Inducible Nitric Oxide Expression Research Articles

Anti-inflammatory effect of Dieffenbachia standleyi Croat extract on lipopolysaccharide-stimulated murine macrophages

ABSTRACT Dieffenbachia standleyi Croat is a plant belonging to Araceae and is native to Central America, and South America. While there have been studies on the Dieffenbachia species, Dieffenbachia standleyi Croat has yet to be studied in depth. Dieffenbachia was used as a folk remedy to prevent inflammation and treat gout, erectile dysfunction, and coldness. Thus, this study investigates the pharmacological function of Dieffenbachia standleyi Croat's methanol extract (DSE). When RAW 264.7 macrophages were treated with DSE and stimulated with lipopolysaccharide, DSE reduced the expression of inducible nitric oxide (NO) synthase and cyclooxygenase-2, thereby decreasing NO and prostaglandin E2 production and pro-inflammatory cytokines including IL-1β, IL-18, IL-6, and TNF-α. By confirming DSE's anti-inflammatory mechanisms, we determined that DSE inhibits IκBα phosphorylation in the nuclear factor κappa B pathway, and inflammasome formation, without affecting MAPK pathways. This study suggests that Dieffenbachia standleyi Croat is a potential anti-inflammatory agent for inflammatory diseases.

Impact of endogenous and exogenous nitrogen species on macrophage extracellular trap (MET) formation by bone marrow-derived macrophages.

Macrophage extracellular traps (METs) represent a novel defense mechanism in the antimicrobial arsenal of macrophages. However, mechanisms of MET formation are still poorly understood and this is at least partially due to the lack of reliable and reproducible models. Thus, we aimed at establishing a protocol of MET induction by bone marrow-derived macrophages (BMDMs) obtained from cryopreserved and then thawed bone marrow (BM) mouse cells. We report that BMDMs obtained in this way were morphologically (F4/80+) and functionally (expression of inducible nitric oxide (NO) synthase and NO production) differentiated and responded to various stimuli of bacterial (lipopolysaccharide, LPS), fungal (zymosan) and chemical (PMA) origin. Importantly, BMDMs were successfully casting METs composed of extracellular DNA (extDNA) serving as their backbone to which proteins such as H2A.X histones and matrix metalloproteinase 9 (MMP-9) were attached. In rendered 3D structure of METs, extDNA and protein components were embedded in each other. Since studies had shown the involvement of oxygen species in MET release, we aimed at studying if reactive nitrogen species (RNS) such as NO are also involved in MET formation. By application of NOS inhibitor - L-NAME or nitric oxide donor (SNAP), we studied the involvement of endogenous and exogenous RNS in traps release. We demonstrated that L-NAME halted MET formation upon stimulation with LPS while SNAP alone induced it. The latter phenomenon was further enhanced in the presence of LPS. Taken together, our findings demonstrate that BMDMs obtained from cryopreserved BM cells are capable of forming METs in an RNS-dependent manner.

Fasudil-modified macrophages reduce inflammation and regulate the immune response in experimental autoimmune encephalomyelitis.

Multiple sclerosis is characterized by demyelination and neuronal loss caused by inflammatory cell activation and infiltration into the central nervous system. Macrophage polarization plays an important role in the pathogenesis of experimental autoimmune encephalomyelitis, a traditional experimental model of multiple sclerosis. This study investigated the effect of Fasudil on macrophages and examined the therapeutic potential of Fasudil-modified macrophages in experimental autoimmune encephalomyelitis. We found that Fasudil induced the conversion of macrophages from the pro-inflammatory M1 type to the anti-inflammatory M2 type, as shown by reduced expression of inducible nitric oxide synthase/nitric oxide, interleukin-12, and CD16/32 and increased expression of arginase-1, interleukin-10, CD14, and CD206, which was linked to inhibition of Rho kinase activity, decreased expression of toll-like receptors, nuclear factor-κB, and components of the mitogen-activated protein kinase signaling pathway, and generation of the pro-inflammatory cytokines tumor necrosis factor-α, interleukin-1β, and interleukin-6. Crucially, Fasudil-modified macrophages effectively decreased the impact of experimental autoimmune encephalomyelitis, resulting in later onset of disease, lower symptom scores, less weight loss, and reduced demyelination compared with unmodified macrophages. In addition, Fasudil-modified macrophages decreased interleukin-17 expression on CD4+ T cells and CD16/32, inducible nitric oxide synthase, and interleukin-12 expression on F4/80+ macrophages, as well as increasing interleukin-10 expression on CD4+ T cells and arginase-1, CD206, and interleukin-10 expression on F4/80+ macrophages, which improved immune regulation and reduced inflammation. These findings suggest that Fasudil-modified macrophages may help treat experimental autoimmune encephalomyelitis by inducing M2 macrophage polarization and inhibiting the inflammatory response, thereby providing new insight into cell immunotherapy for multiple sclerosis.

Vasoplegic Syndrome after Cardiopulmonary Bypass in Cardiovascular Surgery: Pathophysiology and Management in Critical Care.

Vasoplegic syndrome (VS) is a common complication following cardiovascular surgery with cardiopulmonary bypass (CPB), and its incidence varies from 5 to 44%. It is defined as a distributive form of shock due to a significant drop in vascular resistance after CPB. Risk factors of VS include heart failure with low ejection fraction, renal failure, pre-operative use of angiotensin-converting enzyme inhibitors, prolonged aortic cross-clamp and left ventricular assist device surgery. The pathophysiology of VS after CPB is multi-factorial. Surgical trauma, exposure to the elements of the CPB circuit and ischemia-reperfusion promote a systemic inflammatory response with the release of cytokines (IL-1β, IL-6, IL-8, and TNF-α) with vasodilating properties, both direct and indirect through the expression of inducible nitric oxide (NO) synthase. The resulting increase in NO production fosters a decrease in vascular resistance and a reduced responsiveness to vasopressor agents. Further mechanisms of vasodilation include the lowering of plasma vasopressin, the desensitization of adrenergic receptors, and the activation of ATP-dependent potassium (KATP) channels. Patients developing VS experience more complications and have increased mortality. Management includes primarily fluid resuscitation and conventional vasopressors (catecholamines and vasopressin), while alternative vasopressors (angiotensin 2, methylene blue, hydroxocobalamin) and anti-inflammatory strategies (corticosteroids) may be used as a rescue therapy in deteriorating patients, albeit with insufficient evidence to provide any strong recommendation. In this review, we present an update of the pathophysiological mechanisms of vasoplegic syndrome complicating CPB and discuss available therapeutic options.

Yokukansan suppresses neuroinflammation in the hippocampus of mice and decreases the duration of lipopolysaccharide- and diazepam-mediated loss of righting reflex induced by pentobarbital.

Neuroinflammation is associated with the development of hypoactive delirium, which results in poor clinical outcomes. Drugs effective against hypoactive sur have not yet been established. Yokukansan has an anti-neuroinflammatory effect, making it potentially effective against hypoactive delirium. This study aimed to examine the effect of Yokukansan on the pentobarbital-induced loss of righting reflex duration extended with lipopolysaccharide (LPS)-induced neuroinflammation and diazepam-induced gamma-aminobutyric acid receptor stimulation in a mouse model. The active ingredients in Yokukansan and its anti-neuroinflammatory effect on the hippocampus were also investigated. Furthermore, we examined the in vitro anti-inflammatory effects of Yokukansan on LPS-stimulated BV2 cells, a murine microglial cell line. Findings revealed that treatment with Yokukansan significantly decreased the duration of pentobarbital-induced loss of righting reflex by attenuating the LPS-induced increase in interleukin-6 and tumor necrosis factor-alpha levels in the hippocampus. Moreover, treatment with Yokukansan significantly decreased the number of ionized calcium-binding adapter molecule-1-positive cells in the hippocampal dentate gyrus after 24h of LPS administration. In addition, glycyrrhizic acid, an active ingredient in Yokukansan, partially decreased the duration of pentobarbital-induced loss of righting reflex. Treatment with Yokukansan also suppressed the expression of inducible nitric oxide, interleukin-6, and tumor necrosis factor mRNA in LPS-stimulated BV2 cells. Thus, these findings suggest that Yokukansan and glycyrrhizic acid may be effective therapeutic agents for treating neuroinflammation-induced hypoactive delirium.

Abstract 3091: Relevance of iNOS expression in bladder cancer stem cells

Abstract Background: Bladder cancer (BC) is one of the most common tumors of the male urogenital tract and an important worldwide cause of death. Increasing evidence has indicated the presence of cancer stem cells (CSCs) in many types of tumors, associated with aggressiveness, chemoresistance and relapse. The expression of inducible nitric oxide (NO) synthase (iNOS) in human BC is a poor prognostic factor associated with increased invasion and tumor recurrence. Although there is evidence supporting the role of NO/iNOS in the promotion and progression of BC, the possibility that there are CSCs dependent on endogenous NO generation was not yet been clearly defined. In this study we evaluate the role of NO in CSC maintenance, modulating its production, using pharmacological inhibitors or silencing iNOS expression in a murine BC model. Also, we analyzed the correlation between iNOS expression and stemness-related genes (SG) in human BC samples with different invasion grades. Methods: iNOS pharmacological inhibitors (pan-NOS or iNOS specific) or shRNA were used on murine BC model with different iNOS expression and invasiveness grade: MB49 [iNOS+, non-muscle invasive (NMI)] and MB49-I [iNOS++, muscle invasive (MI)], in order to analyze cell proliferation, number of CSC determined as spheres forming efficiency (SFE) and SG (Sox 2, Oct4 and Nanog expression) by qPCR. iNOS expression and SG were also analyzed in human BC samples (16 NMI low grade (LG), 10 NMI high grade (HG) and 17 MI). Results: The number of CSCs was higher in MB49-I than NMI MB49 line (p<0.05), in concordance with the higher expression of iNOS and Sox2, Oct4 and Nanog (p<0.001). iNOS inhibition diminished CSC in both cell lines (p<0.05 vs MB49; p<0.001 vs MB49-I). Inhibition of SG was only observed in MB49-I line (p<0.05). Moreover, the reduction of CSC and SG were more evident in MB49-I-shiNOS (p<0.0001 vs MB49-I scr; p<0.001 vs MB49-I-scr). The in vivo tumorigenic potential of cells derived from MB49-I spheres were higher than the cells growing in monolayers. MB49-I spheres cells treated with iNOS inhibitors did not developed tumors in vivo. Furthermore, MB49-I shiNOS spheres showed an incipient growth and regressed about 5 weeks post inoculation, in correlation with low iNOS and SG expression. Regarding human samples, iNOS was expressed in 78% of MI, 55% HG and 50% LGNMI (p<0.05 vs NMI). SG expression were significantly higher in MI than in NMI LG. Sox2 was expressed 82% in MI, 25% LG and 40% HG in NMI (p=0.0002). 76% of MI samples were positive for Nanog and Oct4 (p<0.05 vs NMI LG). A positive correlation between iNOS and SG expression was found (sox2: p<0.0001; Oct4: p<0.0001 and Nanog: p<0.05). Conclusion: Together, this results show that iNOS plays an important role in maintenance of CSC and in vivo BC growth by regulating stemness and self-renewal ability. Thus, iNOS identification and inhibition could be a potential target to eradicate CSC, responsible for tumor recurrences. Citation Format: Denise Belgorosky, Yanina Langle, Julie Girouard, Jovane Hamelin-Morrissette, Lina Marino, Eduardo I. Agüero, Héctor Malagrino, Carlos Reyes-Moreno, Ana M. Eiján. Relevance of iNOS expression in bladder cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3091.

Ziziphus spina-christi Leaf Extract Mitigates Mercuric Chloride-induced Cortical Damage in Rats.

Mercuric chloride (HgCl2) severely impairs the central nervous system when humans are exposed to it. We investigated the neuroprotective efficiency of Ziziphus spina-christi leaf extract (ZSCLE) on HgCl2-mediated cortical deficits. Twenty-eight rats were distributed equally into four groups: the control, ZSCLE-treated (300 mg/kg), HgCl2-treated (0.4 mg/kg), and ZSCLE+HgCl2-treated groups. Animals received their treatments for 28 days. Supplementation with ZSCLE after HgCl2 exposure prevented the deposition of mercury in the cortical slices. It also lowered malondialdehyde levels and nitrite and nitrate formation, elevated glutathione levels, activated its associated-antioxidant enzymes, glutathione reductase, and glutathione peroxidase, and upregulated the transcription of catalase and superoxide dismutase and their activities were accordingly increased. Moreover, ZSCLE activated the expression of nuclear factor erythroid 2-related factor 2 and heme oxygenase-1 when compared with the HgCl2 group. Notably, post-treatment with ZSCLE increased the activity of acetylcholinesterase and ameliorated the histopathological changes associated with HgCl2 exposure. Furthermore, ZSCLE blocked cortical inflammation, as observed by the lowered mRNA expression and protein levels of interleukin-1 beta and tumor necrosis factor-alpha, as well as decreased mRNA expression of inducible nitric oxide synthase. In addition, ZSCLE decreased neuron loss by preventing apoptosis in the cortical tissue upon HgCl2 intoxication. Based on the obtained findings, we suggest that ZSCLE supplementation could be applied as a neuroprotective agent to decrease neuron damage following HgCl2 toxicity.

The Extraction Solvent Influences the Anti-Inflammatory Effects of Jakyakgamcho-Tang in Lipopolysaccharide-Stimulated Macrophages and Mice with Gouty Arthritis.

Jakyakgamcho-Tang (JGT) is a traditional medicine used to treat muscular tension, spasm, and pain. Several studies have reported its clinical use as an anti-inflammatory and in gynaecological treatment. This study aimed to compare the anti-inflammatory effects of JGT according to extraction solvent, water (JGTW) and 30% EtOH (JGTE) on lipopolysaccharide (LPS)—stimulated macrophages and in mice with monosodium urate (MSU)—induced gouty arthritis. We evaluated the production of inflammatory mediators and cytokines and the expression of inducible nitric oxide (iNOS) and cyclooxygenase-2 (COX-2) in RAW 264.7 cells. We also examined oedema, pain, and inflammation in MSU-induced mice by measuring affected hind paw swelling, weight-bearing, pro-inflammatory cytokines levels, and myeloperoxidase (MPO) activity. In LPS-stimulated RAW264.7 cells, JGTW and JGTE significantly decreased prostaglandin (PG) E2(PGE2) production via suppressing COX-2 expression and cytokines interleukin-1β and interleukin-6. Only JGTE reduced the production of NO and cytokines and the mRNA levels of iNOS and cytokines. In MSU-induced mice, JGTE and JGTW efficiently decreased paw swelling and attenuated joint pain. JGTE (200 and 300 mg/kg) effectively suppressed inflammation by downregulating pro-inflammatory cytokines (tumour necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6) and MPO activity, which were only slightly reduced by JGTW. Our data demonstrate the anti-inflammatory activity of JGT in macrophage and gouty arthritis animal models and show that JGTE is more effective than JGTW at lower concentrations.

The Effects of Crinum asiaticum var. japonicum Baker Seeds on Neuroprotection and Antineuroinflammation in Neuronal Cell Lines

Crinum asiaticum var. japonicum Baker is a Korean herbal medicine that is traditionally prescribed for reducing fever and inflammation. In the present study, we investigated if the ethanol extract of C. asiaticum seeds (ECAS) influences the hallmarks of Alzheimer’s disease (AD) pathogenesis. ECAS markedly inhibited the activity of acetylcholinesterase (AChE). Concurrent treatment with hydrogen peroxide (H2O2) and ECAS significantly prevented the neuronal cell death by regulating phosphorylation of cyclic adenosine monophosphate response element-binding protein and p38 mitogen-activated protein kinase. ECAS revealed antineuroinflammatory effects by inhibiting nitric oxide production and suppressing inducible nitric oxide expression in lipopolysaccharide-stimulated BV-2 microglia. Furthermore, the high-performance liquid chromatographic analysis determined lycorine as a standard compound of ECAS. Our data suggest that ECAS has inhibitory effects on AD pathogenesis such as AChE activation, neuronal damage, and neuroinflammation.

Relevance of iNOS expression in tumor growth and maintenance of cancer stem cells in a bladder cancer model.

The expression of inducible nitric oxide (NO) synthase (iNOS) in human bladder cancer (BC) is a poor prognostic factor associated with invasion and tumor recurrence. Here, we evaluated the relevance of iNOS expression in BC progression and in cancer stem cell (CSC) maintenance in a murine BC model. Also, iNOS expression and CSC markers were analyzed in human BC samples. iNOS inhibitors (L-NAME or 1400W) or shRNA were used on murine BC model with different iNOS expressions and invasiveness grades: MB49 (iNOS+, non-muscle invasive (NMI)) and MB49-I (iNOS++, muscle invasive (MI)), in order to analyzed cell proliferation, tumor growth, angiogenesis, number of CSC, and pluripotential marker expression. iNOS, SOX2, Oct4, and Nanog expressions were also analyzed in human BC samples by qPCR and immunohistochemistry. iNOS inhibtion reduced parameters associated with tumor progression and reduced the number of CSC, wich resulted higher in MB49-I than in MB49, in concordance with the higher expression of SOX2, Oct4, and Nanog. The expression of SOX2 was notoriously diminished, when iNOS was inhibited only in the MI cell line. Similar results were observed in human samples, where MI tumors expressed higher levels of iNOS and pluripotential genes, in comparison to NMI tumors with a positive correlation between those and iNOS, suggesting that iNOS expression is associated with CSC. iNOS plays an important role in BC progression and CSC maintenance. Its inhibition could be a potential therapeutic target to eradicate CSC, responsible fortumor recurrences. KEY MESSAGES: • iNOS expression is involved in bladder tumor development, growth, and angiogenesis. • iNOS expression is involved in bladder cancer stem cell generation and maintenance, playing an important role regulating their self-renewal capacity, especially in muscle invasive murine bladder cancer cells. • iNOS expression is higher in human muscle invasive tumors, in association with a high expression of pluripotential genes, especially of SOX2.

Altered composition of high-lipid diet may generate reactive oxygen species by disturbing the balance of antioxidant and free radicals.

Background In the present era, obesity is increasing rapidly, and high dietary intake of lipid could be a noteworthy risk factor for the occasion of obesity, as well as nonalcoholic fatty liver disease, which is the independent risk factor for type 2 diabetes and cardiovascular disease. For a long time, high-lipid diet (HLD) in "fast food" is turning into part of our everyday life. So, we were interested in fulfilling the paucity of studies by means of preliminary evaluation of these three alternative doses of HLD on a rat model and elucidating the possible mechanism of these effects and divulging the most alarming dose. Methods Thirty-two rats were taken, and of these, 24 were fed with HLD in three distinctive compositions of edible coconut oil and vanaspati ghee in a ratio of 2:3, 3:2 and 1:1 (n = 8), orally through gavage at a dose of 10 mL/kg body weight for a period of 28 days, whereas the other eight were selected to comprise the control group. Results After completion of the experiment, followed by analysis of data it was revealed that hyperlipidemia with increased liver and cardiac marker enzymes, are associated with hepatocellular injury and cardiac damage. The data also supported increased proinflammatory cytokines such as interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α). As oxidative stress parameter increased in both liver and heart, there is also an increased in TNF-α due to an increased expression of inducible nitric oxide (NO) synthase, which led to a high production of NO. Moreover, HLD treatment explicitly weakens reasonability of hepatocytes and cardiomyocytes conceivably through G0/G1 or S stage capture or perhaps by means of enlistment of sub-G0/G1 DNA fragmentation and a sign of apoptosis. Conclusions Based on the outcomes, it tends to be inferred that consequences of the present examination uncovered HLD in combination of 2:3 applies most encouraging systemic damage by reactive oxygen species generation and hyperlipidemia and necroapoptosis of the liver and heart. Hence, outcome of this study may help to formulate health care strategy and warns about the food habit in universal population regarding the use of hydrogenated and saturated fats (vanaspati ghee) in diet.

Anti-Inflammatory Activities of Isogosferol, a Furanocoumarin Isolated from Citrus junos Seed Shells through Bioactivity-Guided Fractionation.

Citrus junos Tanaka is a traditional medicine for treating coughs, dyspepsia, diabetes, asthma, neuralgia, and inflammatory disorders, and is distributed in Asia, especially in Korea, Japan, and China. This study aimed to use bioactivity-guided fractionation to find therapeutic phytochemicals from C. junos seeds, which can attenuate inflammatory responses. Nine coumarins (1–9) were isolated from the methanolic extract of C. junos seed shells and the inhibitory effects against inflammatory mediators were investigated using murine macrophages. Among the coumarins, compound 3, isogosferol (ISO), more potently attenuated the production of nitric oxide (NO) in lipopolysaccharide (LPS)-induced RAW 264.7 cells. ISO also inhibited the expression of inducible nitric oxide (iNOS) and cyclooxygenase-2 (COX-2) in LPS-stimulated macrophages. Additionally, the phosphorylation of extracellular-regulated kinases (pERK)1/2 was reduced by ISO. We confirmed that ISO attenuated the release of interleukin-1 beta (IL-1β), which is a central mediator of the inflammatory response. These results demonstrate that ISO from C. junos seed shells may be a potent therapeutic candidate for the treatment of inflammatory diseases.

Thymoquinone-PLGA-PVA Nanoparticles Ameliorate Bleomycin-Induced Pulmonary Fibrosis in Rats via Regulation of Inflammatory Cytokines and iNOS Signaling.

Pulmonary fibrosis is considered one of the most chronic interstitial illnesses which are not easily treated. thymoquinone's (TQ) benefits are still partly problematic due to poor water solubility; therefore, it was loaded onto PLGA-PVA carriers. This study aimed to evaluate the potential effect of TQ-PLGA-PVA nanoparticles (TQ-PLGA-PVA-NPs) on pulmonary fibrosis induced by bleomycin in albino rats. Forty male rats were randomized into four groups. The first group served as the control group; the second and the third groups received bleomycin intratracheally, whereas the third group received TQ-PLGA-PVA-NPs after 4 weeks from bleomycin administration. The fourth group was administrated TQ-PLGA-PVA-NPs alone. The designed nanoparticles appeared around 20 nm size (10-30 nm), had a spherical shape, and had 80% encapsulation efficiency. The histological examination of rats simultaneously treated with TQ-PLGA-PVA-NPs and bleomycin revealed reduction in the thickness of the alveolar septa and improvement of the other lung structures, with the presence of lymphocytes admixed with exfoliated epithelium in a few lumina remaining. Ultrastructural findings revealed marked collagenolysis and the release of nanoparticles from ruptured pneumocytes within the alveolar septa after 14 days from TQ-PLGA-PVA-NPs administration. Very active pneumocyte types II were seen in the TQ-PLGA-PVANP group. Additionally, immunohistochemical expression of inducible nitric oxide (iNOS) and estimation of inflammatory cytokines in lung tissues including interleukin 10 (IL 10) and transforming growth factor-beta (TGF-β1) confirmed the antioxidant and anti-inflammatory effects of TQ-PLGA-PVANPs. The study concluded that TQ-PLGA-PVA-NPs could attenuate the bleomycin-induced pulmonary fibrosis, through the inhibition of lung inflammation and the suppression of bleomycin- induced oxidative stress.

2,3,7,8-Tetrachlorodibenzo-p-dioxin-induced aryl hydrocarbon receptor activation enhanced the suppressive function of mesenchymal stem cells against splenocyte proliferation

The immunosuppressive function of mesenchymal stem cells (MSCs) is well known. Aryl hydrocarbon receptor (AhR), a transcription factor of the bHLH/PAS family, is widely expressed in several cells and is involved in various physiological and pathological processes. Previously, we found that the expression of AhR was downregulated in MSCs isolated from mice with neutrophilic asthma and that the activation of AhR enhanced the function of MSCs to alleviate neutrophilic asthma. We hypothesized that AhR activation enhanced MSCs for their immunosuppressive function. We aimed to investigate whether AhR activation can augment the suppressive function of MSCs against splenocyte proliferation. We co-cultured MSCs or AhR-activated MSCs with splenocytes at different ratios. The results showed that AhR activation in MSCs upregulated the expression of inducible nitric oxide (iNOS), which promoted the production of nitric oxide (NO), thus enhancing the inhibitory effect on splenocyte proliferation. The NO donor S-nitroso-N-acetylpenicillamine also inhibited the proliferation of splenocytes, and the iNOS inhibitor N(G)-nitro L-arginine methyl ester and NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide partially reversed the immunosuppressive function. Our study indicates that the AhR activation of MSCs might have an important role in the regulation of splenocyte proliferation and might serve as a potential strategy for treating immune-related diseases.

Involvement of inducible nitric oxide synthase and estrogen receptor ESR2 (ERβ) in the vascular dysfunction in female type 1 diabetic rats

AimsInflammation is involved in diabetes-related vascular dysfunction. Estrogen receptor ESR2/ERβ induces the expression of inducible nitric oxide (NO) synthase (iNOS) and inflammation. The present study investigated the effect of alloxan-induced type 1 diabetes on the iNOS and ESR2 expression and the effect of the chronic iNOS inhibition on the vascular smooth muscle dysfunction in diabetic female rats. In addition, we evaluated the involvement of ESR2 in iNOS expression. Main methodsAlloxan-induced diabetic female rats were treated or not with iNOS inhibitor (L-NIL). iNOS and ESR2 immunostaining, S-nitrosylated proteins and IL-1β protein expression in aorta and plasmatic NO levels were analyzed. Contractile response to noradrenaline was analyzed in endothelium-denuded aorta. iNOS mRNA expression was analyzed in isolated aortic smooth muscle cells (ASMCs) of female rats, incubated with 22 mM glucose and an ESR2 antagonist. Key findingsAortic iNOS and ESR2 immunostaining, S-nitrosylated proteins, IL-1β protein expression and plasmatic NO levels were all increased, whereas noradrenaline-induced contraction was reduced in aorta of diabetic female rats. With the exception of iNOS and ESR2 immunostaining, all these parameters were corrected by L-NIL treatment. High glucose increased iNOS mRNA expression in ASMCs, which was reduced by an ESR2 antagonist. SignificanceWe demonstrated that increased iNOS-NO contributed to the impairment of the contractile response of aortic smooth muscle cells in female type 1 diabetic rats and that increased expression of iNOS may involve the participation of ESR2/ERβ.

Effects of Herbal Formulas Bojungikgi-tang and Palmijihwang-hwan on Inflammation in RAW 264.7 Cells and the Activities of Drug-Metabolizing Enzymes in Human Hepatic Microsomes.

In the present study, Bojungikgi-tang (BJIKT: Buzhongyiqi-tang, Hochuekki-to) and Palmijihwang-hwan (PMJHH: Baweidìhuang-wan, Hachimijio-gan), traditional herbal formulas, investigated anti-inflammatory efficacies in murine macrophage cell line and the influence on the activities of drug-metabolizing enzymes (DMEs). The anti-inflammatory potentials of the herbal formulas were evaluated to inhibit the production of the inflammatory mediators and cytokines and the protein expression of inducible nitric oxide and cyclooxygenase-2 (COX-2) in lipopolysaccharide (LPS)-treated RAW 264.7 cells. The activities of the major human DMEs, cytochrome P450 isozymes (CYP450s) and UDP-glucuronosyltransferase isozymes (UGTs), were measured by in vitro enzyme assay systems. BJIKT and PMJHH significantly suppressed the prostaglandin E2 (PGE2) production (IC50 = 317.3 and 282.2 μg/mL, respectively) and the protein expression of COX-2 in LPS-treated RAW264.7 cells. On the human microsomal DMEs, BJIKT inhibited the activities of CYP1A2 (IC50 = 535.05 μg/mL), CYP2B6 (IC50 > 1000 μg/mL), CYP2C9 (IC50 = 800.78 μg/mL), CYP2C19 (IC50 = 563.11 μg/mL), CYP2D6 (IC50 > 1000 μg/mL), CYP2E1 (IC50 > 1000 μg/mL), CYP3A4 (IC50 = 879.60 μg/mL), UGT1A1 (IC50 > 1000 μg/mL), and UGT1A4 (IC50 > 1000 μg/mL), but it showed no inhibition of the UGT2B7 activity at doses less than 1000 μg/mL. PMJHH inhibited the CYP2D6 activity (IC50 = 280.89 μg/mL), but IC50 values of PMJHH exceeded 1000 μg/mL on the activities of CYP1A2, CYP2C19, CYP2E1, and CYP3A4. At concentrations less than 1000 μg/mL, PMJHH did not affect the activities of CYP2B6, CYP2C9, UGT1A1, UGT1A4, and UGT2B7. The results indicate that both BJIKT and PMJHH may be potential candidates to prevent and treat PGE2- and COX-2-mediated inflammatory diseases. In addition, this study will expand current knowledge about herb-drug interactions by BJIKT and PMJHH.

Virgin olive oil and its phenol fraction modulate monocyte/macrophage functionality: a potential therapeutic strategy in the treatment of systemic lupus erythematosus.

Monocytes and macrophages are critical effectors and regulators of inflammation and innate immune response, which appear altered in different autoimmune diseases such as systemic lupus erythematosus (SLE). Recent studies suggested that virgin olive oil (VOO) and particularly its phenol compounds might possess preventive effects on different immune-inflammatory diseases, including SLE. Here, we evaluated the effects of VOO (and sunflower oil) on lipopolysaccharide (LPS)-activated peritoneal macrophages from a model of pristane-induced SLE in BALB/c mice, as well as those of the phenol fraction (PF) from VOO on the immune-inflammatory activity and plasticity in monocytes and monocyte-derived macrophages from healthy volunteers. The release of nitrite and inflammatory cytokines was lower in LPS-treated peritoneal macrophages from pristane-SLE mice fed the VOO diet when compared with the sunflower oil diet. PF from VOO similarly decreased the secretion of nitrite and inflammatory cytokines and expression of inducible nitric oxide, PPARγ and Toll-like receptor 4 in LPS-treated human monocytes. PF from VOO also prevented the deregulation of human monocyte subset distribution by LPS and blocked the genetic signature of M1 macrophages while favouring the phenotype of M2 macrophages upon canonical polarisation of naïve human macrophages. For the first time, our study provides several lines of in vivo and in vitro evidence that VOO and PF from VOO target and counteract inflammatory pathways in the monocyte-macrophage lineage of mice with pristane-induced SLE and of healthy subjects, which is a meaningful foundation for further development and application in preclinical and clinical use of PF from VOO in patients with SLE.

Chronic ethanol consumption increases vascular oxidative stress and the mortality induced by sub-lethal sepsis: Potential role of iNOS

We hypothesized that long-term ethanol consumption would increase the mortality and aggravate the deleterious effects of sub-lethal cecal ligation and puncture (SL-CLP) in the vasculature by inducing the expression of inducible nitric oxide (NO) synthase (iNOS). Male C57BL/6J wild-type (WT) or iNOS-deficient mice (iNOS-/-) were treated with ethanol (20% v/v) for 12 weeks and then subjected to SL-CLP. Mice were killed 24 h post-operatively or followed six days for survival. Septic ethanol-treated mice showed a higher mortality than septic WT mice. However, septic iNOS-deficient mice treated with ethanol showed a decreased mortality rate when compared to ethanol-treated WT mice. Ethanol and SL-CLP augmented superoxide anion (O2-) generation in the mesenteric arterial bed (MAB) of both WT and iNOS-deficient mice. Treatment with ethanol and SL-CLP enhanced lipoperoxidation in the MAB of WT, but not iNOS-deficient mice. SL-CLP enhanced nitrate/nitrite (NOx) concentrations in the MAB of WT, but not iNOS-deficient mice. Both, ethanol and SL-CLP increased TNF-α and IL-6 levels in the MAB. Treatment with ethanol as well as SL-CLP up-regulated the expression of iNOS in the MAB of WT mice. The major finding of our study is that chronic ethanol consumption increases the mortality induced by SL-CLP and that iNOS plays a role in such response. Although ethanol led to vascular alterations, it did not aggravate the vascular injury induced by SL-CLP. Finally, iNOS mediated the increase in oxidative stress and pro-inflammatory cytokines induced by SL-CLP in the vasculature.

Valproic acid attenuates nitric oxide and interleukin-1β production in lipopolysaccharide-stimulated iron-rich microglia.

Iron accumulation in activated microglia has been consistently reported in neurodegenerative diseases. Previous results suggest that these cells facilitate neuroinflammation leading to neuronal cell death. Therefore, chemical compounds that alleviate the activation of iron-rich microglia may result in neuroprotection. In the present study, the effect of valproic acid (VPA) on microglial activation under iron-rich conditions was investigated. BV-2 microglial cells were exposed to lipopolysaccharide (LPS; 1 µg/ml) and iron (300 µg/ml) with or without VPA (1.6 mM). The results demonstrated that VPA attenuated the activation of iron-rich BV2 cells induced by LPS by down-regulating the mRNA expression of inducible nitric oxide (NO) synthase and interleukin 1β (IL-1β; P<0.01), to ultimately reduce the production of NO and IL-1β (P<0.01). These events were accompanied by an attenuation in the nuclear translocation of nuclear factor-κB p65 subunit (P<0.01). These findings suggest that VPA may be therapeutically useful for attenuating the activation of iron-rich microglia.

Phosphatidylinositide 3-Kinase Contributes to the Anti-Inflammatory Effect of Abutilon crispum L. Medik Methanol Extract.

Abutilon crispum L. Medik, better known as bladdermallow, is used as a traditional remedy in India, for its anti-inflammatory effect due to its high content of flavonoids. However, research about its anti-inflammatory effect at the molecular level has not been performed. In this study, we aimed to investigate the mechanism of Abutilon crispum methanol extract (Ac-ME) in inhibiting the inflammatory response by conducting several experiments including cellular and molecular assays. Ac-ME inhibited the production of nitric oxide (NO) in RAW264.7 cells during treatment of LPS and Pam3CSK4 without exhibiting cytotoxicity. Ac-ME also suppressed the mRNA expression of inducible nitric oxide (iNOS) and proinflammatory cytokines such as interleukin (IL)-1β and IL-6. Moreover, Ac-ME was shown to inhibit the NF-κB pathway, according to the luciferase reporter gene assay performed with a NF-κB-Luc construct containing NF-κB-binding promoter regions under MyD88 and TRIF overexpression conditions, and immunoblotting analysis by determining the phospho-form levels of IκBα, IKKα/β, and p85, a regulatory domain of phosphatidylinositide 3-kinase (PI3K). Finally, we observed that the level of phospho-p85 induced by the overexpression of spleen tyrosine kinase (Syk) and Src was decreased by Ac-ME at 200 μg/ml. Therefore, these results suggest that Ac-ME has an anti-inflammatory effect by targeting PI3K in the NF-κB signaling pathway.