scholarly journals The expression of Kruppel-like factor 2 in mice with collagen-induced arthritis and its role in the induction of nitric oxide synthase production

2020 ◽  
Vol 18 ◽  
pp. 205873922092348
Author(s):  
Donglei Wu ◽  
Xinye Wang ◽  
Shuaijun Xu ◽  
Wanjun Zhang ◽  
Shiyi Zeng ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Inos Expression ◽  
Joint Fluid ◽  
Enzyme Linked Immunosorbent Assay ◽  
Collagen Induced Arthritis ◽  
Tnf Α ◽  
Oxide Synthase ◽  
Inos Production

Kruppel-like factor 2 (KLF2) is associated with acute and chronic inflammation. However, the role of KLF2 in rheumatoid arthritis (RA) remains unknown. Here, we investigated the expression of KLF2 in mice with collagen-induced arthritis (CIA) to determine whether KLF2 levels correlate with inducible nitric oxide synthase (iNOS) production in vitro. Hematoxylin and eosin staining was used to assess the synovitis and bone destruction in mice. The concentration of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and nitric oxide (NO) in synovial fluid was determined by enzyme-linked immunosorbent assay, while western blot (WB) analysis was employed to detect the expression of KLF2 and iNOS in the synovium, heart, and kidneys. The expression of iNOS in MH7A cells was analyzed by quantitative polymerase chain reaction (PCR) and WB. The expression of KLF2 and iNOS was significantly elevated in the synovium, heart, and kidneys of CIA mice. This was correlated to an increase in the severity of arthritis and the concentration of inflammatory mediators including TNF-α, IL-6, and NO in joint fluid. KLF2 and iNOS expression in vitro was induced by TNF-α and KLF2 knockdown significantly reduced the TNF-α-induced iNOS expression. These findings indicate that KLF2 influences synovial inflammation in CIA mice by regulating iNOS production in synoviocytes.

scholarly journals Inducible nitric oxide synthase (iNOS) expression in monocytes during acute Dengue Fever in patients and during in vitro infection

2005 ◽  
Vol 5 (1) ◽  
Author(s):  
Patrícia CF Neves-Souza ◽  
Elzinandes L Azeredo ◽  
Sonia MO Zagne ◽  
Rogério Valls-de-Souza ◽  
Sonia RNI Reis ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Dengue Fever ◽  
Inducible Nitric Oxide Synthase ◽  
Inos Expression ◽  
Oxide Synthase ◽  
Inducible Nitric Oxide

Erhöhte NF-κB- und iNOS-Expression in Keratozyten von Keratokonuspatienten – Hinweise auf eine entzündliche Komponente?

2019 ◽  
Author(s):  
Tanja Stachon ◽  
Lorenz Latta ◽  
Krasimir Kolev ◽  
Berthold Seitz ◽  
Achim Langenbucher ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Nuclear Factor Kappa B ◽  
No Synthase ◽  
Inos Expression ◽  
Nuclear Factor ◽  
Nos Inhibitors ◽  
Inos Inhibitor ◽  
Oxide Synthase

Zusammenfassung Hintergrund In den letzten Jahren mehren sich Hinweise auf eine entzündliche Komponente beim Keratokonus (KC). Ein Schlüsselgen bei entzündlichen Prozessen ist der Nuclear Factor Kappa B (NF-κB). NF-κB ist ein Transkriptionsfaktor, der unter anderem das Enzym Nitric Oxide Synthase (NOS), das mit dem konkurrierenden Enzym Arginase (Arg) bei entzündlichen Prozessen involviert ist, aktiviert. Ziel dieser Studie war es, die Isotypen von NOS und Arginase zu analysieren, die Expression NF-κB, NOS und Arginase sowie den regulativen Mechanismus von NOS und Arginase in Keratozyten von Keratokonuspatienten mithilfe des Inhibitors 1400W in vitro zu untersuchen. Methoden Primäre humane Keratozyten wurden durch enzymatische Behandlung mit Kollagenase A aus humanen Korneoskleralscheiben (n = 8) und von Explantaten von geplanten perforierenden Keratoplastiken (KC-Patienten) isoliert (n = 8) und in DMEM/F12-Kulturmedium, versetzt mit 5% fetalem Kälberserum, kultiviert. Die Expression von NF-κB, NOS und Arginase wurden mit quantitativer PCR (qPCR) und Westernblot-Analyse (WB) untersucht. Nitrit- und Ureakonzentrationen im Zellkulturüberstand wurden nach Zugabe des NOS-Inhibitors 1400W (0 – 40 µM) analysiert. Ergebnisse In den Keratozyten wurden ausschließlich die Isotypen iNOS (induzierbare NO-Synthase) und Arg-II nachgewiesen. Die mRNA-Expression von NF-κB und iNOS waren in KC-Keratozyten höher als in normalen Zellen (p = 0,0135 und p = 0,0001), während in der Arg-II-Expression keine Unterschiede messbar waren. Im WB war bei NF-κB eine höhere Bandenintensität messbar (p = 0,0012), bei iNOS konnten keine Unterschiede in der Bandenintensität nachgewiesen werden. Im Überstand der KC-Keratozyten wurden geringere Konzentrationen von Nitrit und Urea nach Zugabe des Inhibitors 1400W gemessen (p = ≤ 0,014), nicht jedoch bei normalen Zellen (p ≥ 0,178). Schlussfolgerung Aufgrund der erhöhten Expression von NF-κB und iNOS muss von einer inflammatorischen Komponente beim Keratokonus ausgegangen werden. Die unterschiedliche Regulation der KC-Keratozyten durch den iNOS-Inhibitor 1400W legt eine veränderte metabolische Aktivität nahe, die durch entzündliche Prozesse hervorgerufen werden kann.

scholarly journals Anti-Inflammatory Effect of Simonsinol on Lipopolysaccharide Stimulated RAW264.7 Cells through Inactivation of NF-κB Signaling Pathway

Molecules ◽  
2020 ◽  
Vol 25 (16) ◽  
pp. 3573
Author(s):  
Lian-Chun Li ◽  
Zheng-Hong Pan ◽  
De-Sheng Ning ◽  
Yu-Xia Fu
Keyword(s):  
Nitric Oxide ◽  
Signaling Pathway ◽  
Morphological Changes ◽  
Raw264.7 Cells ◽  
Enzyme Linked Immunosorbent Assay ◽  
Tnf Α ◽  
Anti Inflammatory ◽  
Factor Α ◽  
Oxide Synthase ◽  
Necrosis Factor

Simonsinol is a natural sesqui-neolignan firstly isolated from the bark of Illicium simonsii. In this study, the anti-inflammatory activity of simonsinol was investigated with a lipopolysaccharide (LPS)-stimulated murine macrophages RAW264.7 cells model. The results demonstrated that simonsinol could antagonize the effect of LPS on morphological changes of RAW264.7 cells, and decrease the production of nitric oxide (NO), tumor necrosis factor α (TNF-α), and interleukin 6 (IL-6) in LPS-stimulated RAW264.7 cells, as determined by Griess assay and enzyme-linked immunosorbent assay (ELISA). Furthermore, simonsinol could downregulate transcription of inducible nitric oxide synthase (iNOS), TNF-α, and IL-6 as measured by reverse transcription polymerase chain reaction (RT-PCR), and inhibit phosphorylation of the alpha inhibitor of NF-κB (IκBα) as assayed by Western blot. In conclusion, these data demonstrate that simonsinol could inhibit inflammation response in LPS-stimulated RAW264.7 cells through the inactivation of the nuclear transcription factor kappa-B (NF-κB) signaling pathway.

scholarly journals Activation of Nuclear Factor κB and Induction of Inducible Nitric Oxide Synthase by Ureaplasma urealyticumin Macrophages

2000 ◽  
Vol 68 (12) ◽  
pp. 7087-7093 ◽  
Author(s):  
Y.-H. Li ◽  
Z.-Q. Yan ◽  
J. Skov Jensen ◽  
K. Tullus ◽  
A. Brauner
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Inducible Nitric Oxide Synthase ◽  
Alveolar Macrophages ◽  
Nuclear Factor Κb ◽  
Inos Expression ◽  
Dependent Manner ◽  
Nuclear Factor ◽  
Oxide Synthase ◽  
Inducible Nitric Oxide

ABSTRACT Chronic lung disease (CLD) of prematurity is an inflammatory disease with a multifactorial etiology. The importance ofUreaplasma urealyticum in the development of CLD is debated, and steroids produce some improvement in neonates with this disease. In the present study, the capability of U. urealyticum to stimulate rat alveolar macrophages to produce nitric oxide (NO), express inducible nitric oxide synthase (iNOS), and activate nuclear factor κB (NF-κB) in vitro was characterized. The effect of NO on the growth of U. urealyticum was also investigated. In addition, the impact of dexamethasone and budesonide on these processes was examined. We found that U. urealyticum antigen (≥4 × 107 color-changing units/ml) stimulated alveolar macrophages to produce NO in a dose- and time-dependent manner (P < 0.05). This effect was further enhanced by gamma interferon (100 IU/ml; P < 0.05) but was attenuated by budesonide and dexamethasone (10−4 to 10−6 M) (P < 0.05). The mRNA and protein levels of iNOS were also induced in response to U. urealyticum and inhibited by steroids.U. urealyticum antigen triggered NF-κB activation, a possible mechanism for the induced iNOS expression, which also was inhibited by steroids. NO induced by U. urealyticum caused a sixfold reduction of its own growth after infection for 10 h. Our findings imply that U. urealyticum may be an important factor in the development of CLD. The host defense response againstU. urealyticum infection may also be influenced by NO. The down-regulatory effect of steroids on NF-κB activation, iNOS expression, and NO production might partly explain the beneficial effect of steroids in neonates with CLD.

Nitric oxide synthase inhibitors 7- and 6-nitroindazole relax smooth muscle in vitro

1994 ◽  
Vol 256 (1) ◽  
pp. R5-R6 ◽  
Author(s):  
Andrew D. Medhurst ◽  
Carol Greenlees ◽  
Andrew A. Parsons ◽  
Susan J. Smith
Keyword(s):  
Nitric Oxide ◽  
Smooth Muscle ◽  
Nitric Oxide Synthase ◽  
Nitric Oxide Synthase Inhibitors ◽  
Oxide Synthase

Abstract 085: CHIP: E3 Ubiquitin Ligase Mediates Proteasomal Degradation of Neuronal Nitric Oxide Synthase in the Paraventricular Nucleus of Rats with Heart Failure

Hypertension ◽  
2017 ◽  
Vol 70 (suppl_1) ◽  
Author(s):  
Neeru M Sharma ◽  
Kenichi Katsurada ◽  
Xuefei Liu ◽  
Kaushik P Patel
Keyword(s):  
Nitric Oxide ◽  
Heart Failure ◽  
Nitric Oxide Synthase ◽  
Paraventricular Nucleus ◽  
Ubiquitin Ligase ◽  
Protein A ◽  
Neuronal Nitric Oxide Synthase ◽  
Proteasomal Degradation ◽  
Oxide Synthase

The exaggerated sympathetic drive is a characteristic of heart failure (HF) due to reduced neuronal nitric oxide synthase (nNOS) within the paraventricular nucleus (PVN). Previously we have shown that there were increased accumulation of nNOS-ubiquitin (nNOS-Ub) conjugates in the PVN of rats with HF (1.0±0.05 Sham vs. 1.29±0.06 HF) due to the increased levels of PIN (a protein inhibitor of nNOS, known to dissociate nNOS dimers into monomers) (0.76±0.10 Sham vs. 1.12±0.09 HF) and decreased levels of tetrahydrobiopterin (BH4): a cofactor required for stabilization of nNOS dimers (0.62±0.02 Sham vs. 0.44±0.03 HF). We also showed that there is blunted nitric oxide-mediated inhibition of sympathetic tone via the PVN in HF. Here we examined whether CHIP(C-terminus of Hsp70 -interacting protein), a chaperone-dependent E3 ubiquitin-protein isopeptide ligase known to ubiquitylate Hsp90-chaperoned proteins could act as an ubiquitin ligase for nNOS in the PVN. Immunofluorescence studies revealed colocalization of nNOS and CHIP in the PVN indicating their possible interaction. CHIP expression was increased by 50% in the PVN of rats with HF(0.96±0.08 Sham vs.1.44±0.10* HF). It is shown that Hsp90 protects nNOS from ubiquitination while Hsp70 promotes the ubiquitination and degradation. We observed significant upregulation of Hsp70 (0.49±0.03 Sham vs. 0.65±0.02* HF) with a trend toward the decrease in Hsp90 expression (0.90±0.07 Sham vs. 0.71±0.06 HF). The opposing effects of the two chaperones could account for the increased CHIP-mediated ubiquitination and degradation of dysfunctional nNOS monomers in the PVN of rats with HF. Furthermore, neuronal NG108-15 cell line transfected with the pCMV3-CHIP-GFP spark (CHIP overexpression plasmid) showed approximately 74% increase in CHIP with concomitant 49% decrease in nNOS expression. In vitro ubiquitination assay in NG108 cells transfected with pCMV-(HA-Ub) 8 and pCMV3-CHIP-GFP spark plasmid reveal increased HA-Ub-nNOS conjugates (1.13 ± 0.09 Scramble vs. 1.65 ± 0.12* CHIP plasmid). Taken together, our results identify CHIP as an E3 ligase for ubiquitination of dysfunctional nNOS and CHIP expression is augmented during HF leading to increased proteasomal degradation of nNOS in the PVN.

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