Molecular activation of NF-κB, pro-inflammatory mediators, and signal pathways in γ-irradiated mice

The effects of γ-irradiation on inflammatory gene expression, including NF-κB activation, in the kidney of C57/BL6 mice exposed to 1–9 Gy doses of ⁶⁰Co γ-irradiation. Radiation enhanced the NF-κB activation and oxidative stress caused a dose-dependent disruption in the redox balance. The significance of this study is the new molecular information gained on γ-irradiation effects through the activation of pro-inflammatory genes by NF-κB via the MAPK signaling pathway. Considering the exquisite sensitivity of NF-κB and other pro-inflammatory mediators to the redox status, we conclude that the activation of inflammatory processes by irradiation is likely initiated by increased oxidative stress.     

Docosahexaenoic acid attenuates VCAM-1 expression and NF-κB activation in TNF-α-treated human aortic endothelial cells

This study was conducted to test the hypothesis that n-3 polyunsaturated fatty acids are able to down-regulate expression of adhesion molecules and nuclear factor-κB (NF-κB) activation in vascular endothelial cells, in addition to reducing atherosclerotic lesions in vivo. We report here that docosahexaenoic acid (DHA) reduces atherosclerotic lesions in the aortic arteries of apolipoprotein E knockout (apoE^-/-) mice. Consistent with the observation in animal study, DHA inhibited THP-1 cell adhesion to tumor necrosis factor α (TNF-α)-activated human aortic endothelial cells (HAECs). Expression of vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) on the cell surface of HAECs was determined by cell-surface enzyme-linked immunosorbent assay. DHA and eicosapentaenoic acid decreased VCAM-1 expression in a dose-dependent manner in TNF-α treated HAECs, while cis-linoleic acid and arachidonic acid did not have any significant effect on either VCAM-1 or ICAM-1 expression. Moreover, DHA significantly reduced VCAM-1 protein expression in the cell lysates of TNF-α-treated HAECs, as determined by Western blot analysis. In line with NF-κB signaling pathway, DHA suppressed the TNF-α-activated IκBα phosphorylation and degradation as well as IκB kinase-β phosphorylation. Subsequently, translocation of the NF-κB (p50/p65) and AP-1 (c-Fos/c-Jun) subunits was down-regulated by DHA in the nucleus of HAECs. These results suggest that DHA negatively regulates TNF-α-induced VCAM-1 expression through attenuation of NF-κB signaling pathway and AP-1 activation. This study provides evidence that DHA may contribute to the prevention of atherosclerosis and inflammatory diseases in vivo.     

Anti-inflammatory activity of myricetin from Diospyros lotus through suppression of NF-κB and STAT1 activation and Nrf2-mediated HO-1 induction in lipopolysaccharide-stimulated RAW264.7 macrophages

Diospyros lotus is traditionally used for the treatment of diabetes, diarrhea, tumor, and hypertension. The purpose of this study was to investigate the anti-inflammatory effect and underlying molecular mechanisms of myricetin in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. Myricetin dose-dependently suppressed the production of pro-inflammatory mediators (NO, iNOS, PGE₂, and COX-2) in LPS-stimulated RAW264.7 macrophages. Myricetin administration decreased the production of NO, iNOS, TNF-α, IL-6, and IL-12 in mice. Myricetin decreased NF-κB activation by suppressing the degradation of IκBα, nuclear translocation of p65 subunit of NF-κB, and NF-κB DNA binding activity in LPS-stimulated RAW264.7 macrophages. Moreover, myricetin attenuated the phosphorylation of STAT1 and the production of IFN-β in LPS-stimulated RAW264.7 macrophages. Furthermore, myricetin induced the expression of HO-1 through Nrf2 translocation. In conclusion, these results suggest that myricetin inhibits the production of pro-inflammatory mediators through the suppression of NF-κB and STAT1 activation and induction of Nrf2-mediated HO-1 expression in LPS-stimulated RAW264.7 macrophages.     

Stercurensin inhibits nuclear factor-κB-dependent inflammatory signals through attenuation of TAK1-TAB1 complex formation

We identified a chalcone, 2',4'-dihydroxy-6'-methoxy-3'-methylchalcone (stercurensin), as an active compound isolated from the leaves of Syzygium samarangense. In the present study, the anti-inflammatory effects and underlying mechanisms of stercurensin were examined using lipopolysaccharide (LPS)-stimulated RAW264.7 cells and mice. To determine the effects of stercurensin in vitro, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression were analyzed by RT-PCR and immunoblotting. Nuclear factor-κB (NF-κB) activation and its upstream signaling cascades were also investigated using a dual-luciferase reporter assay, electrophoretic mobility shift assay, immunoblotting, immunofluorescence, and immunoprecipitation. To verify the effects of stercurensin in vivo, the mRNA expression levels of iNOS and COX-2 were evaluated in isolated mouse peritoneal macrophages by quantitative real-time PCR, and the production of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and IL-1β were assessed in serum samples from mice using a Luminex system. Pretreatment with stercurensin reduced LPS-induced iNOS and COX-2 expression, thereby inhibiting nitric oxide (NO) and prostaglandin E(2) production, respectively. In addition, an inhibitory effect of stercurensin on NF-κB activation was shown by the recovery of LPS-induced inhibitor of κB (I-κB) degradation after blocking the transforming growth factor-β-activated kinase 1 (TAK1)/I-κB kinase signaling pathway. In mouse models, stercurensin negatively regulated NF-κB-dependent pro-inflammatory mediators and cytokines. These results demonstrate that stercurensin modulates NF-κB-dependent inflammatory pathways through the attenuation of TAK1-TAB1 complex formation. Our findings demonstrating the anti-inflammatory effects of stercurensin in vitro and in vivo will aid in understanding the pharmacology and mode of action of stercurensin.     

Critical role of X-box binding protein 1 in NADPH oxidase 4-triggered cardiac hypertrophy is mediated by receptor interacting protein kinase 1

NADPH oxidase 4 (NOX4) and the NOX4-related redox signaling are implicated in cardiac hypertrophy. NOX4 is interrelated with endoplasmic reticulum stress (ERS). Spliced X-box binding protein 1 (Xbp1s) is a key mediator of ERS while its role in cardiac hypertrophy is still poorly understood. Recently, receptor interacting protein kinase 1(RIPK1) has been increasingly reported to be associated with ERS. Therefore, we aimed to test the hypothesis that Xbp1s mediates NOX4-triggered cardiac hypertrophy via RIPK1 signaling. In the heart tissue of transverse aortic constriction (TAC) rats and in primary cultured neonatal cardiomyocytes(NCMs) treated with angiotensinII(AngII) or isoproterenol (ISO), NOX4 expression and reactive oxygen species (ROS) generation, and expression of Xbp1s as well as RIPK1-related phosphorylation of P65 subunit of NF-κB were elevated. Gene silencing of NOX4 by specific small interfering RNA (siRNA) significantly blocked the upregulation of NOX4, generation of ROS, splicing of Xbp1 and activation of the RIPK1-related NF-κB signaling, meanwhile attenuated cardiomyocyte hypertrophy. In addition, ROS scavenger (N-acetyl-L-cysteine, NAC) and NOX4 inhibitor GKT137831 reduced ROS generation and alleviated activation of Xbp1 and RIPK1-related NF-κB signaling. Furthermore, splicing of Xbp1 was responsible for the increase in RIPK1 expression in AngII or ISO-treated NCMs. Upregulated RIPK1 in turn activates NF-κB signaling in a kinase activity-independent manner. These findings suggest that Xbp1s plays an important role in NOX4-triggered cardiomyocyte hypertrophy via activating its downstream effector RIPK1, which may prove significant for the development of future therapeutic strategies.     

Metalloprotease type III effectors that specifically cleave JNK and NF-κB

Two major arms of the inflammatory response are the NF-κB and c-Jun N-terminal kinase (JNK) pathways. Here, we show that enteropathogenic Escherichia coli (EPEC) employs the type III secretion system to target these two signalling arms by injecting host cells with two effector proteins, NleC and NleD. We provide evidence that NleC and NleD are Zn-dependent endopeptidases that specifically clip and inactivate RelA (p65) and JNK, respectively, thus blocking NF-κB and AP-1 activation. We show that NleC and NleD co-operate and complement other EPEC effectors in accomplishing maximal inhibition of IL-8 secretion. This is a remarkable example of a pathogen using multiple effectors to manipulate systematically the host inflammatory response signalling network.     

Mas-related G protein-coupled receptor D participates in inflammatory pain by promoting NF-κB activation through interaction with TAK1 and IKK complex

Mas-related G protein-coupled receptor D (MrgprD) is mainly expressed in small-diameter sensory neurons of the dorsal root ganglion (DRG). Results from previous studies suggest that MrgprD participates in mechanical hyperalgesia and nerve injury-induced neuropathic pain. However, it remains elusive whether and how MrgprD is involved in inflammatory pain. Here, we used a mouse model of chronic inflammatory pain established by intraperitoneal administration of lipopolysaccharide (LPS). The LPS injection induced an evident peripheral neuroinflammation and mechanical hyperalgesia in the mice and increased MrgprD expression in the DRG. The LPS administration also augmented the proportion of MrgprD-expressing neurons in the lumbar 4 DRG. Behaviorally, the LPS-induced hypersensitivities to mechanical and cold stimuli, but not to a heat stimulus, were substantially attenuated in Mrgprd-knockout mice compared with wildtype littermates. Mrgprd deletion in DRGs suppressed the LPS-triggered activation of the NF-κB signaling pathway and attenuated LPS-induced up-regulation of pro-inflammatory factors. Moreover, ectopic overexpression of MrgprD in HEK293 cells stably expressing mouse toll-like receptor 4 (TLR4) markedly promoted the LPS-induced NF-κB activation and enhanced NF-κB's DNA-binding activity. Furthermore, MrgprD physically interacted with TGF-β-activated kinase 1 (TAK1) and I-kappa-B-kinase (IKK) complexes, but not with mitogen-activated protein kinases (MAPKs) in mouse DRGs. In macrophage-like RAW 264.7 cells, MrgprD also interacted with TAK1 and IKK complex, and the treatment of MrgprD agonist elicited the activation of NF-κB signaling, but not of mitogen-activated protein kinases (MAPKs) signaling pathway. Our findings indicate that MrgprD facilitates the development of LPS-triggered persistent inflammatory hyperalgesia by promoting canonical NF-κB activation, highlighting the important roles of MrgprD in NF-κB-mediated inflammation and chronic pain.     

MicroRNA 421 suppresses DPC4/Smad4 in pancreatic cancer

Dihydroavenanthramide D (DHAvD) is a synthetic analog to naturally occurring avenanthramide, which is the active component of oat. Previous study demonstrates that DHAvD strongly inhibits activation of nuclear factor-kappa B (NF-κB), which is a major component in cancer cell invasion. The present study investigated whether DHAvD can modulate MMP-9 expression and cell invasion in MCF-7 human breast cancer cells. MMP-9 expression and cell invasion in response to 12-O-tetradecanoylphorbol-13-acetate (TPA) was increased, whereas these inductions were muted by DHAvD. DHAvD also suppressed activation of mitogen-activated protein kinase (MAPK), and MAPK-mediated nuclear factor-kappa B (NF-κB) and activator protein-1 (AP-1) activations in TPA-treated MCF-7 cells. The results indicate that DHAvD-mediated inhibition of TPA-induced MMP-9 expression and cell invasion involves the suppression of the MAPK/NF-κB and MAPK/AP-1 pathways in MCF-7 cells. DHAvD may have potential value in breast cancer metastasis.