Inhibitor Of Nuclear Factor κB Kinase Research Articles

RNF31-mediated IKKα ubiquitination aggravates inflammation and intestinal injury through regulating NF-κB activation in human and mouse neonates

AimsNeonatal necrotizing enterocolitis (NEC) is a leading cause of intestine inflammatory disease, and macrophage is significantly activated during NEC development. Posttranslational modifications (PTMs) of proteins, particularly ubiquitination, play critical roles in immune response. This study aimed to investigate the effects of ubiquitin-modified proteins on macrophage activation and NEC, and discover novel NEC-related inflammatory proteins. Materials and methodsProteomic and ubiquitin proteomic analyses of intestinal macrophages in NEC/healthy mouse pups were carried out. In vitro macrophage inflammation model and in vivo NEC mouse model, as well as clinical human samples were used for further verification the inhibitor of nuclear factor-κB kinase α (IKKα) ubiquitination on NEC development through Western blot, immunofluorescence, quantitative real-time polymerase chain reaction (qRT-PCR) and flow cytometry. Key findingsWe report here that IKKα was a new ubiquitin-modified protein during NEC through ubiquitin proteomics, and RING finger protein 31 (RNF31) acted as an E3 ligase to be involved in IKKα degradation. Inhibition of IKKα ubiquitination and degradation with siRNF31 or proteasome inhibitor decreased nuclear factor-κB (NF-κB) activation, thereby decreasing the expression of pro-inflammatory factors and M1 macrophage polarization, resulting in reliving the severity of NEC. SignificanceOur study suggests the activation of RNF31-IKKα-NF-κB axis triggering NEC development and suppressing RNF31-mediated IKKα degradation may be therapeutic strategies to be developed for NEC treatment.

Zhitong Jiangu decoction mitigates osteoarthritis in rabbits via regulation of NF-κB signaling pathway


 
 
 
 Purpose: To investigate the effect of Zhitong Jiangu decoction (ZJD) on osteoarthritis rabbits, and the mechanism of action involved.
 Methods: Chondrocytes were obtained from the knees of osteoarthritic rabbits. These chondrocytes were randomly assigned to 7 groups: sham, 5, 10 and 20 % normal serum groups; 5 % ZJD, 10 % ZJD and 20 % ZJD groups. The gross and histopathological features of the rabbit cartilage were examined by microscopy. Each group was treated with a different concentration of rabbit normal serum or rabbit drug-containing serum. The protective effect of different concentrations of ZJD on the cells were determined. Cell proliferation and concentrations of IL-1 and MMP-3 were determined using cell counting kit (CCK) 8 and enzyme-linked immunosorbent assay (ELISA), respectively. The mRNA and protein expressions of inhibitor of nuclear factor-κB kinase α (IKK-α) and nuclear factor kappa B p65 (NF- κB p65) were determined by quantitative reverse transcription polymerase chain reaction (qRT- PCR) and immunoblotting, as appropriate.
 Results: Gross and histopathological examinations of rabbit cartilage showed that osteoarthritis was successfully established in rabbit knee joint. Cell proliferation significantly and time- and concentration-dependently increased in drug-containing serum groups, relative to sham and normal serum-containing groups. However, expressions of NF-κB p65, IL-1, MMP-3 and IKK-α were markedly and time- and concentration-dependently reduced in drug-containing serum groups, relative to sham and normal serum-containing groups (p < 0.05).
 Conclusion: These results indicate that ZJD mitigates osteoarthritis in rabbits via regulation of NF-κB signaling pathway. Thus, it can potentially be developed for the management of osteoarthritis.
 
 
 

Seabuckthorn Berries Extract Attenuates Pulmonary Vascular Hyperpermeability in Lipopolysaccharide-Induced Acute Lung Injury in Mice.

ObjectiveTo investigate the effect of seabuckthorn berries extract (SBE) on pulmonary vascular hyperpermeability in the mice model of acute lung injury (ALI) induced by lipopolysaccharide (LPS).MethodsSixty Kunming mice were allocated into 6 groups by a random number table, including control, LPS, dexamethasone (Dex, 1 mg/kg), and 120, 240 and 480 mg/kg SBE groups, 10 mice in each group. Except the control group, mice were pre-treated with Dex and SBE, respectively, for 7 days before LPS was intraperitoneally injected to induce ALI. Pulmonary vascular hyperpermeability was evaluated by histopathologic observation and transvascular leakage determination. Tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) levels in serum were measured using enzyme-linked immunosorbent assay. The expression of nuclear factor-kappa B (NF-κB) p65 in lung cells was determined by immunofluorescence analysis. The contents of cytoplasmic inhibitor of nuclear factor-κB kinase (IKK) and nuclear p65, as well as downstream proteins of E-selectin (CD62E) and intercellular adhesion molecule-1 (ICAM-1), were determined using Western blot analysis.ResultsHistopathological observation confirmed SBE treatment alleviated morphological lesion induced by LPS. Compared with the LPS group, 480 mg/kg SBE significantly decreased the water content of lung, Evans blue accumulation in lung tissue, and protein concentration and neutrophils count in bronchoalveolar lavage fluid (P<0.01); moreover, 480 mg/kg SBE significantly suppressed release of TNF-α and IL-6, and down-regulated expressions of IKK, nuclear p65, ICAM-1 and CD62E (P<0.01).ConclusionSBE maintained alveolar-capillary barrier integrity under endotoxin challenge in mice by suppressing the key factors in the pathogenesis of ALI.Electronic Supplementary MaterialSupplementary material (Appendix 1) is available in the online version of this article at 10.1007/s11655-021-3346-1.

Assessing the Association of Mitochondrial Function and Inflammasome Activation in Murine Macrophages Exposed to Select Mitotoxic Tri-Organotin Compounds.

Background:Mitochondrial function is implicated as a target of environmental toxicants and found in disease or injury models, contributing to acute and chronic inflammation. One mechanism by which mitochondrial damage can propagate inflammation is via activation of the nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family, pyrin domain-containing receptor (NLRP)3 inflammasome, a protein complex that processes mature interleukin . plays an important role in the innate immune response and dysregulation is associated with autoinflammatory disorders.Objective:The objective was to evaluate whether mitochondrial toxicants recruit inflammasome activation and processing.Method:Murine macrophages (RAW 264.7) exposed to tri-organotins (triethyltin bromide (TETBr), trimethyltin hydroxide (TMTOH), triphenyltin hydroxide (TPTOH), bis(tributyltin)oxide) [Bis(TBT)Ox] were examined for pro-inflammatory cytokine induction. TMTOH and TETBr were examined in RAW 264.7 and bone marrow-derived macrophages for mitochondrial bioenergetics, reactive oxygen species (ROS) production, and inflammasome activation via visualization of aggregate formation, caspase-1 flow cytometry, enzyme-linked immunosorbent assay and Western blots, and microRNA (miRNA) and mRNA arrays.Results:TETBr and TMTOH induced inflammasome aggregate formation and release in lipopolysaccharide (LPS)-primed macrophages. Mitochondrial bioenergetics and mitochondrial ROS were suppressed. Il1a and Il1b induction with LPS or challenge was diminished. Differential miRNA and mRNA profiles were observed. Lower miR-151-3p targeted cyclic adenosine monophosphate (cAMP)-mediated and AMP-activated protein kinase signaling pathways; higher miR-6909-5p, miR-7044-5p, and miR-7686-5p targeted Wnt beta-catenin signaling, retinoic acid receptor activation, apoptosis, signal transducer and activator of transcription 3, IL-22, IL-12, and IL-10 signaling. Functional enrichment analysis identified apoptosis and cell survival canonical pathways.Conclusion:Select mitotoxic tri-organotins disrupted murine macrophage transcriptional response to LPS, yet triggered inflammasome activation. The differential response pattern suggested unique functional changes in the inflammatory response that may translate to suppressed host defense or prolong inflammation. We posit a framework to examine immune cell effects of environmental mitotoxic compounds for adverse health outcomes. https://doi.org/10.1289/EHP8314

Mitochondrial reactive oxygen species mediate the lipopolysaccharide-induced pro-inflammatory response in human gingival fibroblasts

Although periodontal diseases are initiated by bacteria that colonize the tooth surface and gingival sulcus, the host response is believed to play an essential role in the breakdown of connective tissue and bone. Mitochondrial reactive oxygen species (mtROS) have been proposed to regulate the activation of the inflammatory response by the innate immune system. However, the role of mtROS in modulating the response of human gingival fibroblasts (HGFs) to immune stimulation by lipopolysaccharides (LPS) has yet to be fully elucidated. Here, we showed that LPS from Porphyromonas gingivalis stimulated HGFs to increase mtROS production, which could be inhibited by treatment with a mitochondrial-targeted exogenous antioxidant (mito-TEMPO) or transfection with manganese superoxide dismutase (MnSOD). A time-course study revealed that an increase in the concentration of mtROS preceded the expression of inflammatory cytokines in HGFs. Mito-TEMPO treatment or MnSOD transfection also significantly prevented the LPS-induced increase of interleukin (IL)-1β, IL-6, and tumor necrosis factor-α. Furthermore, suppressing LPS-induced mtROS generation inhibited the activation of p38, c-Jun N-terminal kinase, and inhibitor of nuclear factor-κB kinase, as well as the nuclear localization of nuclear factor-κB. These results demonstrate that mtROS generation is a key signaling event in the LPS-induced pro-inflammatory response of HGFs.

Sulforaphane and its methylcarbonyl analogs inhibit the LPS-stimulated inflammatory response in human monocytes through modulating cytokine production, suppressing chemotactic migration and phagocytosis in a NF-κB- and MAPK-dependent manner.

Sulforaphane [SF; 1-isothiocyanato-4-(methylsulfinyl)-butane], an aliphatic isothiocyanate (ITC) naturally derived from cruciferous vegetables and largely known for its chemopreventive potential also appears to possess anti-inflammatory potential. In this study, structural analogs of SF {compound 1 [1-isothiocyanato-4-(methylcarbonyl)-butane] and 2 [1-isothiocyanato-3-(methylcarbonyl)-propane]} containing a carbonyl group in place of the sulfinyl group in SF, were evaluated for their anti-inflammatory activities. In RAW 264.7 cells, the ITCs at non-toxic concentrations caused an inhibition of NO and prostaglandin E2 (PGE2) release through suppressing expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), as well as a reduction in matrix metalloproteinase-9 (MMP-9) expression, secretion and gelatinolytic activity. Further work performed on human monocytes isolated from blood of healthy donors revealed that the ITCs not only suppressed the expression and release of pro-inflammatory mediators IL-1β, IL-6, TNF-α and MMP-9, but also suppressed their antibody-independent phagocytic and chemotactic migratory abilities. These anti-inflammatory activities were mediated through suppression of the NF-κB and MAPK signaling pathways. In addition, the ITCs were revealed to interact with the cysteines in inhibitor of nuclear factor-κB kinase β subunit (IKKβ), which could contribute at least partly to the suppression of NF-κB signaling. In conclusion, results obtained in this study provide deeper insights into the anti-inflammatory properties of SF and its methylcarbonyl analogs and the underlying mechanisms. These compounds thus serve as promising candidates for clinical applications in controlling inflammatory conditions.

PPM1B and P-IKKβ expression levels correlated inversely with rat gastrocnemius atrophy after denervation

Activated inhibitor of nuclear factor-κB kinase β (IKKβ) is necessary and sufficient for denervated skeletal muscle atrophy. Although several studies have shown that Mg2+/Mn2+-dependent protein phosphatase 1B (PPM1B) inactivated IKKβ, few studies have investigated the role of PPM1B in denervated skeletal muscle. In this study, we aim to explore the expression and significance of PPM1B and phosphorylated IKKβ (P-IKKβ) during atrophy of the denervated gastrocnemius. Thirty young adult female Wistar rats were subjected to right sciatic nerve transection and were sacrificed at 0 (control), 2, 7, 14, and 28 days after denervation surgery. The gastrocnemius was removed from both the denervated and the contralateral limb. The muscle wet weight ratio was calculated as the ratio of the wet weight of the denervated gastrocnemius to that of the contralateral gastrocnemius. RT-PCR and Western blot analysis showed that mRNA and protein levels of PPM1B were significantly lower than those of the control group at different times after the initiation of denervation, while P-IKKβ showed the opposite trends. PPM1B protein expression persistently decreased while P-IKKβ expression persistently increased for 28 days after denervation. PPM1B expression correlated negatively with P-IKKβ expression by the Spearman test, whereas decreasing PPM1B expression correlated positively with the muscle wet weight ratio. The expression levels of PPM1B and P-IKKβ were closely associated with atrophy in skeletal denervated muscle. These results suggest that PPM1B and P-IKKβ could be markers in skeletal muscle atrophy.

Clinical study on the relationship between the activation of the IKK-IκB-NFκB pathway and insulin resistance

Objective To explore the difference involved in the activation of inflammation pathway and the plasma level of inflammatory factors in the subjects with different sorts of insulin sensitivity. Methods The study was carried out in 38 women, consisting of obesity (n = 22 ) and control (n = 16 ) groups according to body mass index. The insulin sensitivity was assessed by homeostasis model assessment of insulin resistance (HOMAIR). Plasma concentrations of interleukin-6 (II-6) and IL-1β were determined by enzyme immunoassay. Western blot analysis was used to examine total protein expression and phosphorylation levels of IκB kinase (IKK) ,inhibitor of nuclear factor-κB ( IκB ) in peripheral blood leukcocytes. Electrophoretic mobility shift assay (EMSA)was used to detect the binding activity of NFκB. Results The levels of fasting plasma insulin[62.2 ( 20.0-127. 0) pmol/L vs 19. 15 ( 14. 2-47. 8 ) pmol/L, P<0. 01], HOMA-IR[2. 32 ( 0. 76-5.49 ) vs 0.70(0.53-1.7),P<0.0l], HbA1 C[(5.42±0. 45 ) % vs ( 5.08 ±0. 38) %, P<0. 05], triglyceride[( 1.75 ±0. 68 vs 1.22 ±0. 58 )mmol/L, P<0. 05], plasma IL-6[3. 15 (0. 03-22. 2) pg/ml vs 1.26 (0. 74-6.06 ) pg/ml, P<0. 01], and IL-1 β[6. 53 ( 0. 84-36 ) pg/ml vs 3. 16( 1.48-8. 86 ) pg/ml, P<0. 01]in obesity group were significantly higher than those in control group. Compared with control group, the levels of IKKo, IKKβ expression and IκBα serine phosphorylation in obesity group were markedly increased, while the expression of IκBα was significantly reduced. Accompanied with the degradation of IκBα protein, the binding activity of NFκB in obesity group was significantly increased. Conclusions The plasma levels of IL-6 and IL-1β were significantly raised in obesity group. The activation of IKK-IκB-NFκB pathway is closely associated with the genesis and development of insulin resistance in obese subjects. Key words: Insulin resistance; Interleukin-6; Interleukin-1 ββ; Inhibitor of nuclear factor-κB kinase; NFκB

SiRNA-Mediated Reduction of Inhibitor of Nuclear Factor-κB Kinase Prevents Tumor Necrosis Factor-α–Induced Insulin Resistance in Human Skeletal Muscle

OBJECTIVE—Proinflammatory cytokines contribute to systemic low-grade inflammation and insulin resistance. Tumor necrosis factor (TNF)-α impedes insulin signaling in insulin target tissues. We determined the role of inhibitor of nuclear factor-κB kinase (IKK)β in TNF-α–induced impairments in insulin signaling and glucose metabolism in skeletal muscle.RESEARCH DESIGN AND METHODS—Small interfering RNA (siRNA) was used to silence IKKβ gene expression in primary human skeletal muscle myotubes from nondiabetic subjects. siRNA gene silencing reduced IKKβ protein expression 73% (P < 0.05). Myotubes were incubated in the absence or presence of insulin and/or TNF-α, and effects of IKKβ silencing on insulin signaling and glucose metabolism were determined.RESULTS—Insulin increased glucose uptake 1.7-fold (P < 0.05) and glucose incorporation into glycogen 3.8-fold (P < 0.05) in myotubes from nondiabetic subjects. TNF-α exposure fully impaired insulin-mediated glucose uptake and metabolism. IKKβ siRNA protected against TNF-α–induced impairments in glucose metabolism, since insulin-induced increases in glucose uptake (1.5-fold; P < 0.05) and glycogen synthesis (3.5-fold; P < 0.05) were restored. Conversely, TNF-α–induced increases in insulin receptor substrate-1 serine phosphorylation (Ser312), Jun NH2-terminal kinase phosphorylation, and extracellular signal–related kinase-1/2 mitogen-activated protein kinase (MAPK) phosphorylation were unaltered by siRNA-mediated IKKβ reduction. siRNA-mediated IKKβ reduction prevented TNF-α–induced insulin resistance on Akt Ser473 and Thr308 phosphorylation and phosphorylation of the 160-kDa Akt substrate AS160. IKKβ silencing had no effect on cell differentiation. Finally, mRNA expression of GLUT1 or GLUT4 and protein expression of MAPK kinase kinase kinase isoform 4 (MAP4K4) was unaltered by IKKβ siRNA.CONCLUSIONS—IKKβ silencing prevents TNF-α–induced impairments in insulin action on Akt phosphorylation and glucose uptake and metabolism in human skeletal muscle.

Multiple Functions of the IKK-Related Kinase IKKε in Interferon-Mediated Antiviral Immunity

IKKepsilon is an IKK (inhibitor of nuclear factor kappaBkinase)-related kinase implicated in virus induction of interferon-beta (IFNbeta). We report that, although mice lacking IKKepsilon produce normal amounts of IFNbeta, they are hypersusceptible to viral infection because of a defect in the IFN signaling pathway. Specifically, a subset of type I IFN-stimulated genes are not activated in the absence of IKKepsilon because the interferon-stimulated gene factor 3 complex (ISGF3) does not bind to promoter elements of the affected genes. We demonstrate that IKKepsilon is activated by IFNbeta and that IKKepsilon directly phosphorylates signal transducer and activator of transcription 1 (STAT1), a component of ISGF3. We conclude that IKKepsilon plays a critical role in the IFN-inducible antiviral transcriptional response.

Platycodin D-induced apoptosis through nuclear factor-κB activation in immortalized keratinocytes

Platycodi Radix is the root of Platycodon grandiflorum and it is widely used in the traditional Oriental medicine as an expectorant for pulmonary diseases and a remedy for respiratory disorders. Platycodin D is the major constituent of triterpene saponins in the root. This study investigates apoptosis by platycodin D in immortalized human keratinocytes (HaCaT). Platycodin D-induced apoptosis in HaCaT cells was confirmed by DNA fragmentation, caspase-3 activation, and caspase-8 activation. Platycodin D could activate inhibitor of nuclear factor-κB kinase (IKK)-β in the nuclear factor-kappaB (NF-κB) activation of upstream level, but not IKK-α. Pretreated- N- tosyl- l-phenylalanine chloromethyl ketone (TPCK), a potent NF-κB inhibitor, could suppress the induction of apoptosis and activation of NF-κB of HaCaT cells by platycodin D. We also demonstrated that platycodin D-mediated apoptosis of HaCaT cells upregulates Fas receptor and Fas ligand (FasL) expression, but did not exhibit p53 activation. HaCaT cells were also transfected with pFLF1, which preserves the promoter region of Fas receptor gene containing NF-κB binding site. On incubation with platycodin D, the NF-κB activity related to Fas receptor increased in a dose-dependent manner. Among the major transcription elements on Fas receptor and FasL promoter, NF-κB activation was shown to have an essential role in the expression of the death receptor such as FasL. These results suggest that platycodin D has the ability to induce apoptosis in HaCaT cells through the upregulation of Fas receptor and FasL expression via to NF-κB activation in the transcriptional level. These results demonstrate that the NF-κB activation plays a crucial role in the induction of apoptosis in human HaCaT cells on treatment with platycodin D.

Evaluation of nuclear factor-kappaB, urokinase-type plasminogen activator, and HBx and their clinicopathological significance in hepatocellular carcinoma.

Nuclear factor kappaB (NF-kappaB) signaling pathway is an important regulating pathway in human diseases and cancers. One of its downstream target genes is urokinase plasminogen activator (uPA), which is involved in cancer invasion and metastasis. The purpose of this study was to evaluate NF-kappaB activation, uPA up-regulation, and hepatitis B viral X protein (HBx) expression in human hepatocellular carcinoma (HCC) and to assess their clinicopathological significance. We evaluated NF-kappaB activation, expression of uPA, and presence of HBx in 32 human HCCs. Their clinicopathological significance was assessed by correlation with the clinicopathological features. Aberrant NF-kappaB signaling pathway and uPA up-regulation mediated by HBx were also analyzed in vitro. We found that NF-kappaB activation and uPA up-regulation were frequently (56% and 59%, respectively) observed in HCCs, and particularly in HBx-positive HCCs. NF-kappaB activation and uPA overexpression were closely associated with one another (P < 0.0001). Furthermore, both activation of NF-kappaB and up-regulation of uPA were significantly associated with a more aggressive tumor behavior in terms of venous invasion, direct liver invasion, and absence of tumor encapsulation. In vitro, NF-kappaB activation was induced by HBx transfection in HepG2 cells through inhibitor of nuclear factor-kappaB kinase beta (IKKbeta). HBx also up-regulated uPA and enhanced cell invasion synergistically with IKKbeta. The data indicate that NF-kappaB dysregulation and uPA overexpression may lead to a more aggressive tumor behavior in HCC. In addition, our data suggest that IKKbeta plays a critical role in the HBx-activated NF-kappaB signaling pathway.