Anti-inflammatory, pro-apoptotic, and anti-proliferative effects of a methanolic neem (Azadirachta indica) leaf extract are mediated via modulation of the nuclear factor-κB pathway

Azadirachta indica (neem tree) is used in traditional Indian medicine for its pharmacological properties including cancer prevention and treatment. Here, we studied a neem extract's anti-inflammatory potential via the nuclear factor-κB (NF-κB) signaling pathway, linked to cancer, inflammation, and apoptosis. Cultured human leukemia cells were treated with a methanolic neem leaf extract with or without tumor necrosis factor (TNF)-α stimulation. Inhibition of NF-κB activity was demonstrated by luciferase assay and electrophoretic mobility shift assay (EMSA). Inhibition of viability by neem extracts was assessed by luminescent assays. Western blot analysis allowed assessing the inhibitory effect of the neem extract on TNF-α-induced degradation of inhibitor of κB (IκB) and nuclear translocation of the NF-κB p50/p65 heterodimer. Inhibition of IκB kinase (IKK) activity was shown as well as the effect of neem extract on the induction of apoptotic cell death mechanisms by nuclear fragmentation analysis and flow cytometry analysis. In conclusion, our data provide evidence for a strong effect of the neem extract on pro-inflammatory cell signaling and apoptotic cell death mechanisms, contributing to a better understanding of the mechanisms triggered by Azadirachta indica.     

Identification of a new Mpl-interacting protein,Atp5d

Nuclear factor κB (NF-κB) plays an important role in the regulation of inflammatory proteins. However, it is unclear whether the NF-κB/intercellular adhesion molecule-1 (ICAM-1) pathway is involved in the adhesion of neutrophils and renal injury after hypoxia–ischemia (HI) in neonates. In this report we investigated whether NF-κB and its downstream molecule ICAM-1 were involved in renal injury induced by postasphyxial serum (PS) from neonates. Human renal proximal tubular (HK-2) cells were preincubated with 10 % fetal calf serum (control), 20 % neonatal PS, or 20 % PS plus pyrolidine dithiocarbamate (PDTC). The expression of IκBα, NF-κB p65, and ICAM-1 in HK-2 cells was determined by Western blot and/or immunohistochemistry. Nuclear translocation of NF-κB p65 in HK-2 cells was detected by immunofluorescence and Western blot. The ICAM-1 mRNA was determined by RT-PCR. Then HK-2 cells were cultured with neutrophils from neonates with asphyxia. After HK-2 cells had been cultured with neutrophils, we detected myeloperoxidase (MPO) activity, the leakage rate of lactate dehydrogenase (LDH), and cell viability. We found that PS preincubation resulted in significantly decreased IκBα expression and increased expression of NF-κB and ICAM-1, and facilitated the nuclear translocation of NF-κB in HK-2 cells. PS preincubation increased MPO activity, leading to elevated leakage rates of LDH and decreased cell viability after neutrophil exposure. Furthermore, the inhibition of NF-κB activity by PDTC significantly upregulated IκBα expression, decreased NF-κB and ICAM-1 expression, downregulated the nuclear translocation of NF-κB, and decreased MPO activity. This leads to decreased leakage rates of LDH and increased cell viability after neutrophil exposure. Our findings suggest that NF-κB/ICAM-1 pathway may be involved in neutrophil–endothelial interactions and neonatal renal injury after HI.     

Identification of the RelA domain responsible for action of a new NF-kappaB inhibitor DHMEQ

A new NF-κB inhibitor dehydroxymethylepoxyquinomicin (DHMEQ) has a potential to be applied to clinical medicine as an anti-cancer and anti-inflammatory agent. DHMEQ inhibits localization of NF-κB in the nucleus and the inhibitory effect by DHMEQ is more potent on p50/RelA than on p50 homodimer. However, a molecular target of DHMEQ is unknown. In this study, we identified residues CEGRSAGSI, which appear in RelA (amino acids 38-46), c-Rel (28-36), and RelB (144-152), but not in p50 and p52, as a target of DHMEQ. As a possible mechanism, we propose that DHMEQ accesses CEGRSAGSI domain recognizing RSAGSI structure and directly binds to cysteine. This target domain appears to be unique among mammalian proteins. The results obtained in this study may provide better understanding of the action of DHMEQ and a key for developing a new NF-κB inhibitor with more potent activity.     

Curcumin suppresses T cell activation by blocking Ca2+ mobilization and nuclear factor of activated T cells (NFAT) activation

Curcumin is the active ingredient of the spice turmeric and has been shown to have a number of pharmacologic and therapeutic activities including antioxidant, anti-microbial, anti-inflammatory, and anti-carcinogenic properties. The anti-inflammatory effects of curcumin have primarily been attributed to its inhibitory effect on NF-κB activity due to redox regulation. In this study, we show that curcumin is an immunosuppressive phytochemical that blocks T cell-activation-induced Ca(2+) mobilization with IC(50) = ～12.5 μM and thereby prevents NFAT activation and NFAT-regulated cytokine expression. This finding provides a new mechanism for curcumin-mediated anti-inflammatory and immunosuppressive function. We also show that curcumin can synergize with CsA to enhance immunosuppressive activity because of different inhibitory mechanisms. Furthermore, because Ca(2+) is also the secondary messenger crucial for the TCR-induced NF-κB signaling pathway, our finding also provides another mechanism by which curcumin suppresses NF-κB activation.     

A vaccinia virus deletion mutant reveals the presence of additional inhibitors of NF-kappaB

The classical nuclear factor kappa B (NF-κB) signaling pathway is an important regulator of inflammation and innate immunity that is activated by a wide variety of stimuli, including virus infection, tumor necrosis factor alpha (TNF-α), and interleukin 1β (IL-1β). Poxviruses, including vaccinia virus (VV) and ectromelia virus, encode multiple proteins that function in immune evasion. Recently, a growing number of genes encoded by poxviruses have been shown to target and disrupt the NF-κB signaling pathway. To determine if additional gene products that interfere with NF-κB signaling existed, we used a vaccinia virus deletion mutant, VV811, which is missing 55 open reading frames lacking all known inhibitors of TNF-α-induced NF-κB activation. Immunofluorescence analysis of HeLa cells treated with TNF-α and IL-1β revealed that NF-κB translocation to the nucleus was inhibited in VV811-infected cells. This was further confirmed through Western blotting of cytoplasmic and nuclear extracts for NF-κB. Additionally, VV811 infection inhibited TNF-α-induced IκBα degradation. In contrast to vaccinia virus strain Copenhagen (VVCop)-infected cells, VV811 infection resulted in the dramatic accumulation of phosphorylated IκBα. Correspondingly, coimmunoprecipitation assays demonstrated that the NF-κB-inhibitory IκBα-p65-p50 complex was intact in VV811-infected cells. Significantly, cells treated with 1-β-d-arabinofuranosylcytosine, an inhibitor of poxvirus late gene expression, demonstrated that an additional vaccinia virus late gene was involved in the stabilization of IκBα. Overall, this work indicates that unidentified inhibitors of NF-κB exist in vaccinia virus. The complex inhibition of NF-κB by vaccinia virus illustrates the importance of NF-κB activation in the antiviral response.     

Hsp72对类风湿关节炎滑膜细胞IL-6、IL-8表达及NFκ-B活化的影响

目的:探讨热休克蛋白(Hsp)72对类风湿关节炎患者滑膜细胞IL-6、IL-8表达的影响,从NFκ-B信号通路活化的角度阐明其作用机制。方法:原代培养类风湿关节炎患者的滑膜细胞;采用酶联免疫吸附试验(ELISA)法检测细胞培养上清中IL-6和IL-8的含量;采用Western blot检测滑膜细胞NFκ-B和ΙκBα蛋白的表达变化;采用免疫荧光技术检测NFκ-B核移位的变化。结果:Hsp72抑制TNFα-所诱导的IL-6和IL-8的生成;Hsp72抑制TNFα-所诱导NFκ-B在核内的表达和移位;Hsp72抑制TNFα-所诱导ΙκBα蛋白降解。结论:Hsp72可能通过抑制滑膜细胞IL-6、IL-8表达及抑制NF-κB信号通路活化而对类风湿关节炎发挥抗炎作用。     

Involvement of NF Kappa B in Potentiated Effect of Mn-containing Dithiocarbamates on MPP+ Induced Cell Death

Humans are exposed to various chemical mixtures daily. The toxic response to a mixture of chemicals could be potentiated or suppressed. This study demonstrates that non-toxic doses of pesticides can induce cellular changes that increase cell sensitivity to other toxins or stress. Pesticide exposure is an environmental risk factor for Parkinson’s disease. Manganese (Mn) is essential but high dose exposure may results in neurological dysfunction. Mn-containing dithiocarbamates, maneb (MB) and mancozeb (MZ), are primarily used as pesticides. Studies have shown that MB can augment dopaminergic damage triggered by sub-toxic doses of Parkinsonian mimetic MPTP. However, the mechanism underlying this effect is not clear. Activation of nuclear factor kappa B (NF-κB) has been implicated in MPTP toxicity. Mn stimulates the activation of NF-κB and subsequently induces neuronal injury via an NF-κB dependent mechanism. We speculate that MB and MZ enhance MPTP active metabolite (methyl-4-phenylpyridine ion, MPP⁺) toxicity by activating NF-κB. The activation of NF-κB was observed using Western blot analysis and NF-κB response element driven Luciferase reporter assay. Western blot data demonstrated the nuclear translocation of NF-κB p65 and the degradation of IkBα after MB and MZ 4-h treatments. Results of NF-κB response element luciferase reporter assay confirmed that MB and MZ activated NF-κB. The NF-κB inhibitor (SN50) was also shown to alleviate cytotoxicity induced by co-treatment of MB or MZ and MPP⁺. This study demonstrates that activation of NF-κB is responsible for the potentiated toxic effect of MB and MZ on MPP⁺ induced cytotoxicity.     

STAT-3 activates NF-kappaB in chronic lymphocytic leukemia cells

NF-κB plays a major role in the pathogenesis of B-cell neoplasms. A broad array of mostly extracellular stimuli has been reported to activate NF-κB, to various degrees, in chronic lymphocytic leukemia (CLL) cells. Because CLL cells harbor high levels of unphosphorylated STAT-3 (USTAT-3) and USTAT-3 was reported to activate NF-κB, we sought to determine whether USTAT-3 activates NF-κB in CLL. Using the electrophoretic mobility shift assay (EMSA), we studied peripheral blood low-density cells from 15 patients with CLL and found that CLL cell nuclear extracts from all the samples bound to an NF-κB DNA probe, suggesting that NF-κB is constitutively activated in CLL. Immunoprecipitation studies showed that STAT-3 bound NF-κB p65, and confocal microscopy studies detected USTAT-3/NF-κB complexes in the nuclei of CLL cells, thereby confirming these findings. Furthermore, infection of CLL cells with retroviral STAT-3-short hairpin RNA attenuated the binding of NF-κB to DNA, as assessed by EMSA, and downregulated mRNA levels of NF-κB-regulated genes, as assessed by quantitative PCR. Taken together, our data suggest that USTAT-3 binds to the NF-κB p50/p65 dimers and that the USTAT-3/NF-κB complexes bind to DNA and activate NF-κB-regulated genes in CLL cells.     

15-deoxy-Δ(12,14) -prostaglandin-J2 and ciglitazone inhibit TNF-α-induced matrix metalloproteinase 13 production via the antagonism of NF-κB activation in human synovial fibroblasts

Collagenase-3 (matrix metalloproteinase, MMP-13) plays an important role in the degradation of cartilage in pathologic conditions. MMP-13 is elevated in joint tissues in both rheumatoid arthritis (RA) and osteoarthritis (OA). In addition, inflammation-stimulated synovial fibroblasts are able to release MMP-13 and other cytokines in these diseases. The peroxisome proliferator-activated receptor-γ (PPARγ) ligands are recently considered as new anti-inflammatory compounds and these ligands were reported to ameliorate inflammatory arthritis. The aim of this study is to evaluate the mechanisms how PPARγ ligands inhibit the inflammatory response in synovial fibroblasts. Two PPARγ ligands, cyclopentenone prostaglandin 15-deoxy-Δ(12,14) -prostaglandin-J2 (15d-PGJ2) and synthetic thiazolidinedione compound ciglitazone were examined in this study. Here we found that 15d-PGJ2 and ciglitazone markedly inhibited TNF-α-induced MMP-13 production in human synovial fibroblasts. In addition, activation of nuclear factor κB (NF-κB) is strongly associated with MMP-13 induction by TNF-α and the activation of NF-κB was determined by Western blot, reporter assay, and immunofluorescence. It was found that 15d-PGJ2 markedly attenuated the translocation of NF-κB by direct inhibition of the activation of IKK via a PPARγ-independent manner. Ciglitazone also inhibits TNF-α-induced MMP-13 expression by suppressing NF-κB activation mainly via the modulation of p38-MAPK. Collectively, our data demonstrate that 15d-PGJ2 and ciglitazone attenuated TNF-α-induced MMP-13 expression in synovial fibroblasts primarily through the modulation of NF-κB signaling pathways. These compounds may have therapeutic application in inflammatory arthritis.     

A new regulatory mechanism of NF-kappaB activation by I-kappaBbeta in cancer cells

Transglutaminase 2 (TGase 2) catalyzes covalent isopeptide bond formation between glutamine and lysine residues. Recently, we reported that TGase 2 activates nuclear factor-kappa B (NF-κB) by depleting inhibitor of NF-κBα (I-κBα) levels via polymer formation. Furthermore, TGase 2 expression synergistically increases NF-κB activity with canonical pathway. The major I-κB proteins such as I-κBα and I-κBβ resemble each other in both primary sequence and tertiary structure. However, I-κBβ does not degrade fully, while I-κBα degrades immediately in response to most stimuli. We found that I-κBβ does not contain any of the previously identified TGase 2 target sites. In this study, both an in vitro cross-linking assay and a TGase 2 transfection assay revealed that I-κBβ is independent from TGase 2-mediated polymerization. Furthermore, increased I-κBβ expression reversed NF-κB activation in cancer cells, compensating for the loss of I-κBα via TGase 2 polymerization.     

Glucan phosphate attenuates myocardial HMGB1 translocation in severe sepsis through inhibiting NF-κB activation

Myocardial dysfunction is a major consequence of septic shock and contributes to the high mortality of sepsis. High-mobility group box 1 (HMGB1) serves as a late mediator of lethality in sepsis. We have reported that glucan phosphate (GP) attenuates cardiac dysfunction and increases survival in cecal ligation and puncture (CLP)-induced septic mice. In the present study, we examined the effect of GP on HMGB1 translocation from the nucleus to the cytoplasm in the myocardium of septic mice. GP was administered to mice 1 h before induction of CLP. Sham-operated mice served as control. The levels of HMGB1, Toll-like receptor 4 (TLR4), and NF-κB binding activity were examined. In an in vitro study, H9C2 cardiomyoblasts were treated with lipopolysaccharide (LPS) in the presence or absence of GP. H9C2 cells were also transfected with Ad5-IκBα mutant, a super repressor of NF-κB activity, before LPS stimulation. CLP significantly increased the levels of HMGB1, TLR4, and NF-κB binding activity in the myocardium. In contrast, GP administration attenuated CLP-induced HMGB1 translocation from the nucleus to the cytoplasm and reduced CLP-induced increases in TLR4 and NF-κB activity in the myocardium. In vitro studies showed that GP prevented LPS-induced HMGB1 translocation and NF-κB binding activity. Blocking NF-κB binding activity by Ad5-IκBα attenuated LPS-induced HMGB1 translocation. GP administration also reduced the LPS-stimulated interaction of HMGB1 with TLR4. These data suggest that attenuation of HMGB1 translocation by GP is mediated through inhibition of NF-κB activation in CLP-induced sepsis and that activation of NF-κB is required for HMGB1 translocation.     

Molecular mechanism of the anti-inflammatory activity of a natural diarylnonanoid, malabaricone C

The spice-derived phenolic, malabaricone C (mal C), has recently been shown to accelerate healing of the indomethacin-induced gastric ulceration in mice. In this study, we explored its anti-inflammatory activity and investigated the underlying mechanism of the action. Mal C suppressed the microvascular permeability and the levels of tumor necrosis factor-α, interleukin-1β, and nitric oxide in the lipopolysaccharide (LPS)-administered mice. At a dose of 10 mg/kg, it showed anti-inflammatory activity comparable to that of omeprazole (5 mg/kg) and dexamethasone (50 mg/kg). It also reduced the expression and activities of inducible nitric oxide synthase, cyclooxygenase-2, as well as the pro- vs anti-inflammatory cytokine ratio in the LPS-treated RAW macrophages. Mal C was found to inhibit LPS-induced NF-kB activation in RAW 264.7 cells by blocking the MyD88-dependent pathway. Mal C suppressed NF-κB activation and iNOS promoter activity, which correlated with its inhibitory effect on IκB phosphorylation and degradation, and NF-κB nuclear translocation, in the LPS-stimulated macrophages. It also inhibited LPS-induced phosphorylation of p38 and JNK, which are also upstream activators of NF-κB, without affecting Akt phosphorylation. Mal C also effectively blocked the PKR-mediated activation of NF-κB. These findings indicate that mal C exerts an anti-inflammatory effect through NF-κB-responsive inflammatory gene expressions by inhibiting the p38 and JNK-dependent canonical NF-κB pathway as well as the PKR pathway, and is a potential therapeutic agent against acute inflammation.     

6-Gingerol inhibits ROS and iNOS through the suppression of PKC-α and NF-κB pathways in lipopolysaccharide-stimulated mouse macrophages

Inflammation is involved in numerous diseases, including chronic inflammatory diseases and the development of cancer. Many plants possess a variety of biological activities, including antifungal, antibacterial and anti-inflammatory activities. However, our understanding of the anti-inflammatory effects of 6-gingerol is very limited. We used lipopolysaccharide (LPS)-stimulated macrophages as a model of inflammation to investigate the anti-inflammatory effects of 6-gingerol, which contains phenolic structure. We found that 6-gingerol exhibited an anti-inflammatory effect. 6-Gingerol could decrease inducible nitric oxide synthase and TNF-α expression through suppression of I-κBα phosphorylation, NF-κB nuclear activation and PKC-α translocation, which in turn inhibits Ca²⁺ mobilization and disruption of mitochondrial membrane potential in LPS-stimulated macrophages. Here, we demonstrate that 6-gingerol acts as an anti-inflammatory agent by blocking NF-κB and PKC signaling, and may be developed as a useful agent for the chemoprevention of cancer or inflammatory diseases.