Inhibition Of NF-kappaB Activation Research Articles

TNFAIP3 interacting protein 2 relieves lipopolysaccharide (LPS)-induced inflammatory injury in endometritis by inhibiting NF-kappaB activation.

Endometritis seriously affects the health of women, and it is important to identify new targets for its treatment. This study aimed to explore the role of TNFAIP3 interacting protein 2 (TNIP2) in endometritis through human endometrial epithelial cells (hEECs) stimulated by lipopolysaccharide (LPS). hEECs were induced with LPS to build a cellular model of endometritis. Cell growth and apoptosis were detected by cell counting kit-8 and flow cytometry. The TNIP2 mRNA and protein levels were measured using reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blot analysis, respectively. The caspase3 activity was calculated using a Caspase3 activity kit. Interleukin (IL)-1β, IL-6, and tumor necrosis factor-alpha (TNF-α) levels were determined by enzyme-linked-immunosorbent-assay. The reactive oxygen species (ROS), lactate dehydrogenase (LDH), catalase (CAT), and superoxide dismutase (SOD) levels were determined using the corresponding kits. Nuclear factor-kappaB (NF-κB) pathway was determined by western blot assay. TNIP2 was downregulated in the LPS-induced endometritis cell model. Cell viability was reduced, apoptosis was enhanced, and IL-6, IL-1β, and TNF-α levels increased in LPS-induced hEECs. Additionally, LDH activity and ROS concentration were upregulated, whereas CAT and SOD activities were downregulated in LPS-induced hEECs. These results were reversed by TNIP2 overexpression. Moreover, the results hinted that NF-κB was involved in the effects of TNIP2 on the LPS-induced endometritis cell model. TNIP2 alleviated endometritis by inhibiting the NF-κB pathway, suggesting a potential therapeutic target for endometritis.

Diosmectite inhibits the interaction between SARS-CoV-2 and human enterocytes by trapping viral particles, thereby preventing NF-kappaB activation and CXCL10 secretion

SARS-CoV-2 enters the intestine by the spike protein binding to angiotensin-converting enzyme 2 (ACE2) receptors in enterocyte apical membranes, leading to diarrhea in some patients. Early treatment of COVID-19-associated diarrhea could relieve symptoms and limit viral spread within the gastrointestinal (GI) tract. Diosmectite, an aluminomagnesium silicate adsorbent clay with antidiarrheal effects, is recommended in some COVID-19 management protocols. In rotavirus models, diosmectite prevents pathogenic effects by binding the virus and its enterotoxin. We tested the trapping and anti-inflammatory properties of diosmectite in a SARS-CoV-2 model. Trapping effects were tested in Caco-2 cells using spike protein receptor-binding domain (RBD) and heat-inactivated SARS-CoV-2 preparations. Trapping was assessed by immunofluorescence, alone or in the presence of cells. The effect of diosmectite on nuclear factor kappa B (NF-kappaB) activation and CXCL10 secretion induced by the spike protein RBD and heat-inactivated SARS-CoV-2 were analyzed by Western blot and ELISA, respectively. Diosmectite bound the spike protein RBD and SARS-CoV-2 preparation, and inhibited interaction of the spike protein RBD with ACE2 receptors on the Caco-2 cell surface. Diosmectite exposure also inhibited NF-kappaB activation and CXCL10 secretion. These data provide direct evidence that diosmectite can bind SARS-CoV-2 components and inhibit downstream inflammation, supporting a mechanistic rationale for consideration of diosmectite as a management option for COVID-19-associated diarrhea.

Mono-macrophage-Derived MANF Alleviates Bacterial Myocarditis by Inhibiting NF-kappaB Activation and Myocardial Inflammation.

Bacterial myocarditis is a key cause leading to myocardial damage and cardiac dysfunction. Mesencephalic astrocyte-derived neurotrophic factor (MANF) has been found to be an anti-inflammatory factor. This study is to explore the effect of MANF on LPS-induced myocardial inflammation and macrophage differentiation. The myocarditis mouse model was constructed by LPS treatment. Myocardial damage and serum inflammatory factors were evaluated by ELISA. RT-qPCR was used to detect mRNA of M1/M2 macrophage markers. Western blot, immunohistochemical, and immunofluorescent staining were used to examine myocardial M1/M2 macrophages and NF-κB activation. Mono-macrophage-derived MANF deficiency enhanced LPS-induced inflammatory response and increased M1 macrophages in myocardium tissues, further causing more severe myocardial injury and lower survival rate of mice. Also, LPS-induced myocardial NF-κB activation was strengthened after mono-macrophage-derived MANF knockout. Mono-macrophage-derived MANF inhibits bacterial myocarditis and myocardial M1 macrophage differentiation, which is potential to be used for bacterial myocarditis treatment clinically.

PA-X protein of H5N1 avian influenza virus inhibits NF-kappaB activity, a potential mechanism for PA-X counteracting the host innate immune responses

PA-X is a fusion protein of influenza virus which plays a crucial role in modulating influenza virus-induced host innate immune response and subsequent pathogenicity. However, the potential mechanism of PA-X regulation of the host innate immune response remains largely unknown. It is well known that NF-κB signal pathway is crucial for the immediate early step of immune responses activation, while the specific role of PA-X in NF-κB transcriptional activity is totally unknown. In this study, we initially showed that PA-X inhibits NF-κB transcription that stimulated by poly(I:C). We then further determined that the inhibitory effect on NF-κB activation mediated by PA-X was characterized by restricting NF-κB p65 nuclear translocation and nuclear NF-κB p65 activity but not by impeding the phosphorylation of NF-κB p65. Correspondingly, PA-X decreases the amount of NF-κB signaling pathway-associated genes, including TNF-α, Nos2, IL-6 and IL-2. Moreover, PA-X also suppresses both the mRNA and protein expression level of IFN-β, suggesting the direct contribution of PA-X to the inhibition of NF-κB-regulated IFN-β expression. Together, our study sheds light on the potential molecular mechanisms underlying the regulation of host NF-κB activity by PA-X and also identifies a novel functional role for PA-X in counteracting the host innate immune response. However, further exploration of the more elaborate mechanism of PA-X-mediated inhibition of NF-κB activity and the associated signaling pathway may help to elucidate its precise mechanism of evading and subverting the host immune response.

The Anti-inflammatory Effect of Honeysuckle Flowers (Lonicera japonica Thunb., Caprifoliaceae) in Combination with Oroxylum Indicum (Oroxylum indicum Vent. Bignoniaceae)


 This study investigates the anti-inflammatory effect of the honeysuckle flower in combination with Oroxylum indicum in search of a new kind of anti-inflammatory therapy drugs with stronger anti-inflammatory effects and without unwanted adverse reactions. The study results show that a honeysuckle flower dose of 0.72 g/kg/day, Oroxylum indicum dose of 1.2 g/kg/day together with honeysuckle flower combined with Oroxylum indicum with 1:1 ratio had an acute anti-inflammatory effect on the white rat on a mouse Leg Edema Model with carrageenin and the model causing mouse peritonitis. The results also show that a honeysuckle flower dose of 1.44 g/kg/day, Oroxylum indicum the dose of 2.4 g/kg/day together with honeysuckle flower combined with Oroxylum indicum with 1:1 ratio had a chronic anti-inflammation effect on the white mouse on the model causing chronic inflammation of the granuloma. These results open up a subsequent research direction on the possibility of combining various anti-inflammatory medicinal herbs to obtain better anti-inflammatory efficacy and contribute to the promoting of the effect of the available domestic medicinal resources.
 Keywords
 Honeysuckle flowers, Oroxylum indicum, anti-inflammatory effect
 References
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Anti-psoriatic properties of paeoniflorin: suppression of the NF-kappaB pathway and Keratin 17.

Psoriasis is a common inflammatory skin disease characterized by hyperproliferation of epidermal keratinocytes, however, there is still no curative therapy for psoriasis. Paeoniflorin (PF), a Chinese herbal medicine, has shown anti-inflammatory effects. We aimed to illustrate the effect and associated mechanism of PF on keratinocyte proliferation using the IMQ-induced psoriasis mouse model. The anti-psoriatic effect of PF in vivo and in vitro was assessed by western blot, RT-PCR, immunofluorescence, cell counting kit-8 and haematoxylin/eosin staining. In vivo, epidermal thickness, dermal infiltrated lymphocytes and the level of several antimicrobial peptides, pro-inflammatory cytokines, and K17 were significantly reduced in mice with topical application of PF. In vitro, PF inhibited the proliferation of HaCaT cells in a dose-dependent manner, down-regulated K17 expression, and suppressed NF-kappaB activation. Our findings indicate that PF may inhibit the proliferation of keratinocytes by targeting K17, suggesting that PF might be a potential target in the treatment of psoriasis.

Nicardipine Inhibits Priming of the NLRP3 Inflammasome via Suppressing LPS-Induced TLR4 Expression.

The Nod-like receptor protein 3 (NLRP3) inflammasome is a multi-protein complex composed of NLRP3, pro-caspase-1, and apoptosis-associated speck-like protein that contains a caspase recruitment domain (ASC). After NLRP3 priming by lipopolysaccharide (LPS), the ligand of toll-like receptor 4 (TLR4), activation of the NLRP3 inflammasome triggers caspase-1 maturation, leading to pyroptosis and release of interleukin-1beta (IL-1beta). Expression of TLR4 modulates LPS-triggered inflammatory cascades as well as the NLRP3 signaling. L-type calcium channel antagonists are widely used as anti-hypertensive drugs and also exert anti-inflammatory effects through inhibiting release of cytokines including IL-1beta. However, few studies reveal effects of L-type calcium channel antagonists on the NLRP3 inflammasome. In this study, we investigated the effects of nicardipine and verapamil, both L-type calcium channel antagonists, on the NLRP3 inflammasome using differentiated THP-1 cells. Pyroptosis or levels of IL-1beta and caspase-1 were assayed by flow cytometry or enzyme-linked immunosorbent assay, respectively. ASC oligomerization was assayed by immunofluorescence microscopy. Expression of NLRP3 or TLR4 was assayed by polymerase chain reaction and immunoblotting. Nuclear factor-kappaB (NF-kappaB) pathway was also studied. Our results showed that pyroptosis and IL-1beta release were attenuated by nicardipine, but not verapamil. Nicardipine also mitigated caspase-1 activation, inhibited ASC oligomerization, and reduced NLRP3 expression. Furthermore, nicardipine downregulated phosphorylation or nuclear translocation of NF-kappaB p65, consistent with the inhibitory effect of nicardipine on LPS-induced TLR4 expression. In conclusion, nicardipine exerted anti-inflammatory effects through inhibiting NLRP3 inflammasome pathway. Nicardipine may mitigate NLRP3 priming via inhibiting NF-kappaB activation, mediated by suppressing LPS-induced TLR4 expression.

Development of new treatment using CMG for oxaliplatin-resistant colorectal cancer with KRAS mutation

Abstract Background KRAS mutations are known as poor prognostic factor for patients with colon cancer who receive chemotherapy. KRAS mutations have been shown to activate NF-kappa B (NF-kB) signaling pathway, that plays a pivotal role in cancer progression or chemoresistance. In this study, we tested whether KRAS mutations could confer chemoresistance to oxaliplatin (L-OHP) in vitro. In addition, we tested the efficacy of the newly developed water-soluble injectable type of curcumin monoglucuronide (CMG) in vivo, that is transformed into an active form of curcumin after the injection and potentially suppresses NF-kB activation. Methods Sensitivity of L-OHP was tested using HCT116/DLD1 colon cancer cell lines with/without KRAS mutations (KRAS+/- cells). Efficacy of CMG was tested using xenograft mouse models. Results IC50 of L-OHP was significantly higher in KRAS+ cells compared to KRAS- cells. Addition of p53 deletion in KRAS+ HCT 116 cells (KRAS+p53- cells) significantly increased chemoresistance to L-OHP compared to KRAS+ cells. Curcumin showed the similar anti-tumor effects regardless of KRAS/p53 mutation status in vitro. Higher NF-kB activity in KRAS+ cells compared to KRAS- cells was detected by using a reporter gene assay and NF-kB activation was suppressed by curcumin in vitro. In xenograft models using KRAS+p53- HCT116 cell line, intraperitoneal administration of CMG demonstrated significantly superior anti-tumor effects compared to L-OHP monotherapy. Notably, no adverse events were noted in the CMG group, whereas bodyweight loss, severe myelosuppression, and liver damage were observed in the L-OHP group. Combination of CMG and L-OHP showed synergic anti-tumor effects. Conclusion These results indicate that CMG could be a novel anti-cancer drug with minimal toxicities for colorectal cancer patients with KRAS mutations.

Immunosuppression of Human Adipose-Derived Stem Cells on T Cell Subsets via the Reduction of NF-kappaB Activation Mediated by PD-L1/PD-1 and Gal-9/TIM-3 Pathways.

Adipose-derived stem cells (ADSCs) are a type of multipotent mesenchymal stem cells with immunosuppressive capacities. However, the underlying mechanisms involved in the inhibitory effects of ADSCs on T cells are not completely elucidated. In this study, human peripheral blood mononuclear cells (PBMCs) stimulated with anti-CD3/CD28 antibody-coated beads were cultured with or without allogeneic ADSCs (ADSC-to-PBMC ratio, 1:5). Surface marker levels, violet-labeled cell proliferation, apoptosis, interferon-gamma (IFN-gamma) production, and nuclear factor-kappaB (NF-kappaB) phosphorylation of CD4+ and CD8+ T cells were detected using flow cytometry. It was observed that ADSCs significantly suppressed the proliferation and IFN-gamma production but enhanced apoptosis of both CD4+ and CD8+ T cells in T cell receptor (TCR)-stimulated PBMCs. The expressions of programmed death-ligand 1 (PD-L1) and galectin 9 (Gal-9) on ADSCs were significantly upregulated and induced during coculture with PBMCs. TCR-stimulated CD4+ and CD8+ T cells cultured with ADSCs had higher expression levels of programmed death-1 (PD-1) and T cell immunoglobulin and mucin-containing protein-3 (TIM-3) than those in cells cultured without ADSCs. Moreover, the suppressive effects of ADSCs on T cells in terms of proliferation and IFN-gamma production were significantly reversed in the presence of anti-PD-L1 and anti-Gal-9 antibodies. Importantly, the phosphorylation of NF-kappaB in CD4+ and CD8+ T cells cocultured with ADSCs was significantly inhibited, and this inhibition was significantly attenuated via the PD-L1 and Gal-9 blockades. In conclusion, human ADSCs perform immunoregulatory functions partially through the inhibition of NF-kappaB activation in T cells via the PD-L1/PD-1 and Gal-9/TIM-3 pathways, which provide new insights into the mechanism of human ADSC-mediated immunomodulation.

Novel Small Molecule Inhibitors of Protein Kinase D Suppress NF-kappaB Activation and Attenuate the Severity of Rat Cerulein Pancreatitis.

Nuclear factor-kappa B (NF-κB) activation is a key early signal regulating inflammatory and cell death responses in acute pancreatitis. Our previous in vitro studies with molecular approaches on AR42J cell showed that protein kinase D (PKD/PKD1) activation was required in NF-κB activation induced by cholecystokinin 8 (CCK) or carbachol (CCh) in pancreatic acinar cells. Recently developed small molecule PKD inhibitors, CID755673 and CRT0066101, provide potentially important pharmacological approaches to further investigate the effect of PKD in pancreatitis therapy. The aim of this study was to explore whether CID755673 and CRT0066101 block NF-κB activation with in vitro and in vivo models of experimental pancreatitis and whether the small molecule PKD inhibitors have therapeutic effects when given before or after the initiation of experimental pancreatitis. Freshly prepared pancreatic acini were incubated with CID755673 or CRT006101, followed by hyperstimulation with CCK or CCh. For in vivo experimental pancreatitis, rats were treated with intraperitoneal injection of CID755673 or CRT0066101 prior to or after administering cerulein or saline. PKD activation and NF-κB-DNA binding activity in nuclear extracts from pancreatic acini and tissue were measured. The effects of PKD inhibitors on pancreatitis responses were evaluated. Our results showed that both CID755673 or CRT0066101 selectively and specifically inhibited PKD without effects on related protein kinase Cs. Inhibition of PKD resulted in significantly attenuation of NF-κB activation in both in vitro and in vivo models of experimental pancreatitis. NF-κB inhibition by CID755673 was associated with decreased inflammatory responses and attenuated severity of the disease, which were indicated by less inflammatory cell infiltration, reduced pancreatic interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1), decreased intrapancreatic trypsin activation, and alleviation in pancreatic necrosis, edema and vacuolization. Furthermore, PKD inhibitor CID755673, given after the initiation of pancreatitis in experimental rat model, significantly attenuated the severity of acute pancreatitis. Therapies for acute pancreatitis are limited. Our results indicate that small chemical PKD inhibitors have significant potential as therapeutic interventions by suppressing NF-κB activation.

Sneaking ligands inhibiting NF-kappaB activation selectively in endothelial cells improve experimental arthritides

The activation of intracellular signaling pathways such as the classical nuclear factor kappaB (NF-kappaB) pathway is related to the pathogenesis of several inflammatory autoimmune diseases including rheumatoid arthritis (RA)

Piperlongumine attenuates experimental autoimmune encephalomyelitis through inhibition of NF-kappaB activity

Multiple sclerosis (MS) is a chronic, autoimmune and neurodegenerative disease in which demyelination sporadically and repeatedly occurs in the central nervous system (CNS). The activity of nuclear factor kappa B (NF-κB), a family of transcription factors, was increased in the cerebrospinal fluid (CSF) and/or the serum and brain and/or spinal cord of MS patients than in a healthy donors. In our study, we investigated whether piperlongumine (PL), which is known to have inhibitory effect on activity of NF-κB, can alleviate an experimental autoimmune encephalomyelitis (EAE). The mice were immunized with myelin oligodendrocyte glycoprotein 35–55 (MOG35–55), and then we injected PL (1.5mg/kg/day or 3.0mg/kg/day) into the mice intraperitoneally on every second day from days 2 to 28. For in vitro study, we treated PL (0.5, 1 and 2.5μM) to RAW 264.7 and Jurkat cells with each stimulator. We observed that the paralytic severity and neuropathology of EAE in PL-treated group were decreased compared with the EAE group. PL showed a suppressed effect on demyelination, immune cells infiltration, astrocytes/microglials activation, level of inflammatory cytokines and proteins as well as NF-κB activity. Production of inflammatory cytokines and proteins as well as translocation of NF-κB into nucleus by treatment stimulators in RAW 264.7 and Jurkat cells were reduced by PL. Moreover, treatment of NF-κB inhibitor further inhibited production of inflammatory cytokines and proteins. These results suggest that PL can mitigate MOG-induced EAE symptoms and activation of macrophages and T cells by inhibiting NF-κB signaling. Therefore, PL could be useful for the treatment for MS.

Pyrrolidine Dithiocarbamate Inhibits NF-KappaB Activation and Upregulates the Expression of Gpx1, Gpx4, Occludin, and ZO-1 in DSS-Induced Colitis.

Inflammatory bowel disease (IBD) correlates with oxidative stress, inflammation, and alteration in several signal pathways, including nuclear transcription factor-kappaB (NF-κB). Pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-κB, has been widely demonstrated to exhibit an antioxidant and anti-inflammatory function. This study aimed to test the hypothesis that NF-κB inhibitor PDTC confers a beneficial role in a colitis model induced by dextran sodium sulfate (DSS) in mouse. The results showed that DSS decreased daily weight gain, induced colonic inflammation, suppressed the expression of antioxidant enzymes and tight junctions, and activated NF-κB and nuclear factor erythroid 2-related factor 2/Kelch-like ECH-associated protein 1 (Nrf2/Keap1) signaling pathways. PDTC significantly upregulated (P<0.05) Gpx1, Gpx4, occludin, and ZO-1 expressions in the DSS-induced colitis model. Meanwhile, PDTC reversed (P<0.05) the activation of NF-κB signal pathway caused by DSS treatment. In conclusion, PDTC could serve as an adjuvant therapy for the patient with IBD.

Anticancer effect of tectochrysin in colon cancer cell via suppression of NF-kappaB activity and enhancement of death receptor expression.

BackgroundFlavonoids are a diverse family of natural phenolic compounds commonly found in fruits and vegetables. Epidemiologic studies showed that flavonoids also reduce the risk of colon cancer. Tectochrysin is one of the major flavonoids of Alpinia oxyphylla Miquel. However, the anti-cancer effects and the molecular mechanisms of tectochrysin in colon cancer cells have not yet been reported. We investigated whether tectochrysin could inhibit colon cancer cell growth at 1, 5, 10 μg/ml. In in vivo study, we injected a tectochrysin treatment dose of 5 mg/kg to each mouse.ResultsTectochrysin suppressed the growth of SW480 and HCT116 human colon cancer cells. The expression of DR3, DR4 and Fas were significantly increased, and pro-apoptotic proteins were also increased. Tectochrysin treatment also inhibited activity of NF-κB. A docking model indicated that tectochrysin binds directly to the p50 unit. In in vivo, tumor weights and volumes in mice were reduced when treated with tectochrysin. Tectochrysin leads to apoptotic cell death in colon cancer cells through activation of death receptors expression via the inhibition of NF-κB.ConclusionsTectochrysin can be a useful agent for the treatment of colon cancer cell growth as well as an adjuvant agent for chemo-resistant cancer cells growth.

Withaferin A inhibits NF-kappaB activation by targeting cysteine 179 in IKKβ

The transcription factor NF-κB is one of the main players involved in inflammatory responses during which NF-κB becomes rapidly activated. However to maintain homeostasis, this NF-κB activation profile is only transient. Nevertheless deregulation of NF-κB activity is often observed and can lead to chronic inflammatory diseases as well as cancer. Therefore various research projects focus on the development of therapeutics that target the NF-κB activation pathway. One such compound is Withaferin A from the Ayurvedic plant Withania somnifera. Several reports already described the NF-κB inhibiting, anti-inflammatory capacity of WA, either in vitro as well as in vivo. However the underlying molecular mechanism remains largely unknown. In this paper we demonstrate a direct interaction of WA with the IKK-complex, more specifically with IKKβ, a kinase which is indispensable for the nuclear translocation of NF-κB. Hereby WA directly inhibits IKK catalytic activity. By mutation of Cys179 in IKKβ we could demonstrate loss of interaction between IKKβ and WA indicating that WA exerts its anti-inflammatory effects by targeting the crucial Cys179 residue located in the catalytic site of IKKβ. Upon docking of WA to a IKKβ homology structure model, WA was found to fit nicely into the groove of IKKβ where it can form hydrogen bond to stabilize its interaction with Cys179.

Anti-Inflammatory Effects of New Flavonoids from Streptomyces sp. BT01 in Lipopolysaccharide-Stimulated RAW 264.7 Murine Macrophages via Inhibition of NF-KappaB Activation

Most flavonoids are known to have anti-oxidant, anti-bacterial and analgesic properties. In this study, the new flavonoids, 7-methoxy-3,3’,4’,6-tetrahydroxyflavone (1) and 2’,7dihydroxy-4’5’-dimethoxyisoflavone (2) isolated from Streptomyces sp. BT01 inhibited the pro-inflammatory mediators including cytokines by blocking nuclear factor-kappaB (NFκB) signalling in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. These flavonoids suppressed mRNA and protein expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in LPS-stimulated RAW 264.7 cells. The molecular mechanism was associated with the inhibition of NF-κB activation. These results suggest that these flavonoids have anti-inflammatory effects by suppressing expression of Original Research Article British Journal of Applied Science & Technology, 4(29): 4201-4222, 2014 4202 iNOS, COX-2 and cytokines by blocking the NF-κB signalling in LPS-stimulated RAW 264.7 cells.

The biology of A20-binding inhibitors of NF-kappaB activation (ABINs).

The family of A20-Binding Inhibitors of NF-kappaB (ABINs) consists of three proteins, ABIN-1, ABIN-2 and ABIN-3, which were originally identified as A20-binding proteins and inhibitors of cytokines and Lipopolysaccharide (LPS) induced NF-kappaB activation. ABIN family members have limited sequence homology in a number of short regions that mediate A20-binding, ubiquitin-binding, and NF-kappaB inhibition. The functional role of A20 binding to ABINs remains unclear, although an adaptor function has been suggested. ABIN-1 and ABIN-3 expression is upregulated when cells are triggered by NF-kappaB-activating stimuli, suggesting a role for these ABINs in a negative feedback regulation of NF-kappaB signaling. Additional ABIN functions have been reported such as inhibition of TNF-induced hepatocyte apoptosis, regulation of HIV-1 replication for ABIN-1, and Tumor Progression Locus 2 (TPL-2)-mediated Extracellular signal-Regulated Kinase (ERK) activation for ABIN-2. In mice, ABIN-1 overexpression reduces allergic airway inflammation and TNF-mediated liver injury, ABIN-2 overexpression delays liver regeneration, and ABIN-3 overexpression partially protects against LPS-induced acute liver failure. Analysis of mice deficient in ABIN-1 or ABIN-2 demonstrates the important immune regulatory function of ABINs. Future studies should clarify the functional implication of the A20-ABIN interaction in supporting ABINs' mechanisms of action.

HSV-1 activates NF-kappaB in mouse astrocytes and increases TNF-alpha and IL-6 expression via Toll-like receptor 3

Objective: To investigate the effect of HSV-1 infection via TLR3 on the transcriptional activity of NF-kappaB and the expression of cytokines TNF-alpha and IL-6 in astrocytes.Methods: HSV-1-infected primary astrocytes were cultured until the third passage and the mRNA and protein levels of TLR3, NF-kappaB, TNF-alpha, and IL-6 were assessed by immunofluorescence, RT-PCR, and Western blot. The effects of the NF-kappaB inhibitor pyrrolidine dithiocarbamate (PDTC) and TLR3-neutralizing antibody on the expression of NF-kappaB, TNF-alpha, and IL-6 were investigated.Results: Uninfected astrocytes expressed TLR3 and NF-kappaB at the mRNA and protein levels. After infection with HSV-1, the TLR3 mRNA and protein levels were up-regulated and NF-kappaB protein was highly expressed. Also, the mRNA and protein levels of TNF-alpha and IL-6 were up-regulated. Pyrrolidine dithiocarbamate inhibited NF-kappaB activation, resulting in the down-regulation of nuclear NF-kappaB protein, which led to the down-regulation of the mRNA and protein levels of TNF-alpha and IL-6. After blocking astrocyte membrane TLR3, the nuclear NF-kappaB protein expression was down-regulated and the mRNA and protein levels of TNF-alpha and IL-6 were increased. The antiviral functions of astrocytes were weaker, as reflected by higher HSV-1 glycoprotein D (gD) mRNA expression and increased HSV-1 titers.Conclusion: Astrocytes infected with HSV-1 can activate NF-kappaB via TLR3 so as to up-regulate the expression of TNF-alpha and IL-6 that have antiviral functions.

ASSA13-03-13 Inhibitory Effects of Vinpocetine on the Progression of Atherosclerosis Are Mediated by Akt/NF-kB Dependent Mechanisms

<h3>Objective</h3> Recent studies have found additional role for vinpocetine, a potent phosphodiesterase type I inhibitor, in anti-proliferation and anti-inflammation of vascular smooth muscle cells and cancer cells via different mechanisms. In this study, we attempted to investigate whether vinpocetine protected against atherosclerotic development in apoE^-/- mice and explore the underlying anti-atherogenic mechanisms in macrophages. <h3>Methods and Results</h3> Vinpocetine markedly decreased atherosclerotic lesion size in apoE^-/- mice measured by oil red O. Masson’s trichrome staining and immunohistochemical analyses revealed that vinpocetine significantly increased the thickness of fibrous cap, reduced the size of lipid-rich necrotic core and attenuated the expression of TNF-alpha and matrix metalloproteinase-9 (MMP-9) within plaque area. <i>In vitro</i> experiments exhibited a significant decrease in monocyte adhension treated with vinpocetine. In parallel with few changes in ox-LDL uptake and foam cell formation, the expression levels of scavenger receptor A and CD 36 were not altered after treatment with vinpocetine. Further, active TNF-alpha, IL-6, monocyte chemoattractant protein-1 and MMP-9 expression induced by ox-LDL was attenuated by vinpocetine in a dose-dependent manner. Similarly, ox-LDL-induced reactive oxygen species were significantly repressed by vinpocetine. Both western blot and luciferase activity assay showed that vinpocetine restored the enhanced Akt, IKK-alpha/beta, IkappaB-alpha phosphorylation and NF-kappaB activity induced by ox-LDL. Cotreatment with LY294002, a specific Akt inhibitor, augmented the inhibitory effects of vinpocetine on IKK-alpha/beta and IkappaB-alpha phosphorylation, suggesting that inhibition of NF-kappaB activity was partly caused by Akt dephosphorylation. Knockdown of PDE1B resulted in an increase of intracellular cGMP contents, however, did not affect Akt, IKK-alpha/beta and IkappaB-alpha phosphorylation. <h3>Conclusions</h3> Vinpocetine exerts anti-atherogenic effects through inhibition of monocyte adhension, oxidative stress and inflammatory response, which are mediated by Akt/NF-kappaB dependent pathway but independent of PDE1 blockade in macrophages.

CDA-2, a Urinary Preparation, Inhibits Lung Cancer Development through the Suppression of NF-kappaB Activation in Myeloid Cell

CDA-2 (cell differentiation agent 2), a urinary preparation, has potent anti- proliferative and pro-apoptotic properties in cancer cells. However, the mechanisms of tumor inhibitory action of CDA-2 are far from clear, and especially there was no report on lung cancer. Here we demonstrate that CDA-2 and its main component phenylacetylglutamine (PG) reduce the metastatic lung tumor growth, and increases survival time after inoculation with Lewis lung carcinoma (LLC) cells in a dose-dependent manner in C57BL6 mice. Proliferative program analysis in cancer cells revealed a fundamental impact of CDA-2 and PG on proliferation and apoptosis, including Bcl-2, Bcl-XL, cIAP1, Survivin, PCNA, Ki-67 proteins and TUNEL assays. CDA-2 and PG significantly reduced NF-κB DNA-binding activity in lung cancer cells and in alveolar macrophages of tumor bearing mice and especially decreased the release of inflammatory factors including TNFα, IL-6, and KC. Furthermore, CDA-2 and PG decrease the expressions of TLR2, TLR6, and CD14, but not TLR1, TLR3, TLR4, and TLR9 in bone-marrow-derived macrophages (BMDM) of mice stimulated by LLC-conditioned medium (LLC-CM). Over-expressing TLR2 in BMDM prevented CDA-2 and PG from inhibiting NF-κB activation, as well as induction of TNFα and IL-6. TLR2:TLR6 complexes mediate the effect of NF-κB inactivation by CDA-2. In conclusion, CDA-2 potently inhibits lung tumor development by reduction of the inflammation in lung through suppression of NF-κB activation in myeloid cells, associating with modulation of TLR2 signaling.