model of PD, as well as in in vitro (LPS-exposed BV2 cells and M-213 cells treated with conditioned media generated from LPS-exposed BV2 cells) cellular models. The type of interaction of VCE-003.2 at the PPARγ receptor was furtherly investigated in bone marrow-derived human mesenchymal stem cells (MSCs) and sustained with transcriptional assays and in silico docking studies.ResultsVCE-003.2 has no activity at the cannabinoid receptors, a fact that we confirmed in this study using competition studies. The administration of VCE-003.2 to LPS-lesioned mice attenuated the loss of tyrosine hydroxylase (TH)-containing nigrostriatal neurons and, in particular, the intense microgliosis provoked by LPS in the substantia nigra, measured by Iba-1/Cd68 immunostaining. The analysis by qPCR of proinflammatory mediators such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and inducible nitric oxide synthase (iNOS) in the striatum showed they were markedly elevated by the LPS lesion and strongly reduced by the treatment with VCE-003.2. The effects of VCE-003.2 in LPS-lesioned mice implied the activation of PPARγ receptors, as they were attenuated when VCE-003.2 was co-administered with the PPARγ inhibitor T0070907. We then moved to some in vitro approaches, first to confirm the anti-inflammatory profile of VCE-003.2 in cultured BV2 cells exposed to LPS. VCE-003.2 was able to attenuate the synthesis and release of TNF-α and IL-1β, as well as the induction of iNOS and cyclooxygenase-2 (COX-2) elicited by LPS in these cells. However, we found such effects were not reversed by GW9662, another classic PPARγ antagonist. Next, we investigated the neuroprotective effects of VCE-003.2 in cultured M-213 neuronal cells exposed to conditioned media generated from LPS-exposed cultured BV2 cells. VCE-003.2 reduced M-213 cell death, but again, such effects were not reversed by T0070907. Using docking analysis, we detected that VCE-003.2 binds both the canonical and the alternative binding sites in the PPARγ ligand-binding pocket (LBP). Functional assays further showed that T0070907 almost abolished PPARγ transcriptional activity induced by rosiglitazone (RGZ), but it did not affect the activity of VCE-003.2 in a Gal4-Luc system. However, T0070907 inhibited the effects of RGZ and VCE-003.2 on the expression of PPARγ-dependent genes upregulated in MSCs.ConclusionsWe have demonstrated that VCE-003.2 is neuroprotective against inflammation-driven neuronal damage in an in vivo model of PD and in in vitro cellular models of neuroinflammation. Such effects might involve PPARγ receptors, although in silico and in vitro experiments strongly suggest that VCE-003.2 targets PPARγ by acting through two binding sites at the LBP, one that is sensitive to T0070907 (canonical binding site) and other that is not affected by this PPARγ antagonist (alternative binding site).

Some Phytochemicals and Anti-inflammation Effect of Juice from <i>Tiliacora triandra</i> Leaves

Tiliacora triandra (TT), native to Southeast Asia, has been widely used as folk medicine and in cuisines in many areas. Although little scientific evidence supports the health benefits of TT leaves, the juice extracted from TT leaves has become popular for consumers among all socioeconomic classes. Thus, this study aims to evaluate the phytochemical profile and anti-inflammatory properties of juice extracted from TT leaves. The result revealed that, the highest total phenolic contents (199.92 mg GAE/g), total flavonoid contents (29.76 mg RUE/g), and extraction yield (61.2%) of the lyophilized TT leaves juice powder were found in the solvent mixture of ethanol, water, and acetone extracts. The highest DPPH radical scavenging value (90.95%) of the lyophilized TT leaves juice powder was found in hot water extract. By HPLC analysis, total phenolic compounds of the lyophilized TT leaves juice powder was 3,938.1 mg/kg. Tannic acid, gallic acid, and rutin are the major phenolic compound and the juice is a rich source of chlorophyll compound (3,551.6 mg/kg). The lyophilized TT leaves juice down-regulated the induction of inflammatory iNOS and COX-2 proteins in LPS-stimulated macrophages. The results suggest that intake of TT leaves juice, providing various phenolic and chlorophyll compounds, has great potential for reducing the inflammatory process.

Salmonella small RNA fragment Sal-1 facilitates bacterial survival in infected cells via suppressing iNOS induction in a microRNA manner

Salmonella can hijack host atypical miRNA processing machinery to cleave its small non-coding RNA into a ~22-nt RNA fragment, Sal-1, which facilitates Salmonella survival in the infected host. The mechanism through which Sal-1 promotes Salmonella survival, however, remains unknown. In the present study, we reported that Sal-1 targets cellular inducible nitric oxide synthase (iNOS) in a miRNA manner, leading to attenuation of host cell iNOS/NO-mediated anti-microbial capacity. First, depletion of Sal-1 in Salmonella-infected epithelial cells significantly increased the iNOS level but not the levels of various inflammatory cytokines. Bioinformatics analysis and mutagenesis strategies were consistent with the identification of mRNA of iNOS as a target of Sal-1 in both human and mice. Second, western blot and immunohistochemical analysis confirmed that Sal-1 suppressed iNOS expression in vitro and in vivo, thus reducing the production of NO. Finally, Sal-1 facilitating Salmonella survival through suppressing iNOS induction was confirmed in mouse model by expressing mutated iNOS that is not targeted by Sal-1 in mice colon. In conclusion, our study provides new insight into the pathogenic mechanism of intracellular bacteria to modulate host innate immune response.

Allele-Specific Effects of ecSOD in Bacterial Infection

Extracellular superoxide dismutase (ecSOD) is the only enzyme that scavenges superoxide specifically in the extracellular compartment. Recent studies indicate that ecSOD is important for protecting tissues from oxidative damage by dampening immune responses but imply that the immunosuppressive effect may be counterproductive during acute infections against invading microorganisms. Although many studies have revealed the importance of the reactive oxygen species (ROS) in bacterial killing, the role of ecSOD during bacterial infection is beginning to be understood. In this study, Listeria monogytogene and Streptococcus pneumoniae were used to infect mice expressing various level of ecSOD: congenic mice sod3129 (relatively high), sod3wt and sod3 KO mice (none). ecSOD expression is inversely related to bacterial clearance. On the other hand, bacterial infection significantly decreases ecSOD activity in infected tissues, while the protein levels are not affected. The induction of iNOS expression is significantly lower in the liver and spleen of sod3 KO mice when compared to those of congenic sod3129 and sod3wt mice. The NO levels in liver and spleen were independent on the level of iNOS induction. However, the NO levels in plasma were significantly induced regardless of the genotype. The oxidative stress levels measured by nitrotyrosine immunoblotting in plasma and liver were significantly higher in sod3 KO mice compared to the congenic sod3129 and sod3wt mice. In conclusion, high ecSOD expression impairs the bacterial clearance and the infection diminishes the ecSOD activity. These data provide a potential target for therapeutic intervention for acute infection.

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.