Inhibitor Of NF-κB Kinase Research Articles

Houttuynia cordata Thunb repairs steroid-induced avascular necrosis of the femoral head through regulating NF-κB signaling pathway

To investigate the influences of Houttuynia cordata Thunb (HCT) in steroid-induced avascular necrosis of the femoral head (SANFH), we conducted a comprehensive study evaluating the effects of HCT on various aspects. Cell Counting Kit-8 assay was used to examine bone marrow stem cells (BMSCs) cell viability. Flow cytometry and lactate dehydrogenase detection assay were conducted to determine cell apoptosis. The levels of apoptosis-related proteins, osteogenic-related markers, inflammatory factors, and nuclear factor kappa B (NF-κB) pathway-associated proteins were determined via western blotting. Hematoxylin and eosin and terminal deoxynucleotidyl transferase dUTP nick-end labeling assays were utilized to verify the effects of HCT in SANFH rats. Our findings revealed that HCT could enhanced cell viability and arrested cell apoptosis in dexamethasone (Dex)-treated BMSCs. Dex increased the levels of cleaved caspase-3, Bcl2-associated X, interleukin (IL)-1β, IL-18, IL-6, p65, and inhibitor of NF-κB kinase β (IKKβ), while this promoting trend was weakened by HCT. Moreover, pyrrolidine dithiocarbamate (PDTC, an inhibitor of NF-κB signaling pathway) further increased the inhibitory role of apoptosis and the levels of IL-1β, IL-18, and IL-6 and the promotional effect of the levels of RUNX2 and ALP in Dex-treated BMSCs. The in-vivo assays showed that HCT decreased the percentage of empty lacunae, apoptosis, and the levels of IL-1β, IL-18, IL-6, p65, and IKKβ in SANFH rats. In conclusion, our study demonstrated that HCT relieved SANFH, which might be possibly achieved by NF-κB pathway.

TLR5S negatively regulates the TLR5M-mediated NF-κB signaling pathway in Epinephelus coioides

Nuclear factor kappa-B (NF-κB) pathway is a key mediator of inflammation response that plays a role in host defense for pathogen elimination, but excessive activation may lead to tissue damage or pathogen transmission. The negative regulation of NF-κB in lower vertebrates is largely unknown, hindering further understanding of immune signaling evolution. Here, we provided evidence that Epinephelus coioides soluble toll-like receptor 5 (TLR5S), a member of the TLR5 subfamily, has been newly identified as a negative regulator of NF-κB signaling. EcTLR5S was a cytoplasmic protein consisting of 17 leucine-rich repeat domains, which specifically responded to Vibrio flagellin and suppressed flagellin-induced NF-κB signaling activation and cytokine expression. The amino-terminal LRR 1–5 region was necessary for its negative regulatory function. Dual-luciferase reporter assay showed that EcTLR5S significantly inhibited the NF-κB-luc activity induced by inhibitor of NF-κB kinase α (IKKα) and IKKβ. Subsequently, the functional relationship between EcTLR5M and EcTLR5S was analyzed, revealing that the negative regulatory function of EcTLR5S targeted the activation of the NF-κB pathway mediated by EcTLR5M. The above results reveal that EcTLR5S negatively regulates the flagellin-induced EcTLR5M-NF-κB pathway activation, which may prevent over-activation of immune signaling and restore homeostasis.

Anti-rheumatic effect of KMU-11361 in various rheumatoid arthritis-related cells targeting multiple protein kinase

Abstract Protein kinases important for regulating protein phosphorylation, which is a promising therapeutic target for inflammatory disease, such as rheumatoid arthritis (RA). In this study, we synthesized a novel multi-protein kinase inhibitor, KMU-11361, a derivative of indolin-2-one and investigated the mechanisms of anti-inflammatory and anti-rheumatic effects using THP-1 cells, RAW264.7 cells, Jurkat T cells, and human RA fibroblast-like synoviocytes (RA-FLS). Our results revealed that KMU-11361 inhibited LPS-induced upregulation of inducible iNOS, and COX-2, and suppressed LPS-induced phosphorylation of TAK1, JNK, inhibitor of NF-κB kinase α/β (IKKα/β), IκBα, and NF-κB p65 as well as nuclear translocation of NF-κB p65 in THP-1 cells. Furthermore, KMU-11361 attenuated proinflammatory cytokines such as IL-1β, TNF-α, and IL-6, and, remarkably, inhibited LPS-induced activation of the NLRP3 inflammasome in THP-1 cells. Additionally, in activated Jurkat T cells, KMU-11361 inhibited the TAK1-IKKα-mediated NF-κB pathway as well as the phosphorylation of MAPKs. Moreover, KMU-11361 inhibited anti-CD3/CD28 antibodies-induced upregulation of proinflammatory cytokine IL-2. Moreover, it suppressed LPS-induced upregulation of proinflammatory cytokines, p-TAK1, p-IKKα/β, and p-NF-κB p65 in RA-FLS. Surprisingly, KMU-11361 also inhibited the receptor activator of NF-κB-induced osteoclast differentiation and up-regulation of nuclear factor of activated T-cells, NFATc-1 and c-Fos in RAW264.7 cells. Taken together, our results suggest that KMU-11361 has anti-inflammatory and anti-rheumatic effects in various RA-related cells and could be developed as a potent anti-rheumatic drug.

Epstein-Barr Virus Envelope Glycoprotein gp110 Inhibits IKKi-Mediated Activation of NF-κB and Promotes the Degradation of β-Catenin.

Epstein-Barr virus (EBV) infects host cells and establishes a latent infection that requires evasion of host innate immunity. A variety of EBV-encoded proteins that manipulate the innate immune system have been reported, but whether other EBV proteins participate in this process is unclear. EBV-encoded envelope glycoprotein gp110 is a late protein involved in virus entry into target cells and enhancement of infectivity. Here, we reported that gp110 inhibits RIG-I-like receptor pathway-mediated promoter activity of interferon-β (IFN-β) as well as the transcription of downstream antiviral genes to promote viral proliferation. Mechanistically, gp110 interacts with the inhibitor of NF-κB kinase (IKKi) and restrains its K63-linked polyubiquitination, leading to attenuation of IKKi-mediated activation of NF-κB and repression of the phosphorylation and nuclear translocation of p65. Additionally, gp110 interacts with an important regulator of the Wnt signaling pathway, β-catenin, and induces its K48-linked polyubiquitination degradation via the proteasome system, resulting in the suppression of β-catenin-mediated IFN-β production. Taken together, these results suggest that gp110 is a negative regulator of antiviral immunity, revealing a novel mechanism of EBV immune evasion during lytic infection. IMPORTANCE Epstein-Barr virus (EBV) is a ubiquitous pathogen that infects almost all human beings, and the persistence of EBV in the host is largely due to immune escape mediated by its encoded products. Thus, elucidation of EBV's immune escape mechanisms will provide a new direction for the design of novel antiviral strategies and vaccine development. Here, we report that EBV-encoded gp110 serves as a novel viral immune evasion factor, which inhibits RIG-I-like receptor pathway-mediated interferon-β (IFN-β) production. Furthermore, we found that gp110 targeted two key proteins, inhibitor of NF-κB kinase (IKKi) and β-catenin, which mediate antiviral activity and the production of IFN-β. gp110 inhibited K63-linked polyubiquitination of IKKi and induced β-catenin degradation via the proteasome, resulting in decreased IFN-β production. In summary, our data provide new insights into the EBV-mediated immune evasion surveillance strategy.

Turmeronols (A and B) from <i>Curcuma longa</i> have anti-inflammatory effects in lipopolysaccharide-stimulated BV-2 microglial cells by reducing NF-κB signaling

Turmeronols (A and B), bisabolane-type sesquiterpenoids found in turmeric, reduce inflammation outside the brain in animals; however, their effects on neuroinflammation, a common pathology of various neurodegenerative diseases, are not understood. Inflammatory mediators produced by microglial cells play a key role in neuroinflammation, so this study evaluated the anti-inflammatory effects of turmeronols in BV-2 microglial cells stimulated with lipopolysaccharide (LPS). Pretreatment with turmeronol A or B significantly inhibited LPS-induced nitric oxide (NO) production; mRNA expression of inducible NO synthase; production of interleukin (IL)-1β, IL-6, and tumor necrosis factor α and upregulation of their mRNA expression; phosphorylation of nuclear factor-κB (NF-κB) p65 proteins and inhibitor of NF-κB kinase (IKK); and nuclear translocation of NF-κB. These results suggest that these turmeronols may prevent the production of inflammatory mediators by inhibiting the IKK/NF-κB signaling pathway in activated microglial cells and can potentially treat neuroinflammation associated with microglial activation.

Abstract P3071: Ikkε Deficiency Reduces P38 Activity Of Macrophages To Increases Cardiac Injury

Background: Inhibitor of NF-κB kinase ε (IKKε) is a non-classical modulator of nuclear factor-kappa B (NF-κB), and is reported to regulate inflammation in various diseases. In this study, the involvement of IKKε in pathophysiological responses was examined in a mouse myocardial infarction model. Methods: MI was induced in IKKε-/- mice by coronary artery ligation. Neutrophils and macrophages were isolated from the heart tissues or bone marrows for further comparison studies by Western blot, PCR, and flow cytometry. Cell phenotypes were identified by immunofluorescence staining. Results: The IKKε-/- group showed poor early survival rate, high circulating IL-6 level, large cardiac fibrosis, and low ejection fraction (30.33±5.25% vs. 34.02±5.53%, p<0.05) compared with the wild type group. Further study revealed that the numbers of neutrophils and macrophages in the heart were higher in the IKKε-/- group than in the PBS group. Macrophage phenotype was more inflammatory along with lower p38 activity in the IKKε-/- group. Importantly, we identified macrophage-myofibroblast transition (MMT) in the infarcted myocardium and found that the number of MMT F4/80(+)SMA(+) cells was 2-fold higher in the IKKε-/- group. The inactivation of p38 accelerated MMT in human macrophages, and the level of phosphorylated p38 was lower in infarct zone of human failing heart tissues. Conclusion: Collectively, IKKε-p38 axis may lead macrophages to aggravated inflammation and accelerated MMT, resulting in poor outcome after myocardial infarction. Here, we suggest that restoring IKKε or p38 in macrophages could be the target to attenuate cardiac inflammation.

MicroRNA-155-5p Targets SKP2, Activates IKKβ, Increases Aβ Aggregation, and Aggravates a Mouse Alzheimer Disease Model.

The nuclear factor kappa B (NF-κB) pathway and inhibitor of NF-κB kinase β (IKKβ) are involved in Alzheimer disease (AD) pathogenesis. This study explored the mechanisms underlying IKKβ-mediated Aβ aggregation and neuron regeneration in APP.PS1 mice. Adenoviral transduction particles were injected into the hippocampal CA1 region of the mice to knock down or inhibit target genes. Morris water maze was performed to evaluate the cognitive function of the mice. Aβ deposition was determined by histological examination. sh-IKKβ plasmids and microRNA (miR)-155-5p inhibitor were transfected into Aβ1-42-induced N2a cells. The expressions of AD-related proteins were detected by Western blot. The interaction between S-phase kinase-associated protein 2 (SKP2) and IKKβ was assessed by co-immunoprecipitation. IKKβ knockdown (KD) and miR-155-5p inhibition ameliorated cognitive impairment, improved neuron regeneration, and attenuated Aβ deposition in APP/PS1 mice. SKP2 KD aggravated cognitive impairment, inhibited neuron regeneration, and promoted Aβ deposition in the mice. SKP2 regulated the stability of IKKβ protein via ubiquitination. MiR-155-5p regulates Aβ deposition and the expression of Aβ generation-related proteins in N2a cells via targeting SKP2. These results indicate that the miR-155-5p/SKP2/IKKβ axis was critical for pathogenesis in this AD model and suggest the potential of miR-155-5p as a target for AD treatment.

The Anti-Cancer Effects of a Zotarolimus and 5-Fluorouracil Combination Treatment on A549 Cell-Derived Tumors in BALB/c Nude Mice.

Zotarolimus is a semi-synthetic derivative of rapamycin and a novel immunosuppressive agent used to prevent graft rejection. The pharmacological pathway of zotarolimus restricts the kinase activity of the mammalian target of rapamycin (mTOR), which potentially leads to reductions in cell division, cell growth, cell proliferation, and inflammation. These pathways have a critical influence on tumorigenesis. This study aims to examine the anti-tumor effect of zotarolimus or zotarolimus combined with 5-fluorouracil (5-FU) on A549 human lung adenocarcinoma cell line implanted in BALB/c nude mice by estimating tumor growth, apoptosis expression, inflammation, and metastasis. We established A549 xenografts in nude mice, following which we randomly divided the mice into four groups: control, 5-FU (100 mg/kg/week), zotarolimus (2 mg/kg/day), and zotarolimus combined with 5-FU. Compared the results with those for control mice, we found that mice treated with zotarolimus or zotarolimus combined with 5-FU retarded tumor growth; increased tumor apoptosis through the enhanced expression of cleaved caspase 3 and extracellular signal-regulated kinase (ERK) phosphorylation; decreased inflammation cytokines levels (e.g., IL-1β, TNF-α, and IL-6); reduced inflammation-related factors such as cyclooxygenase-2 (COX-2) protein and nuclear factor-κB (NF-κB) mRNA; enhanced anti-inflammation-related factors including IL-10 and inhibitor of NF-κB kinase α (IκBα) mRNA; and inhibited metastasis-related factors such as transforming growth factor β (TGF-β), CD44, epidermal growth factor receptor (EGFR), and vascular endothelial growth factor (VEGF). Notably, mice treated with zotarolimus combined with 5-FU had significantly retarded tumor growth, reduced tumor size, and increased tumor inhibition compared with the groups of mice treated with 5-FU or zotarolimus alone. The in vivo study confirmed that zotarolimus or zotarolimus combined with 5-FU could retard lung adenocarcinoma growth and inhibit tumorigenesis. Zotarolimus and 5-FU were found to have an obvious synergistic tumor-inhibiting effect on lung adenocarcinoma. Therefore, both zotarolimus alone and zotarolimus combined with 5-FU may be potential anti-tumor agents for treatment of human lung adenocarcinoma.

KMU-1170, a Novel Multi-Protein Kinase Inhibitor, Suppresses Inflammatory Signal Transduction in THP-1 Cells and Human Osteoarthritic Fibroblast-Like Synoviocytes by Suppressing Activation of NF-κB and NLRP3 Inflammasome Signaling Pathway.

Protein kinases regulate protein phosphorylation, which are involved in fundamental cellular processes such as inflammatory response. In this study, we discovered a novel multi-protein kinase inhibitor, KMU-1170, a derivative of indolin-2-one, and investigated the mechanisms of its inflammation-inhibiting signaling in both THP-1 cells and human osteoarthritic fibroblast-like synoviocytes (FLS). We demonstrated that in THP-1 cells, KMU-1170 inhibited lipopolysaccharide (LPS)-induced upregulation of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), and, furthermore, suppressed LPS-induced phosphorylation of transforming growth factor-β-activated kinase 1, JNK, ERK, inhibitor of NF-κB kinase α/β (IKKα/β), and NF-κB p65 as well as nuclear translocation of NF-κB p65. Moreover, KMU-1170 suppressed LPS-induced upregulation of proinflammatory cytokines such as IL-1β, TNF-α, and IL-6, and, notably, inhibited LPS-induced upregulation of the NLRP3 inflammasome in THP-1 cells. Importantly, KMU-1170 attenuated LPS-mediated inflammatory responses in human osteoarthritic FLS, such as the upregulation of IL-1β, TNF-α, IL-6, iNOS, and COX-2 and the phosphorylation of IKKα/β and NF-κB p65. Collectively, these results suggest that KMU-1170 inhibits inflammatory signal transduction and could be developed as a potential anti-inflammatory agent.

Oyster TBK1/IKKε responds to bacterial and viral challenges and participates in the innate immune signaling

The Pacific oyster, Crassostrea gigas, is distributed worldwide and has important economic value. However, in recent years, oyster farming has been seriously plagued by diseases, and research on the innate immune system of oysters is scarce. The IKK (Inhibitor of NF-κB kinases)-related kinases TBK1 (TANK-binding kinase-1) and IKKε are crucial signal transduction proteins, which play important roles in NF-κB and IRF3/7 activation. In this study, a TBK1/IKKε homolog (termed CgTBK1/IKKε) was obtained from the Pacific oyster. The open reading frame of CgTBK1/IKKε is 2259-bp and encodes 752 amino acids. Quantitative real-time polymerase chain reaction results revealed that CgTBK1/IKKε transcripts could be detected in all test tissues, and its expression was induced by lipopolysaccharide and polyinosinic–polycytidylic acid challenge. The results of co-immunoprecipitation assays showed that CgTBK1/IKKε could interact with oyster mitochondria antiviral signaling (MAVS) and myeloid differential protein-88 (MyD88), which are the key adaptor proteins of the retinoic acid–inducible gene-I-like receptor and toll-like receptor signaling pathways, respectively. Additionally, CgTBK1/IKKε, CgTBK1, and CgIKKε-like can combine with each other and form a complex, which may be critical for the immune signaling transduction and downstream target protein activation. Finally, CgTBK1/IKKε transfection into human cell lines inhibited the activation activity of interferon-stimulated response element (ISRE) and NF-κB reporter genes caused by CgIKKε-like transfection. In summary, these results indicate that CgTBK1/IKKε can respond to pathogen infection, participate in immune signaling, and regulate the activation of NF-κB and ISRE reporter genes. Therefore, CgTBK1/IKKε may be a key signal transduction molecule and immune activity regulator, and may play an important role in the immune system of oysters.

Abstract 430: Immature Cardiac Fibroblasts With Upregulated P53 Contributed to Fibrosis in Ikke Deficient Mouse Myocardial Infarction Model

Background: Inhibitor of NF-κB kinase (IKK), an upstream of nuclear factor-kappa B (NF-κB), is a critical modulator for pathophysiological inflammation. IKKε is a non-classical IKK and has been studied in infectious diseases and cancers. However, the role of IKKε in a myocardial infarction (MI) has not been addressed. Methods and Results: In this study, we used IKKε knockout (KO) mice to induce MI by coronary artery ligation. The IKKε KO group showed poor early survival rate, large cardiac fibrosis (14.7±4.8% in KO vs. 31.1±10.2% in WT, p <0.05), and low fractional shortening (13.47±1.21% in KO vs. 16.36±4.46% in WT, p <0.05) compared with WT group. Next, we investigated the inflammatory responses and found that inflammatory markers such as inducible nitric oxide synthase (iNOS) and CD80 were much higher in both cardiac macrophages and bone marrow-derived macrophages (BMDM) in the IKKε KO group than in the wild type (WT) group. To explore the responsible mediator, we performed phosphorylated protein array and found phosphorylated p38 was significantly downregulated in the IKKε knockout BMDM. Conversely, both knockdown of p38 by siRNA and inhibition of p38 by SB203580 treatment in RAW264.7 cells upregulated iNOS. More interestingly, IKKε deficient cardiac fibroblasts showed highly accumulated nuclear p53 and exhibited immature differentiation. The levels of myofibroblast markers containing α-smooth muscle actin, periostin, and transforming growth factor-β1 were lower, and functional contractility was substantially impaired in the cardiac fibroblasts isolated from IKKε KO mice. Conclusion: Our data showed excessive inflammation was associated with p38 inactivation in macrophages and pathological fibrosis was resulted from immature myofibroblast phenotype with p53 upregulation. Collectively, IKKε is involved in the control of inflammation resolution and wound healing process in the infarcted myocardium.

Ethyl acetate extract of herpetospermum pedunculosum alleviates α-naphthylisothiocyanate-induced cholestasis by activating the farnesoid x receptor and suppressing oxidative stress and inflammation in rats

BackgroundTraditionally, seeds of Herpetospermum pedunculosum were used to treat liver disease or cholepathy. Up to date, their protecting effect against cholestasis was remain unclarified. PurposeTo investigate the efficacy, possible mechanisms, and active constituents of the ethyl acetate extract from the seeds of Herpetospermum pedunculosum (HPEAE), studies were carried out using cholestasis rat model induced by α-naphthylisothiocyanate (ANIT). MethodsMale rats were intragastrically treated with HPEAE (100, 200 or 400 mg/kg) once a day for 7 days and were modeled with ANIT (60 mg/kg). The levels of serum indicators, bile flow, and histopathology were evaluated. Indices of oxidative stress and inflammatory mediators were detected using the enzyme-linked immunosorbent assay. Western blotting method was employed for analyzing the protein levels in the signal pathways of farnesoid X receptor (FXR), kelch ech associating protein 1/nuclear factor erythroid 2-related factor 2 (Keap1/Nrf2) and nuclear factor κB (NF-κB). The chemical compositions of HPEAE was analyzed by HPLC, and partially chemical components of HPEAE were identified by comparisons of their retention times with the standards. The FXR agonistic activity of the identified compounds was evaluated in l-02 cells induced by guggulsterone using a high-content screening system. ResultsThe cholestasis caused by ANIT can be significantly ameliorated by restoring the liver function indexes of alanine transaminase, aspartate transaminase, alkaline phosphatase, gamma-glutamyltransferase, total bilirubin, direct bilirubin and total bile acid, which are dose-dependent, as well as pathological liver injury and bile flow. Mechanical studies suggested that HPEAE can activate the expression of FXR and then up regulate its downstream proteins (multidrug resistance-associated protein 2, bile salt export pump and Na+/taurocholate cotransporting polypeptide). Moreover, the levels of the active oxygen index glutathione, superoxide dismutase, glutathione peroxidase, catalase and malondialdehyde were markedly restored by treatment with HPEAE. Western blotting further confirmed that HPEAE up regulated the expression of quinone oxidoreductase 1, heme oxygenase 1 and Keap1, lowered the expression of Nrf2 and reduced oxidative stress. HPEAE also up regulated P-glycoprotein 65, phosphorylated P-glycoprotein 65 and inhibitor of NF-κB kinase α expression, down regulated inhibitor of NF-κB (IκB), restored inflammatory mediator tumor necrosis factor-α, interleukin-1β (IL-1β), IL-6 and IL-10, and reduced inflammatory response. Fifteen compounds were identified (12 lignans and 3 coumarins). Among them, five lignans exhibited the significant FXR agonistic activity in vitro. ConclusionHPEAE may alleviate the cholestasis and liver injury caused by ANIT in rats by activating FXR, as well as suppressing the Keap1/Nrf2 and NF-κB signaling pathways and lignans may be its main active components.

Astragaloside IV regulates NF-κB-mediated cellular senescence and apoptosis of hepatic stellate cells to suppress PDGF-BB-induced activation.

Activated hepatic stellate cells (HSCs) are the principal effectors during hepatic fibrosis, which is characterized by the accumulation of extracellular matrix. Therefore, present therapies and investigations into hepatic fibrosis mainly focus on the suppression of activated HSCs. Astragaloside IV (ASIV) is an effective constituent extracted from the plant Astragalus membranaceus and has exhibited anti-fibrotic properties in hepatic fibrosis. However, its protective mechanism against hepatic fibrosis is not fully understood. The present study aimed to investigate the mechanistic role of ASIV on rat HSC-T6 cells activated with platelet-derived growth factor (PDGF)-BB. HSC-T6 cells were activated using PDGF-BB and subsequently treated with ASIV (final concentrations of 20 and 40 µg/ml) for 48 h. ASIV treatment decreased the expression of α1 type I collagen, α-smooth muscle actin and fibronectin on mRNA and protein levels, suggesting that ASIV suppresses PDGF-BB-induced HSC-T6 activation. Senescence-associated β-galactosidase activity, p21, high-mobility group AT-hook 1 and p53, common biomarkers of senescence, were upregulated by ASIV treatment. In addition, the expression of telomerase reverse transcriptase was reduced. ASIV promoted apoptosis of PDGF-BB-activated HSC-T6 cells. The NF-κB signaling pathway, which controls cellular senescence and apoptosis, was demonstrated to be stimulated by ASIV by increasing p65, p52, p50 and inhibitor of NF-κB kinase α expression levels, and by suppressing the expression of NF-κB inhibitor α. Taken together, these results demonstrated that ASIV promoted cellular senescence and apoptosis by activating the NF-κB pathway to suppress PDGF-BB-induced HSC-T6 activation; with potential implications for the treatment of hepatic fibrosis.

FM0807 decelerates experimental arthritis progression by inhibiting inflammatory responses and joint destruction via modulating NF-κB and MAPK pathways

Background: Rheumatoid arthritis (RA) is a chronic articular synovial inflammatory disease. The precise etiology underlying the pathogenesis of RA remains unknown. We aimed to investigate the inhibitory effect of curcumin analog FM0807 (curcumin salicylate monoester, 2-hydroxy-, 4-[(1E,6E)-7-(4-hydroxy-3-methoxyphenyl)-3,5-dioxo-1,6-heptadien-1-yl]-2-methoxyphenyl ester) on experimental RA and investigate its possible mechanisms of action.Method: Rats with Freund’s complete adjuvant (FCA)-induced arthritis (AIA) were administered aspirin (0.1 mmol.kg−1), curcumin (0.1 mmol.kg−1), FM0807 (0.1, 0.2 mmol.kg−1) and vehicle via gastric gavage, from days 7 to 21, once daily. The hind paw volume and arthritis index (AI) were measured, and radiographic and histological examinations were performed. Twenty-one days later, the animals were killed and left ankle joints were removed to measure protein expression of the elements of the nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) pathway by Western blot analysis. The enzyme-linked immunosorbent assay (ELISA) was employed to measure synovial fluid levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-6, IL-1β and IL-10.Results: Compared with AIA group, FM0807 reduced the AI and swelling of the injected hind paw in a dose-dependent manner, and inhibited increases in inflammatory cell infiltration, pannus formation and cartilage destruction. FM0807 also potently attenuated the increase in the expression of inflammatory factors TNF-α, IL-6 and IL-1β in synovial fluid, while IL-10 levels were also elevated. FM0807 significantly suppressed phosphorylation of extracellular-signal-regulated kinase (ERK) 1/2 (ERK1/2), c-Jun-N-terminal kinase (JNK) 1/2 (JNK1/2), p38MAPK, inhibitor of NF-κB kinase (IKK), IκB and NF-κB p65 protein, (all P<0.05), which displayed more potential effects compared with those of the aspirin and curcumin groups.Conclusion: FM0807 exerts its therapeutic effects on RA by inhibiting cartilage degeneration. FM0807 treatment might be an effective therapeutic approach for RA.

GSK-3β Inhibitor Induces Expression of the TLR4/MyD88/NF-κB Signaling Pathway to Protect Against Renal Ischemia-Reperfusion Injury During Rat Kidney Transplantation.

Ischemia-reperfusion injury (IRI) is an inevitable consequence of kidney transplantation (KT). The aim of our study was to investigate the protective effect of a glycogen synthase kinase 3β (GSK-3β) inhibitor against cold IRI in a rat renal transplantation (RT) model and a rat cold-IRI model through the toll-like receptor 4 (TLR4)/myeloid differentiation factor 88 (MyD88)/nuclear factor κ-light-chain-enhancer of the activated B cell (NF-κB) signaling pathway. We treated Sprague Dawley (SD) rats in the RT and cold-IRI models with 5mg/kg and 1mg/kg, respectively, of the GSK-3β inhibitor 4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (TDZD-8). We then measured inflammatory factors, i.e., tumor necrosis factor alpha (TNF-α) and interleukins-1β and IL-6 (IL-1β, IL-6), as well as oxidative stress markers, i.e., superoxide dismutase (SOD) and malondialdehyde (MDA), in serum and kidneys. Renal function tests and pathological examinations were performed at 0, 1, 2, 3, and 7days after RT or cold IRI. We measured expression of TLR4, MyD88, inhibitor of NF-κB kinase (IκB), phosphorylated IκB (p-IκB), NF-κB p65, p-p65, GSK-3β, and phosphorylated GSK-3β (p-GSK-3β) by Western blot and immunohistological staining. After intervention with the GSK-3β inhibitor, renal function was improved; oxidative stress injury was reduced; expression of p-GSK-3β was upregulated; expression of p-IκB, TLR4, MyD88, and p-p65 was downregulated; pathological damage was significantly reduced; and expression of TNF-α, IL-1β, and IL-6 messenger ribonucleic acid (mRNA) was downregulated. These results strongly suggested that GSK-3β might be a key target for the treatment of IRI in KT. The GSK-3β inhibitor inhibited phosphorylation of NF-κB p65 and IκB by inhibiting the TLR/MyD88 pathway, reducing oxidative stress injury and the production of downstream inflammatory factors.

Abstract 221: IKKe Deficiency Aggravates Cardiac Inflammation with Dysregulation of p52 and p38 in Myocardial Infarction

Inhibitor of NF-κB kinase (IKK), an upstream of nuclear factor-kappa B (NF-κB), is a critical modulator for pathophysiological inflammation. IKKε is a non-classical IKK and has been studied in infectious diseases and cancers. However, the role of IKKε in a myocardial infarction (MI) has not been addressed. In this study, we used IKKε knockout mice to induce MI by coronary artery ligation. Cardiac function was analyzed by echocardiograph after 2 weeks, and fractional shortening (FS) was 16.36±4.46% in the wild type group and 13.47±1.21% in the knockout group. Cardiac fibrosis and macrophage infiltration deteriorated in the knockout group. Next, we investigated the inflammatory responses and found that the expression of inducible nitric oxide synthase (iNOS), an inflammatory marker, was much higher in both infarcted heart tissues and bone marrow-derived macrophages (BMDM) isolated from knockout mice than from wild type mice. Besides, cardiac macrophages displayed more inflammatory phenotype in the IKKε knockout group than in the wild type group. To explore the responsible mediator, we performed phosphorylated protein array and found phosphorylated p38 was significantly downregulated in the IKKε knockout BMDM. Conversely, both knockdown of p38 by siRNA and inhibition of p38 by SB203580 treatment in RAW264.7 cells upregulated iNOS induction. In the infarcted heart tissue, non-canonical NF-κB2 (p52) protein was dramatically upregulated in the IKKε knockout group than in the wild type group, while mRNA level was not different in the both groups. Immunohistochemical analysis showed nuclear accumulation of p52 in cardiomyocytes and fibroblasts in the peri-infarct lesion. Our data showed excessive inflammation in IKKε knockout mice was associated with inactivation of p38 in macrophages and upregulated p52 in the infarcted myocardium. Collectively, IKKε is involved in the control of inflammation resolution through modulating p38 activity and p52 post-translational modification in the infarcted myocardium.

IκB Kinase α Is Required for Development and Progression of KRAS-Mutant Lung Adenocarcinoma.

Although oncogenic activation of NFκB has been identified in various tumors, the NFκB-activating kinases (inhibitor of NFκB kinases, IKK) responsible for this are elusive. In this study, we determined the role of IKKα and IKKβ in KRAS-mutant lung adenocarcinomas induced by the carcinogen urethane and by respiratory epithelial expression of oncogenic KRASG12D Using NFκB reporter mice and conditional deletions of IKKα and IKKβ, we identified two distinct early and late activation phases of NFκB during chemical and genetic lung adenocarcinoma development, which were characterized by nuclear translocation of RelB, IκBβ, and IKKα in tumor-initiated cells. IKKα was a cardinal tumor promoter in chemical and genetic KRAS-mutant lung adenocarcinoma, and respiratory epithelial IKKα-deficient mice were markedly protected from the disease. IKKα specifically cooperated with mutant KRAS for tumor induction in a cell-autonomous fashion, providing mutant cells with a survival advantage in vitro and in vivo IKKα was highly expressed in human lung adenocarcinoma, and a heat shock protein 90 inhibitor that blocks IKK function delivered superior effects against KRAS-mutant lung adenocarcinoma compared with a specific IKKβ inhibitor. These results demonstrate an actionable requirement for IKKα in KRAS-mutant lung adenocarcinoma, marking the kinase as a therapeutic target against this disease.Significance: These findings report a novel requirement for IKKα in mutant KRAS lung tumor formation, with potential therapeutic applications. Cancer Res; 78(11); 2939-51. ©2018 AACR.

IKKs and tumor cell plasticity.

Nuclear factor κB (NF-κB) transcription factors are the central hubs of signaling pathways connecting proinflammatory signals to cell survival, proliferation and cytokine production. In cancers, NF-κB signaling influences many aspects of tumor development, from initiation to metastasis. These functions are mediated by tumor-induced plasticity that allows tumor cells to adapt and survive in changing conditions within the tumor microenvironment. Tumor cell plasticity is shaped by the inflammatory microenvironment in tumors. This review focuses on inhibitor of NF-κB kinases, the direct upstream elements of NF-κB regulation, specifically on their conventional and non-conventional functions in animal models of tumorigenesis from the recent literature.

Sustained high-fat diet modulates inflammation, insulin signalling and cognition in mice and a modified xenin peptide ameliorates neuropathology in a chronic high-fat model.

To demarcate pathological events in the brain as a result of short-term to chronic high-fat-diet (HFD) feeding, which leads to cognitive impairment and neuroinflammation, and to assess the efficacy of Xenin-25[Lys(13)PAL] in chronic HFD-fed mice. C57BL/6 mice were fed an HFD or a normal diet for 18 days, 34 days, 10 and 21 weeks. Cognition was assessed using novel object recognition and the Morris water maze. Markers of insulin signalling and inflammation were measured in brain and plasma using immunohistochemistry, quantitative PCR and multi-array technology. Xenin-25[Lys(13)PAL] was also administered for 5 weeks in chronic HFD-fed mice to assess therapeutic potential at a pathological stage. Recognition memory was consistently impaired in HFD-fed mice and spatial learning was impaired in 18-day and 21-week HFD-fed mice. Gliosis, oxidative stress and IRS-1 pSer616 were increased in the brain on day 18 in HFD-fed mice and were reduced by Xenin-25[Lys(13)PAL] in 21-week HFD-fed mice. In plasma, HFD feeding elevated interleukin (IL)-6 and chemokine (C-X-C motif) ligand 1 at day 34 and IL-5 at week 10. In the brain, HFD feeding reduced extracellular signal-regulated kinase 2 (ERK2), mechanistic target of rapamycin (mTOR), NF-κB1, protein kinase C (PKC)θ and Toll-like receptor 4 (TLR4) mRNA at week 10 and increased expression of glucacon-like peptide-1 receptor, inhibitor of NF-κB kinase β, ERK2, mTOR, NF-κB1, PKCθ and TLR4 at week 21, elevations that were abrogated by Xenin-25[Lys(13)PAL]. HFD feeding modulates cognitive function, synapse density, inflammation and insulin resistance in the brain. Xenin-25[Lys(13)PAL] ameliorated markers of inflammation and insulin signalling dysregulation and may have therapeutic potential in the treatment of diseases associated with neuroinflammation or perturbed insulin signalling in the brain.

IKK2/NF‐κB signaling protects neurons after traumatic brain injury

Traumatic brain injury (TBI) is the leading cause of death in young adults. After the initial injury, a poorly understood secondary phase, including a strong inflammatory response determines the final outcome of TBI. The inhibitor of NF-κB kinase (IKK)/NF-κB signaling system is the key regulator of inflammation and also critically involved in regulation of neuronal survival and synaptic plasticity. We addressed the neuron-specific function of IKK2/NF-κB signaling pathway in TBI using an experimental model of closed-head injury (CHI) in combination with mouse models allowing conditional regulation of IKK/NF-κB signaling in excitatory forebrain neurons. We found that repression of IKK2/NF-κB signaling in neurons increases the acute posttraumatic mortality rate, worsens the neurological outcome, and promotes neuronal cell death by apoptosis, thus resulting in enhanced proinflammatory gene expression. As a potential mechanism, we identified elevated levels of the proapoptotic mediators Bax and Bad and enhanced expression of stress response genes. This phenotype is also observed when neuronal IKK/NF-κB activity is inhibited just before CHI. In contrast, neuron-specific activation of IKK/NF-κB signaling does not alter the TBI outcome. Thus, this study demonstrates that physiological neuronal IKK/NF-κB signaling is necessary and sufficient to protect neurons from trauma consequences.—Mettang, M., Reichel, S. N., Lattke, M., Palmer, A., Abaei, A., Rasche, V., Huber-Lang, M., Baumann, B., Wirth, T. IKK2/NF-κB signaling protects neurons after traumatic brain injury.