Blood Level of Nitric Oxide Metabolites and Expression of Genes Regulating NO Synthesis in Early Forms of Non-Alcoholic Fatty Liver Disease.

The levels of NO metabolites in the plasma and mRNA of the NOS3, ATG9B, and NOS2 genes in peripheral blood leukocytes of healthy people and patients with early forms of non-alcoholic fatty liver disease (steatosis and weak activity non-alcoholic steatohepatitis) were studied. In patients with steatohepatitis, the concentration of NO metabolites in the blood and the level of mRNA of the NOS2 gene were higher than in patients with steatosis and healthy people. These differences can be of diagnostic value for distinguishing between steatosis and weak activity steatohepatitis in non-alcoholic fatty liver disease. A correlation between the levels of NO metabolites and the expression of the NOS2 gene in weak activity steatohepatitis was established, which indicates activation of NO synthesis in non-alcoholic steatohepatitis due to the expression of the inducible NO synthase gene. The level of the NOS2 gene mRNA in peripheral blood leukocytes of patients with weak activity steatohepatitis correlated with the level of TNFα and IL-6 cytokines. An increase in the level of NO in the blood in weak activity steatohepatitis correlated with the level of MDA, an indicator of oxidative stress.

iNOS regulates hematopoietic stem and progenitor cells via mitochondrial signaling and is critical for bone marrow regeneration.

Hematopoietic stem cells (HSCs) replenish blood cells under steady state and on demand, that exhibit therapeutic potential for Bone marrow failures and leukemia. Redox signaling plays key role in immune cells and hematopoiesis. However, the role of reactive nitrogen species in hematopoiesis remains unclear and requires further investigation. We investigated the significance of inducible nitric oxide synthase/nitric oxide (iNOS/NO) signaling in hematopoietic stem and progenitor cells (HSPCs) and hematopoiesis under steady-state and stress conditions. HSCs contain low levels of NO and iNOS under normal conditions, but these increase upon bone marrow stress. iNOS-deficient mice showed subtle changes in peripheral blood cells but significant alterations in HSPCs, including increased HSCs and multipotent progenitors. Surprisingly, iNOS-deficient mice displayed heightened susceptibility and delayed recovery of blood progeny following 5-Fluorouracil (5-FU) induced hematopoietic stress. Loss of quiescence and increased mitochondrial stress, indicated by elevated MitoSOX and MMPhi HSCs, were observed in iNOS-deficient mice. Furthermore, pharmacological approaches to mitigate mitochondrial stress rescued 5-FU-induced HSC death. Conversely, iNOS-NO signaling was required for demand-driven mitochondrial activity and proliferation during hematopoietic recovery, as iNOS-deficient mice and NO signaling inhibitors exhibit reduced mitochondrial activity. In conclusion, our study challenges the conventional view of iNOS-derived NO as a cytotoxic molecule and highlights its intriguing role in HSPCs. Together, our findings provide insights into the crucial role of the iNOS-NO-mitochondrial axis in regulating HSPCs and hematopoiesis.

Fish arginase constrains excessive production of nitric oxide and limits mitochondrial damage during Aeromonas hydrophila infection.

Bacteria-enhanced inducible nitric oxide synthase (iNOS) overproduces nitric oxide (NO) leading to mitochondrial and cellular damage. In mammals, arginase (ARG), the enzyme consuming the same substrate l-arginine with iNOS, was believed to inhibit iNOS activity by competing the substrate. But in fish, this conception has been widely challenged. In this study, the gene expression using real-time quantitative PCR (RT-qPCR) technology showed that when stimulated by Aeromonas hydrophila (A. hydrophila), grass carp (gc) iNOS was up-regulated in head kidney monocytes/macrophages (M0/MФ), and its changes were not detected in the whole tissue of liver or spleen, showing a high degree of cell-specific expression pattern. At the same time, gcARG2 had a high basal expression in tissues and was up-regulated by A. hydrophila stimulation. Next, phthalaldehyde-primaquine reaction was first used in the determination of intracellular urea in fish cells. It was found that the induced gcARG2 led to an increase in the intracellular urea content. Moreover, urea and NO production in M0/MФ were increased in a substrate dose-dependent manner from 30 to 100 µM of l-arginine and reached the highest yield at 300 and 3000 µM of l-arginine, respectively. Furthermore, head kidney M0/MФ was cultured in RPMI1640 medium containing physiological concentration (500 µM) of l-arginine to evaluate the effect of ARG. Under A. hydrophila stimulation, treatment with the arginase inhibitor S-(2-boronoethyl)-l-cysteine (BEC) showed that inhibition of arginase could further enhance the NO production stimulated by A. hydrophila. This in turn led to a cumulation in peroxynitrite (ONOO-) content and an injury of the mitochondrial membrane potential. Our study showed for the first time that fish ARG in head kidney M0/MФ can limit excessive production of NO and harmful products by iNOS to maintain mitochondrial and cellular homeostasis.

Uncovering Novel Protein Partners of Inducible Nitric Oxide Synthase in Human Testis.

Peroxidative damage to human spermatozoa has been shown to be the primary cause of male infertility. The possible role of nitric oxide (NO) in affecting sperm motility, capacitation, and acrosome reaction has been reported, too. The overproduction of NO by the enzyme inducible nitric oxide synthase (iNOS) could be responsible as it has been implicated in the pathogenesis of many diseases. There have been many studies on regulating iNOS function in various tissues, especially by protein-protein interaction; however, no study has looked for iNOS-interacting proteins in the human testis. Here, we have reported the identification of two proteins that interact with iNOS. We initially undertook a popular yeast two-hybrid assay to screen a human testis cDNA library in yeast using an iNOS-peptide fragment (amino acids 181-335) as bait. We verified our data using the mammalian chemiluminescent co-IP method; first, employing the same peptide and, then, a full-length protein co-expressed in HEK293 cells in addition to the candidate protein. In both cases, these two protein partners of iNOS were revealed: (a) sperm acrosome-associated 7 protein and (b) retinoblastoma tumor-suppressor binding protein.

Valosin-containing protein acts as a target and mediator of S-nitrosylation in the heart through distinct mechanisms.

S-nitrosylation (SNO) is an emerging paradigm of redox signaling protecting cells against oxidative stress in the heart. Our previous studies demonstrated that valosin-containing protein (VCP), an ATPase-associated protein, is a vital mediator protecting the heart against cardiac stress and ischemic injury. However, the molecular regulations conferred by VCP in the heart are not fully understood. In this study, we explored the potential role of VCP in cardiac protein SNO using multiple cardiac-specific genetically modified mouse models and various analytical techniques including biotin switch assay, liquid chromatography, mass spectrometry, and western blotting. Our results showed that cardiac-specific overexpression of VCP led to an overall increase in the levels of SNO-modified cardiac proteins in the transgenic (TG) vs. wild-type (WT) mice. Mass spectrometry analysis identified mitochondrial proteins involved in respiration, metabolism, and detoxification as primary targets of SNO modification in VCP-overexpressing mouse hearts. Particularly, we found that VCP itself underwent SNO modification at a specific cysteine residue in its N-domain. Additionally, our study demonstrated that glyceraldehyde 3-phosphate dehydrogenase (GAPDH), a key enzyme in glycolysis, also experienced increased SNO in response to VCP overexpression. While deletion of inducible nitric oxide synthase (iNOS) in VCP TG mice did not affect VCP SNO, it did abolish SNO modification in mitochondrial complex proteins, suggesting a dual mechanism of regulation involving both iNOS-dependent and independent pathways. Overall, our findings shed light on post-translational modification of VCP in the heart, unveiling a previously unrecognized role for VCP in regulating cardiac protein SNO and offering new insights into its function in cardiac protection.

iNOS is necessary for GBP-mediated T. gondii clearance in murine macrophages via vacuole nitration and intravacuolar network collapse.

Toxoplasma gondii is an obligate intracellular parasite of rodents and humans. Interferon-inducible guanylate binding proteins (GBPs) are mediators of T. gondii clearance, however, this mechanism is incomplete. Here, using automated spatially targeted optical micro proteomics we demonstrate that inducible nitric oxide synthetase (iNOS) is highly enriched at GBP2+ parasitophorous vacuoles (PV) in murine macrophages. iNOS expression in macrophages is necessary to limit T. gondii load in vivo and in vitro. Although iNOS activity is dispensable for GBP2 recruitment and PV membrane ruffling; parasites can replicate, egress and shed GBP2 when iNOS is inhibited. T. gondii clearance by iNOS requires nitric oxide, leading to nitration of the PV and collapse of the intravacuolar network of membranes in a chromosome 3 GBP-dependent manner. We conclude that reactive nitrogen species generated by iNOS cooperate with GBPs to target distinct structures in the PV that are necessary for optimal parasite clearance in macrophages.

Structure-activity relationship of anti-inflammatory meroterpenoids isolated from Dictyopteris polypodioides in RAW264 cells.

In this study, we explored anti-inflammatory compounds from the brown alga Dictyopteris polypodioides and isolated 7 meroterpenoids. Their anti-inflammatory activities were evaluated using the lipopolysaccharide-stimulated mouse macrophage cell line, RAW264. Yahazunol (1) exhibited similar nitric oxide (NO) production inhibitory activity as zonarol (2), which has previously been shown to be an anti-inflammatory compound. Yahazunol (1), zonarol (2), and isozonarol (3) inhibited not only NO production but also inducible nitric oxide synthase, interleukin-6, and C-C motif chemokine ligand 2 mRNA expression in RAW264 cells. The structure-activity relationships of the 11 compounds, including their synthetic analogs, revealed the significance of the hydroquinone moiety in the anti-inflammatory activity of these sesquiterpenoids in RAW264 cells. Diacetylated zonarol (9) exhibited an activity comparable to that of zonarol as a result of intracellular deacetylation. These results provide new insights into the anti-inflammatory activity of hydroquinone-containing natural products.

Depth of invasion of oral squamous cell carcinoma in Nos2-knockout mice correlated to alterations in systemic inflammatory markers following 4NQO treatment.

BACKGROUND: Peripheral blood analysis is a non-invasive and low-cost technique of prognostic value for several diseases, including oral cancer. Considering the role of inducible nitric oxide synthase in tumor-associated inflammation, this study purposed to evaluate the influence of this enzyme on peripheral blood parameters and systemic inflammatory biomarkers during murine oral carcinogenesis. METHODS: A 50 µg/mL solution of 4-nitroquinoleine-N-oxide was provided to 15 C57BL/6J (Nos2+/+ ) and 16 B6.129P2-Nos2tm1Lau /J (Nos2-/- ) for 16 weeks. Animals were followed for 8 weeks after treatment. Blood samples and tongues were collected for hematological and histopathological analyses. Red blood cells, white blood cells, and platelet cell parameters were analyzed. The neutrophil-to-lymphocyte ratio, platelet-to-lymphocyte ratio, and the systemic immune-inflammation index were also calculated. The depth of invasion of all carcinomas was measured. RESULTS: Differences were found in several blood parameters. The depth of invasion in Nos2-/- was lower than in Nos2+/+ (p = 0.009), and strong correlations were found between depth of invasion and neutrophil count (ρ = -0.68, p = 0.017), lymphocyte count (ρ = 0.72, p = 0.011), neutrophil-to-lymphocyte ratio (ρ = -0.65, p = 0.025), platelet-to-lymphocyte ratio (ρ = -0.73, p = 0.013), and systemic immune-inflammation index (ρ = -0.67, p = 0.037) in Nos2-/- mice. CONCLUSION: Inducible nitric oxide synthase seems to have an important role in OSCC invasion and progression, which might be associated to alterations in immune-inflammatory cell dynamics evidenced by peripheral blood and systemic inflammatory biomarkers.

Innate Immune-Enhancing Effect of Pinus densiflora Pollen Extract via NF-κB Pathway Activation.

Considering the emergence of various infectious diseases, including the coronavirus disease 2019 (COVID-19), people's attention has shifted towards immune health. Consequently, immune-enhancing functional foods have been increasingly consumed. Hence, developing new immune-enhancing functional food products is needed. Pinus densiflora pollen can be collected from the male red pine tree, which is commonly found in Korea. P. densiflora pollen extract (PDE), obtained by water extraction, contained polyphenols (216.29 ± 0.22 mg GAE/100 g) and flavonoids (35.14 ± 0.04 mg CE/100 g). PDE significantly increased the production of nitric oxide (NO) and reactive oxygen species (ROS) but, did not exhibit cytotoxicity in RAW 264.7 cells. Western blot results indicated that PDE induced the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2. PDE also significantly increased the mRNA and protein levels of cytokines and the phosphorylation of IKKα/ß and p65, as well as the activation and degradation of IκBα. Additionally, western blot analysis of cytosolic and nuclear fractions and immunofluorescence assay confirmed that the translocation of p65 to the nucleus after PDE treatment. These results confirmed that PDE increases the production of cytokines, NO, and ROS by activating NF-κB. Therefore, PDE is a promising nutraceutical candidate for immune-enhancing functional foods.

Antioxidant and Anti-Inflammatory Activities of Lindera glauca Extracts.

INTRODUCTION: The current study investigated the antioxidant and anti-inflammatory effects of ethanol extracts from Lindera glauca twig (LGT) and leaf/stem (LGLS). METHODS: The antioxidant activities were measured by total content of polyphenol and flavonoid, DPPH radical scavenging, and ABTS+ radical scavenging activity. To evaluate the anti-inflammatory effect in the LPS-induced RAW 264.7 cells, protein and mRNA expression of major inflammatory factors were analyzed using Western blot analysis and RT-PCR. RESULTS: The total polyphenol content of LGT and LGLS was 88.45 ± 11.74 and 115.75 ± 7.87 GA mg/g, respectively. The total flavonoid content was 66 ± 2.89 and 74.33 ± 2.89 QE mg/g. Both LGT and LGLS showed high DPPH and ABTS+ radical scavenging activities. Neither LGT nor LGLS was cytotoxic to RAW 264.7 cells. The anti-inflammatory activities were measured by LPS-induced RAW 264.7 cells. LGT and LGLS showed inhibition of the LPS-induced production of nitric oxide (NO), inducible NO synthase, cyclooxygenase-2 at the protein and mRNA levels, as determined by Western blotting and RT-PCR, respectively. In addition, the release of tumor necrosis factor-α and interleukin-6 mRNA expression levels of these cytokines was reduced by LGT and LGLS. CONCLUSION: These results suggest that LGT and LGLS extracts have potential for use as a functional antioxidant and anti-inflammatory ingredient in cosmetic industry.

Capsicum annuum var. abbreviatum Induces a Macrophage M2-Like Phenotype Mediated by Nrf2/HO-1 in LPS-Stimulated Macrophages.

Capsicum annuum var. abbreviatum (CAAE), which is in the genus Capsicum L. (Solanaceae), was found to be richer in polyphenols and flavonoids than other prevalent peppers of Capsicum annuum var. angulosum and Capsicum annuum. L. Yet, it is still unclear how CAAE reduces inflammation. In this study, we used the lipopolysaccharide-stimulated RAW264.7 macrophage cell line and bone marrow-derived macrophages to assess its anti-inflammatory activities. Initially, we discovered that CAAE decreased the levels of nitric oxide and inducible nitric oxide synthase. In addition, CAAE decreased the intracellular reactive oxygen species levels and increased the nuclear factor-erythroid 2-related factor 2 and heme oxygenase-1 compared with the phenotype of M2 macrophages. CAAE inhibited the activation of mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinases, c-Jun N-terminal kinases, and p38 MAPKs. CAAE also inhibited the translocation of nuclear factor kappa B into nuclear, hence preventing the production of proinflammatory cytokines. Therefore, we suggest that CAAE might have potential as a candidate therapeutic agent for inflammatory diseases.

Nilotinib exhibits less toxicity than imatinib and influences the immune state by modulating iNOS, p-p38 and p-JNK in LPS/IFN gamma-activated macrophages.

In this study, we aimed to analyze the effects of first and second-generation Bcr-Abl tyrosine kinase inhibitors, imatinib and nilotinib on LPS/IFN gamma activated RAW 264.7 macrophages. Our data revealed that imatinib was less effective on nitrite levels and more toxic on macrophages compared to nilotinib. Therefore, we further analysed the effect of nilotinib on various inflammatory markers including iNOS, COX-2, NFkB, IL-6, p-ERK, p-p38 and p-JNK in LPS/IFN gamma activated RAW264.7 macrophages. Spectrophotometric viability test and Griess assay,western blot, RT-PCR and luciferase reporter assays were used to analyze the biological activity of nilotinib. Our findings revealed that nilotinib decreases nitrite levels, iNOS mRNA, iNOS and p-p38 protein expressions significantly whereas induces IL-6 mRNA and p-JNK protein expressions at particular doses. We did not find significant effect of nilotinib on COX-2, p-ERK and nuclear p65 proteins and NFkB transcriptional activity. In addition, the binding mode of nilotinib to iNOS protein was predicted by molecular docking. According to the docking analyses, nilotinib exhibited hydrophobic interactions between MET349, ALA191, VAL346, PHE363, TYR367, MET368, CYS194, TRP366 residues at the binding pocket and the molecule as well as van der Waals interactions at specific residues. In conclusion, our results reveal that, in addition to its anticancer activity, nilotinib can exhibit immune modulatory effects on macrophages through its effects on iNOS, IL-6, p-p38 and p-JNK.

Identification of Major Bioactive Anti-inflammatory Compounds of Derris scandens Stem Using RAW 264.7 Cells and HPLC-UV Analysis.

Derris scandens (DS) is widely recognized for its therapeutic properties, specifically its analgesic effects, which significantly alleviate muscle pain. The chemical constituents of DS stem include various isoflavone derivatives. However, there is currently a lack of specified anti-inflammatory chemical markers and analytical methods for quality control. The present study aimed to evaluate the anti-inflammatory activity of DS and its constituents using the RAW 264.7 cell model. The expression of inflammatory genes such as inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), interleukin-6 (IL-6), and 5-lipoxygenase (5-LOX) was examined using quantitative RT-PCR. An high-performance liquid chromatography with a UV detection method was developed to quantitatively analyze genistein-7-O-[α-rhamnopyranosyl-(1 → 6)]-ß-glucopyranoside, genistein, derrisisoflavone A, lupalbigenin, and 6,8-diprenylgenistein in DS stem. The developed HPLC-UV method demonstrated high sensitivity with limits of detection and quantification ranging from 0.01 to 0.06 µg/mL and 0.03 to 0.18 µg/mL, respectively. The accuracy of the method ranged from 93.3 to 109.6%. Furthermore, the repeatability and reproducibility of the method were suitable, as indicated by the relative standard deviations of ≤ 3.02% and ≤ 6.22%, respectively. The DS extract notably inhibited NO production, exhibiting effects comparable to those of 500 µM diclofenac, and substantially suppressed the expression of iNOS, COX-2, IL-6, and 5-LOX of lipopolysaccharide (LPS)-induced genes. As to the pure isoflavone derivatives, the order of NO production inhibition was found to be genistein > lupalbigenin > derrisisoflavone A > 6,8-diprenylgenistein > genistein-7-O-[α-rhamnopyranosyl-(1 → 6)]-ß-glucopyranoside. Genistein, derrisisoflavone A, and 6,8-diprenylgenistein significantly suppressed the upregulation of all LPS-induced genes. Consequently, these compounds are recommended as anti-inflammatory markers for the quantitative chemical analysis of DS.

Superoxide, nitric oxide and peroxynitrite production by macrophages under different physiological oxygen tensions.

Macrophages count on two O2-consuming enzymes to form reactive radical species: NAPDH oxidase 2 (Nox2) and nitric oxide synthase 2 (inducible isoform, iNOS) that produce superoxide radical (O2•-) and nitric oxide (•NO), respectively. If formed simultaneously, the diffusion-controlled reaction of O2•- and •NO yields peroxynitrite, a potent cytotoxic oxidant. In human tissues and cells, the oxygen partial pressure (pO2) normally ranges within 2-14 %, with a typical average pO2 value for most tissues ca. 5 %. Given that O2 is a substrate for both Nox2 and iNOS, its tissue and cellular concentration can affect O2•- and •NO production. Also, O2 is a modulator of the macrophage adaptative response and may influence iNOS expression in a hypoxia inducible factor 1-α (HIF1α-)-dependent manner. However, most of the reported experiments in cellula, analyzing the formation and effects of O2•- and •NO during macrophage activation and cytotoxicity towards pathogens, have been performed in cells exposed to atmospheric air supplemented with 5 % CO2; under these conditions, most cells are exposed to supraphysiologic oxygen tensions (ca. 20 % O2) which are far from the physiological pO2. Here, the role of O2 as substrate in the oxidative response of J774A.1 macrophages was explored upon exposure to different pO2 and O2•- and •NO formation rates were measured, obtaining a KM of 26 and 42 µM O2 for Nox2 and iNOS, respectively. Consequently, peroxynitrite formation was influenced by pO2, reaching a maximum at ≥ 10 % O2, but even at levels as low as 2 % O2, a substantial formation rate of this oxidant was detected. Indeed, the cytotoxic capacity of immunostimulated macrophages against the intracellular parasite T. cruzi was significant, even at low pO2 values, confirming the role of peroxynitrite as a potent oxidizing cytotoxin within a wide range of physiological oxygen tensions.

Influence of microRNAs on iNOS expression in postmortem human infarction hearts.

MicroRNAs (miRNAs) are important post-transcriptional regulators in several diseases, including cancer, immunologic and cardiovascular diseases. A growing list of miRNAs are dysregulated in cardiac arrhythmias, contractility diseases, myocardial infarction (MI), sudden cardiac death (SCD), chronic heart failure and hypertrophy. However, the exact regulatory pathways, through which miRNAs exert their effects are often unclear. In this study, we measured the expression patterns of miR-21, miR-939 and miR-30e in postmortem human MI. The aim of the study was to examine the influence of these miRNAs on cardiac inducible nitric oxide synthase (iNOS) mRNA levels. We measured iNOS mRNA and miRNA expression patterns by means of qPCR. Further we used correlation analyses to determine causality between miRNA expression and cardiac iNOS levels. iNOS mRNA, miR-21, miR-939 and miR-30e were significantly upregulated in infarcted and non-infarcted regions of postmortem human MI hearts in comparison to healthy controls. While miR-21 and miR-939 showed their strongest expression in infarcted regions, miR-30e peaked in the non-infarcted myocardium. Further, we found a significant correlation between miR-939 and iNOS expression levels in controls and infarcted regions. The results indicate, that miR-939 is a regulator of cardiac iNOS expression. However, a massive iNOS activation might exceed the capability of miR-939 to keep its expression in balance. miR-21 and miR-30e do not seem to influence cardiac iNOS levels in MI. Further studies are needed to evaluate downstream targets of these miRNAs and their signaling pathways to clarify their role in human MI.

NRF2 interacts with distal enhancer and inhibits nitric oxide synthase 2 expression in KRAS-driven pancreatic cancer cells.

Nitric oxide is a pleiotropic free radical produced by three nitric oxide synthases (NOS1-3), of which inducible NOS2 is involved in tumor initiation and progression. In this study, RNA-seq, ChIP-seq and qRT-PCR experiments combined with bioinformatic analyses showed that NRF2 is a repressor of NOS2 gene by maintaining a distal enhancer located 22 kb downstream of TSS in an inactive state. Deletion of NRF2 leads to activation of the enhancer, which exerts a pioneering function before it is fully activated. Specifically, NRF2 controls the expression of NOS2 in response to intracellular oxidative stress and extracellular oxygen pressure. We found that abrogation of NOS2 expression by siRNAs partially reduced the ability of WT Panc-1 cells to form 3D spheroids, but strongly reduced the formation of 3D spheroids by NRF2-depleted Panc-1 cells. Mechanistically, this effect correlates with the finding that NOS2 and nitric oxide stimulate epithelial-to-mesenchymal transition in NRF2-depleted Panc-1 and MIA PaCa-2 cells. We also found that knockdown of NOS2 leads to blockade of 3D matrigel invasion of NRF2-depleted PDAC cells, demonstrating that a short-circuit in the reciprocal regulation of NOS2 and NRF2 attenuates the malignancy of PDAC cells. In summary, we show for the first time that: (i) NRF2 is a suppressor of NOS2 in pancreatic cancer cells; (ii) NRF2 binds to and inactivates an enhancer located 22 kb downstream of TSS of the NOS2 gene; (iii) activation of NOS2 requires suppression of NRF2; (iv) NOS2 is required for NRF2-depleted Panc-1 cells to maintain their malignancy and invasiveness.

Inducible nitric oxide synthase accelerates nonalcoholic fatty liver disease progression by regulating macrophage autophagy.

BACKGROUND: Cells and tissues, such as macrophages, express inducible nitric oxide synthase (INOS) after stimulation by certain factors. INOS helps mediate the macrophage inflammatory reaction, but few studies have explored how INOS affects macrophage function in nonalcoholic fatty liver disease (NAFLD). OBJECTIVE: This study investigated the role of INOS-mediated macrophage activity in NAFLD. METHODS: A high-fat diet was used to establish an NAFLD mouse model. After 12 weeks, blood was collected for immune cell and lipid analyses, and liver tissues were collected for pathological analyses with hematoxylin and eosin and Oil Red O staining. Peritoneal macrophages were extracted in situ, cultured in Dulbecco's modified Eagle's medium, and stimulated with palmitic acid to mimic in vivo conditions for further assays. Real-time polymerase chain reaction, western blot analysis, and immunofluorescence were used to verify the expression of target genes or proteins. RESULTS: In the NAFLD model, INOS expression in macrophages increased, and INOS knockdown significantly decreased the number of macrophages. Pathological examinations confirmed that INOS knockdown slowed NAFLD progression and macrophage infiltration during inflammation. INOS knockdown also enhanced phagocytosis and lipid transport by macrophages, and increased the expression of autophagy-related molecules in macrophages, which improved the autophagy level, promoted apoptotic cell degradation, and maintained intracellular environment homeostasis. CONCLUSIONS: These results indicate a correlation between INOS expression and macrophage function in NAFLD.

Transplacental infection by bovine alphaherpesvirus type 1 induces protein expression of COX-2, iNOS and inflammatory cytokines in fetal lungs and placentas.

Bovine alphaherpesvirus type 1 (BoAHV-1) is associated with respiratory and reproductive syndromes. Until present the immunologic mechanisms involved in BoAHV-1 abortion are partially known. We studied key elements of the innate immune response in the placentas and fetal lungs from cattle experimentally-inoculated with BoAHV-1. These tissues were analyzed by histopathology. Furthermore, virus identification was performed by qPCR and the expression of the inflammatory cytokines such as tumor necrosis factor-alpha, interleukin 1-alpha and inflammatory mediators like inducible nitric oxide synthase and cyclooxeganse-2 was evaluated by immunohistochemistry. The viral transplacental infection was confirmed by the detection of BoAHV-1 by qPCR in the placenta and fetal organs, which revealed mild inflammatory lesions. Inducible nitric oxide synthase immunolabelling was high in the lungs of infected fetuses and placentas, as well as for tumor necrosis factor-alpha in the pulmonary parenchyma and cyclooxeganse-2 in fetal annexes. However, the expression of interleukin 1-alpha was weak in these organs. To our knowledge, this is the first study that provides strong evidence of an early immune response to BoAHV-1 infection in the conceptus. Advances in the knowledge of the complex immunological interactions at the feto-maternal unit during BoAHV-1 infection are needed to clarify the pathogenesis of abortion.

Anti-neuroinflammatory Effects of Active Compound SPA1413 via Suppression of the MAPK and JAK/STAT Signaling Pathways.

Isoflavones and their derivatives possess neuroprotective activities against neurological disorders. Recently, the active compound SPA1413 (dehydroequol) derived from S-equol, an isoflavone-derived metabolite produced by human intestinal bacteria, was identified as a potent anti-amyloidogenic and neuroinflammatory candidate against Alzheimer's disease. However, its detailed modes of action, associated signaling pathways, and comparison with potential isoflavone derivatives have not yet been studied. Hence, the current study aimed to identify signaling pathways associated with SPA1413 using lipopolysaccharides (LPS)-stimulated BV2 cells as the experimental model via biological assays, Western blotting, and quantitative (q)RT-PCR. The results indicate that the SPA1413 anti-neuroinflammatory effect arises due to suppression of the nitric oxide (NO), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and mitogen-activated protein kinase (MAPK) signaling networks, including those of p38 and c-Jun N-terminal kinase (JNK). Interestingly, SPA1413 inhibited IL-11 through the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway. In addition, SPA1413 inhibited neuronal cell death by reducing LPS-activated microglia in neuronal N2a cells. Our findings suggest that SPA1413 may act as a strong anti-neuroinflammatory candidate by suppressing the MAPK and JAK/STAT signaling pathways.

Nitric Oxide Synthase Inhibition Prevents Cell Proliferation in Glioblastoma.

Glioblastoma multiforme (GBM) is a prevalent and aggressive primary brain tumor, presenting substantial treatment challenges and high relapse rates. GBM is characterized by alterations in molecular signaling and enzyme expression within malignant cells. This tumor exhibits elevated nitric oxide (NO.) levels. NO. is a crucial signaling molecule involved in the regulation of neuronal functions, synaptic transmission, and cell proliferation. It is primarily synthesized from L-arginine by nitric oxide synthase (NOS) enzymes. The increased levels of NO. in GBM stem from dysregulated activity and expression of clinically relevant NOS isoforms, particularly inducible NOS (iNOS) and neuronal NOS (nNOS). Based on this knowledge, we hypothesize that targeted pharmacological intervention with N6-(1-iminoethyl)-L-lysine (L-NIL), an iNOS inhibitor, and 7-Nitroindazole (7-NI), an nNOS inhibitor, may suggest a promising therapeutic strategy for the treatment of GBM. To test our hypothesis, we utilized the U87-MG cell line as an in vitro model of GBM. Our results showed that treatment with L-NIL and 7-NI led to a reduction in NO. levels, NOS activity, and clonogenic proliferation in U87-MG cells. These findings suggest that NO. and NOS enzymes might be prospective therapeutic targets for GBM.