kappaB Activation Research Articles

Enterovirus-71 virus-like particles induce the activation and maturation of human monocyte-derived dendritic cells through TLR4 signaling.

Enterovirus 71 (EV71) causes seasonal epidemics of hand-foot-and-mouth disease and has a high mortality rate among young children. We recently demonstrated potent induction of the humoral and cell-mediated immune response in monkeys immunized with EV71 virus-like particles (VLPs), with a morphology resembling that of infectious EV71 virions but not containing a viral genome, which could potentially be safe as a vaccine for EV71. To elucidate the mechanisms through which EV71 VLPs induce cell-mediated immunity, we studied the immunomodulatory effects of EV71 VLPs on human monocyte-derived dendritic cells (DCs), which bind to and incorporate EV71 VLPs. DC treatment with EV71 VLPs enhanced the expression of CD80, CD86, CD83, CD40, CD54, and HLA-DR on the cell surface; increased the production of interleukin (IL)-12 p40, IL-12 p70, and IL-10 by DCs; and suppressed the capacity of DCs for endocytosis. Treatment with EV71 VLPs also enhanced the ability of DCs to stimulate naïve T cells and induced secretion of interferon (IFN)-γ by T cells and Th1 cell responses. Neutralization with antibodies against Toll-like receptor (TLR) 4 suppressed the capacity of EV71 VLPs to induce the production of IL-12 p40, IL-12 p70, and IL-10 by DCs and inhibited EV71 VLPs binding to DCs. Our study findings clarified the important role for TLR4 signaling in DCs in response to EV71 VLPs and showed that EV71 VLPs induced inhibitor of kappaB alpha (IκBα) degradation and nuclear factor of kappaB (NF-κB) activation.

Beta-arrestin-2 negatively modulates inflammation response in mouse chondrocytes induced by 4-mer hyaluronan oligosaccharide

Beta-arrestin-2 is an adaptor protein that terminates G protein activation and seems to be involved in the modulation of the inflammatory response. Small hyaluronan (HA) fragments, such as 4-mer HA oligosaccharides, are known to interact with the toll-like receptor-4 (TLR-4) with consequent activation of the nuclear factor kappaB (NF-kB) that in turn stimulates the inflammation response. NF-kB activation is mediated by different pathways, in particular by the transforming growth factor-activated kinase-1 (TAK-1). Conversely, increased levels of protein kinase A (PKA), induced by cyclic adenosine monophosphate (cAMP), seem to inhibit NF-kB activation. We studied the involvement and role of beta-arrestin-2 in mouse chondrocytes stimulated with 4-mer HA fragments. The exposure of chondrocytes to 4-mer HA produced a significant up-regulation in TLR-4, cAMP, beta-arrestin-2, TAK-1, protein 38 mitogen-activated protein kinase (p38MAPK), and PKA, both in terms of mRNA expression and of the related protein levels. NF-kB was significantly activated, thereby producing the transcription of pro-inflammatory mediators, including tumor necrosis factor alpha, interleukin-6, and interleukin-17. The treatment of 4-mer HA-stimulated chondrocytes with antibodies against beta-arrestin-2 and/or a specific PKA inhibitor, significantly increased the inflammatory response, while the treatment with a specific p38MAPK inhibitor significantly reduced the inflammatory response. Interestingly, the anti-inflammatory action exerted by beta-arrestin-2 appeared to be mediated in part through the direct inhibition of p38MAPK, preventing NF-kB activation, and in part through cAMP and PKA activation primed by G protein signaling, which exerted an inhibitory effect on NF-kB. Taken together, these results could be useful for future anti-inflammatory strategies.

Systemic inflammation induces a profound long term brain cell injury in rats.

The long term impact of neuroflammation induced by systemic inflammation on astroglial transcription factor nuclear factor (NF)-kappaB activation in the sub-region of hippocampus as well as its' association with the ultra-structural changes in brain cells are yet unknow. In this study, rats received 10 mg/kg of lipopolysaccharide (LPS, ip injection) and NF-kappaBp65 expression on the astroglia as well as the neuronal and astroglial ultra-structural changes and/or death and microvasculature damage were assessed with immunofluorescence and transmission electron microscopy respectively up to 16 days after treatment. The results showed that in the CA1 and CA3 region of the hippocampus, the fluorescence intensity of NF-kappaBp65 was increased from day 1 after LPS injection, reaching a peak at day 3, and stayed at higher level when compared to the control up to 16 days after LPS treatment (P<0.05). Electron microscopy studies revealed sustainable substructural alterations, injury or even death of astrocytes, neurons and capillaries even after 16 days post LPS injection. Our study demonstrated that long-term sustainable activation of astroglial NF-kappaB following systemic inflammation was associated with brain cell and microvasculature injury in the sub-region of the hippocampus which ultimately likely results in brain functional impairment.

DIABETES MELLITUS TIPO 2: QUAL O PAPEL DA INSULINA NA EXPRESSÃO DE NF-KappaB, PPARϒ E CD36?

No diabetes mellitus tipo 2 (DM2) e na síndrome de resistência à insulina, as complicações cardiovasculares resultam de um conjunto de processos aterogênicos envolvendo hiperglicemia crônica, excessiva glicação de proteínas (AGEs), ativação do fator nuclear kappa B (NKκB) associada com o aumento da expressão de citocinas inflamatórias e estresse oxidativo, observando-se ainda alteração de LDL e expressão do receptor de scavenger CD36. A contribuição da hiperinsulinemia nesta sequência não é completamente elucidada. Nesta revisão, relata-se como a insulina pode modular a expressão proteica de NFκB, PPAR gama (PPARγ) e CD36 em células da musculatura lisa vascular (CMLV) da aorta de ratos estimuladas pelos AGE.

Intestinal anti-inflammatory activity of Sasa quelpaertensis leaf extract by suppressing lipopolysaccharide-stimulated inflammatory mediators in intestinal epithelial Caco-2 cells co-cultured with RAW 264.7 macrophage cells.

BACKGROUND/OBJECTIVESInflammatory bowel disease (IBD), including Crohn's disease and ulcerative colitis, involves chronic inflammation of the gastrointestinal tract. Previously, Sasa quelpaertensis leaves have been shown to mediate anti-inflammation and anti-cancer effects, although it remains unclear whether Sasa leaves are able to attenuate inflammation-related intestinal diseases. Therefore, the aim of this study was to investigate the anti-inflammatory effects of Sasa quelpaertensis leaf extract (SQE) using an in vitro co-culture model of the intestinal epithelial environment.MATERIALS/METHODSAn in vitro co-culture system was established that consisted of intestinal epithelial Caco-2 cells and RAW 264.7 macrophages. Treatment with lipopolysaccharide (LPS) was used to induce inflammation.RESULTSTreatment with SQE significantly suppressed the secretion of LPS-induced nitric oxide (NO), prostaglandin E2 (PGE2), IL-6, and IL-1β in co-cultured RAW 264.7 macrophages. In addition, expressions of inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2, and tumor necrosis factor (TNF)-α were down-regulated in response to inhibition of IκBα phosphorylation by SQE. Compared with two bioactive compounds that have previously been identified in SQE, tricin and P-coumaric acid, SQE exhibited the most effective anti-inflammatory properties.CONCLUSIONSSQE exhibited intestinal anti-inflammatory activity by inhibiting various inflammatory mediators mediated through nuclear transcription factor kappa-B (NF-kB) activation. Thus, SQE has the potential to ameliorate inflammation-related diseases, including IBD, by limiting excessive production of pro-inflammatory mediators.

Intermittent hypoxia and diet-induced obesity: effects on oxidative status, sympathetic tone, plasma glucose and insulin levels, and arterial pressure.

Obstructive sleep apnea (OSA) consists of sleep-related repetitive obstructions of upper airways that generate episodes of recurrent or intermittent hypoxia (IH). OSA commonly generates cardiovascular and metabolic pathologies defining the obstructive sleep apnea syndrome (OSAS). Literature usually links OSA-associated pathologies to IH episodes that would cause an oxidative status and a carotid body-mediated sympathetic hyperactivity. Because cardiovascular and metabolic pathologies in obese patients and those with OSAS are analogous, we used models (24-wk-old Wistar rats) of IH (applied from weeks 22 to 24) and diet-induced obesity (O; animals fed a high-fat diet from weeks 12 to 24) to define the effect of each individual maneuver and their combination on the oxidative status and sympathetic tone of animals, and to quantify cardiovascular and metabolic parameters and their deviation from normality. We found that IH and O cause an oxidative status (increased lipid peroxides and diminished activities of superoxide dismutases), an inflammatory status (augmented C-reactive protein and nuclear factor kappa-B activation), and sympathetic hyperactivity (augmented plasma and renal artery catecholamine levels and synthesis rate); combined treatments worsened those alterations. IH and O augmented liver lipid content and plasma cholesterol, triglycerides, leptin, glycemia, insulin levels, and HOMA index, and caused hypertension; most of these parameters were aggravated when IH and O were combined. IH diminished ventilatory response to hypoxia, and hypercapnia and O created a restrictive ventilatory pattern; a combination of treatments led to restrictive hypoventilation. Data demonstrate that IH and O cause comparable metabolic and cardiovascular pathologies via misregulation of the redox status and sympathetic hyperactivity.

Cancer cell growth inhibitory effect of bee venom via increase of death receptor 3 expression and inactivation of NF-kappa B in NSCLC cells.

Our previous findings have demonstrated that bee venom (BV) has anti-cancer activity in several cancer cells. However, the effects of BV on lung cancer cell growth have not been reported. Cell viability was determined with trypan blue uptake, soft agar formation as well as DAPI and TUNEL assay. Cell death related protein expression was determined with Western blotting. An EMSA was used for nuclear factor kappaB (NF-κB) activity assay. BV (1–5 μg/mL) inhibited growth of lung cancer cells by induction of apoptosis in a dose dependent manner in lung cancer cell lines A549 and NCI-H460. Consistent with apoptotic cell death, expression of DR3 and DR6 was significantly increased. However, deletion of DRs by small interfering RNA significantly reversed BV induced cell growth inhibitory effects. Expression of pro-apoptotic proteins (caspase-3 and Bax) was concomitantly increased, but the NF-κB activity and expression of Bcl-2 were inhibited. A combination treatment of tumor necrosis factor (TNF)-like weak inducer of apoptosis, TNF-related apoptosis-inducing ligand, docetaxel and cisplatin, with BV synergistically inhibited both A549 and NCI-H460 lung cancer cell growth with further down regulation of NF-κB activity. These results show that BV induces apoptotic cell death in lung cancer cells through the enhancement of DR3 expression and inhibition of NF-κB pathway.

RhoA/rho kinase signaling reduces connexin43 expression in high glucose-treated glomerular mesangial cells with zonula occludens-1 involvement

RhoA/Rho kinase (ROCK) signaling has been suggested to be involved in diabetic nephropathy (DN) pathogenesis. Altered expression of connexin43 (Cx43) has been found in kidneys of diabetic animals. Both of them have been found to regulate nuclear factor kappa-B (NF-κB) activation in high glucose-treated glomerular mesangial cells (GMCs). The aim of this study was to investigate the relationship between RhoA/ROCK signaling and Cx43 in the DN pathogenesis. We found that upregulation of Cx43 expression inhibited NF-κB p65 nuclear translocation induced by RhoA/ROCK signaling in GMCs. Inhibition of RhoA/ROCK signaling attenuated the high glucose-induced decrease in Cx43. F-actin accumulation and an enhanced interaction between zonula occludens-1 (ZO-1) and Cx43 were observed in high glucose-treated GMCs. ZO-1 depletion or disruption of F-actin formation also inhibited the reduction in Cx43 protein levels induced by high glucose. In conclusion, activated RhoA/ROCK signaling induces Cx43 degradation in GMCs cultured in high glucose, depending on F-actin regulation. Increased F-actin induced by RhoA/ROCK signaling promotes the association between ZO-1 and Cx43, which possibly triggered Cx43 endocytosis, a mechanism of NF-κB activation in high glucose-treated GMCs.

Microglia change from a reactive to an age-like phenotype with the time in culture

Age-related neurodegenerative diseases have been associated with chronic neuroinflammation and microglia activation. However, cumulative evidence supports that inflammation only occurs at an early stage once microglia change the endogenous characteristics with aging and switch to irresponsive/senescent and dystrophic phenotypes with disease progression. Thus, it will be important to have the means to assess the role of reactive and aged microglia when studying advanced brain neurodegeneration processes and age-associated related disorders. Yet, most studies are done with microglia from neonates since there are no adequate means to isolate degenerating microglia for experimentation. Indeed, only a few studies report microglia isolation from aged animals, using either short-term cultures or high concentrations of mitogens in the medium, which trigger microglia reactivity. The purpose of this study was to develop an experimental process to naturally age microglia after isolation from neonatal mice and to characterize the cultured cells at 2 days in vitro (DIV), 10 DIV, and 16 DIV. We found that 2 DIV (young) microglia had predominant amoeboid morphology and markers of stressed/reactive phenotype. In contrast, 16 DIV (aged) microglia evidenced ramified morphology and increased matrix metalloproteinase (MMP)-2 activation, as well as reduced MMP-9, glutamate release and nuclear factor kappa-B activation, in parallel with decreased expression of Toll-like receptor (TLR)-2 and TLR-4, capacity to migrate and phagocytose. These findings together with the reduced expression of microRNA (miR)-124, and miR-155, decreased autophagy, enhanced senescence associated beta-galactosidase activity and elevated miR-146a expression, are suggestive that 16 DIV cells mainly correspond to irresponsive/senescent microglia. Data indicate that the model represent an opportunity to understand and control microglial aging, as well as to explore strategies to recover microglia surveillance function.

Propylene-glycol aggravates LPS-induced sepsis through production of TNF-α and IL-6.

Propylene glycol (1,2-propanediol, PG) is a commonly used solvent for oral, intravenous, as well as topical pharmaceutical preparations. While PG is generally considered to be safe, it has been known that large intravenous doses given over a short period of time can be toxic. To evaluate the effect of PG in sepsis induced by the bacterial endotoxin lipopolysaccharide (LPS). Balb/c mice were treated with LPS (1 mg/kg b.w., i.p.) with or without PG (5 g/kg b.w. i.v.). The survival rate and the production of inflammatory cytokines were measured. In RAW264.7 mouse macrophages encoding NF-kB-luc reporter gene, the nuclear transcription factor kappa-B (NF-kB) activation was measured. We found that intravenous PG increased the mortality rate in sepsis induced by the bacterial endotoxin lipopolysaccharide (LPS) in mice. In accordance with that, PG enhanced LPS-induced production of inflammatory cytokines, including tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in vivo. PG also increased the LPS-induced macrophage activation in vitro as detected by measuring NF-kB activation. Our results indicate that drugs containing high doses of PG can pose a risk when administered to patients suffering from or prone to Gram negative bacterial infection.

Mesenchymal Stem Cell Paracrine Activity Is Modulated by Platelet Lysate: Induction of an Inflammatory Response and Secretion of Factors Maintaining Macrophages in a Proinflammatory Phenotype

Wound healing is achieved through distinct programmed phases: hemostasis, inflammation, mesenchymal cell proliferation and migration, and tissue remodeling. At the injury site, clot formation and platelet degranulation release cytokines and growth factors and actively participating in the healing process and regulating the migration of inflammatory cells, such as neutrophils, macrophages, and lymphocytes. We previously demonstrated that, in an inflammatory environment, prostaglandin E2 (PGE2) secreted by mesenchymal stem cells (MSCs) promoted the macrophage switch from a proinflammatory to a proresolving phenotype. Using an in vitro model, we here evaluated the role carried out by the two main players of the wound healing process, the platelet degranulation content mimicked by the platelet lysate (PL) and the inflammatory stimulus, on the modulation of mouse bone-marrow-derived MSC paracrine activity. We demonstrated that, in MSCs, PL induced nuclear factor kappaB (NF-κB) activation, expression of COX-2 and mPGE synthase, and PGE2 production; in an inflammatory microenvironment, PL increased the inflammatory response and promoted the secretion of the proinflammatory cytokine IL-6. We assayed on mouse primary macrophages the paracrine activity of MSCs exposed to the different microenvironments and we observed that PL-treated MSC-conditioned medium maintained macrophages in a proinflammatory state. The involved factors were granulocyte macrophage-colony stimulating factor induced by PL in MSCs and TNF-α induced by PL-MSC-conditioned medium in macrophages. Our findings indicate that PL triggers an inflammatory response in MSCs and induces the secretion of factors maintaining macrophages in a proinflammatory state thus enhancing the initial inflammatory response to the injury, a key element in the activation of wound healing.

Berberine regulates AMP-activated protein kinase signaling pathways and inhibits colon tumorigenesis in mice.

Colorectal cancer, a leading cause of cancer death, has been linked to inflammation and obesity. Berberine, an isoquinoline alkaloid, possesses anti-inflammatory, anti-diabetes and anti-tumor properties. In the azoxymethane initiated and dextran sulfate sodium (AOM/DSS) promoted colorectal carcinogenesis mouse model, berberine treated mice showed a 60% reduction in tumor number (P = 0.009), a 48% reduction in tumors <2 mm, (P = 0.05); 94% reduction in tumors 2-4 mm, (P = 0.001), and 100% reduction in tumors >4 mm (P = 0.02) compared to vehicle treated mice. Berberine also decreased AOM/DSS induced Ki-67 and COX-2 expression. In vitro analysis showed that in addition to its anti-proliferation activity, berberine also induced apoptosis in colorectal cancer cell lines. Berberine activated AMP-activated protein kinase (AMPK), a major regulator of metabolic pathways, and inhibited mammalian target of rapamycin (mTOR), a downstream target of AMPK. Furthermore, 4E-binding protein-1 and p70 ribosomal S6 kinases, downstream targets of mTOR, were down regulated by berberine treatment. Berberine did not affect Liver kinase B1 (LKB1) activity or the mitogen-activated protein kinase pathway. Berberine inhibited Nuclear Factor kappa-B (NF-κB) activity, reduced the expression of cyclin D1 and survivin, induced phosphorylation of p53 and increased caspase-3 cleavage in vitro. Berberine inhibition of mTOR activity and p53 phosphorylation was found to be AMPK dependent, while inhibition NF-κB was AMPK independent. In vivo, berberine also activated AMPK, inhibited mTOR and p65 phosphorylation and activated caspase-3 cleavage. Our data suggests that berberine suppresses colon epithelial proliferation and tumorigenesis via AMPK dependent inhibition of mTOR activity and AMPK independent inhibition of NF-κB.

Chemopreventive effects of a curcumin-like diarylpentanoid [2,6-bis(2,5-dimethoxybenzylidene)cyclohexanone] in cellular targets of rheumatoid arthritis in vitro.

Synovial fibroblast has emerged as a potential cellular target in progressive joint destruction in rheumatoid arthritis development. In this study, BDMC33 (2,6-bis[2,5-dimethoxybenzylidene]cyclohexanone), a curcumin analogue with enhanced anti-inflammatory activity has been synthesized and the potency of BDMC33 on molecular and cellular basis of synovial fibroblasts (SF) were evaluated in vitro. Synovial fibroblast cells (HIG-82) were cultured in vitro and induced by phorbol-12-myristate acetate (PMA) to stimulate the expression of matrix metalloproteinase (MMPs) and pro-inflammatory cytokines. The protective effects of BDMC33 were evaluated toward MMP activities, pro-inflammatory cytokine expression and nuclear factor kappa-B (NF-κB) activation by using various bioassay methods, including zymography, Western blotting, reverse transcription polymerase chain reaction, immunofluorescense microscopy and electrophoretic mobility shift assay. The results showed that BDMC33 significantly inhibited the pro-gelatinase B (pro-MMP-9) and collagenase activities via suppression of MMP-1 in activated SF. In addition, BDMC33 strongly suppressed MMP-3 gene expression as well as inhibited COX-2 and IL-6 pro-inflammatory gene expression. We also demonstrated that BDMC33 abolished the p65 NF-κB nuclear translocation and NF-κB DNA binding activity in PMA-stimulated SF. BDMC33 represents an effective chemopreventive agent and could be used as a promising lead compound for further development of rheumatoid arthritis therapeutic intervention.

Cholinergic signaling in the gut: a novel mechanism of barrier protection through activation of enteric glia cells.

Enteric glia cells (EGCs) play an important role in maintaining proper intestinal barrier function. We have shown that vagal nerve stimulation (VNS) increases EGC activation, which is associated with better gut barrier integrity. Enteric neurons communicate with EGCs through nicotinic cholinergic signaling, which may represent a pathway by which VNS activates EGCs. This study sought to define further the mechanism by which VNS prevents intestinal barrier failure using an in vitro model. We hypothesized that a nicotinic cholinergic agonist would increase EGC activation, prevent intestinal nuclear factor kappa-B (NF-κB) activation, and result in better intestinal barrier function. Cultured EGCs were exposed to the nicotinic cholinergic agonist nicotine. Expression of glial fibrillary acidic protein (GFAP) was measured by immunoblot to determine changes in EGC activation. Caco-2 cells were grown to confluence and incubated alone or in co-culture with EGCs. Cells were then stimulated with Cytomix for 24 h in the presence or absence of nicotine, and barrier integrity was assessed by permeability to 4-kDa FITC-dextran. Changes in phosphorylated inhibitor of NF-κb (P-IκBα) and phosphorylated NF-κB (P-NF-κB) were assessed by immunoblot. Stimulation with nicotine resulted in EGC activation, as demonstrated by an increase in GFAP expression. Cytomix stimulation increased permeability in Caco-2 cells cultured alone or with EGCs. Treatment of stimulated Caco-2/EGC co-cultures with nicotine reduced permeability similar to control. Nicotine failed to prevent barrier permeability in Caco-2 cells alone. Co-culture of stimulated Caco-2 cells with nicotine-activated EGCs prevented Cytomix-induced increases in P-IκBα and P-NF-κB expression. A pharmacologic nicotinic cholinergic agonist increased EGC activation and improved intestinal epithelial barrier function in an in vitro model of intestinal injury. Nicotine-activated EGCs appear to modulate barrier function by preventing the activation of the NF-κB pathway. Therapies aimed at activating EGCs may have important clinical applications for improving intestinal barrier function after injury.

Immunodeficiency and disseminated mycobacterial infection associated with homozygous nonsense mutation of IKKβ

A, Genealogical tree for the kindred. Arrow highlights index case. Asterisks denote family members who died within the first 2 months of life with febrile illnesses. B, Histology showing granuloma in skin by hematoxylin and eosin staining.

Insights in the mechanism underlying the protective effect of α-lipoic acid against acetaminophen-hepatotoxicity

Acetaminophen (APAP) is one of the most widely used analgesic antipyretic drugs and is a major cause of acute liver failure at overdose. The aim of this study is to investigate the possible protective effect of α-lipoic acid (α-LA, 20 or 100mg/kg administered simultaneously or after 1.5h) against APAP-induced hepatotoxicity in rats. Administration of APAP (1.5g/kg i.p.) resulted in elevation of serum ALT and hepatic malondialdehyde (MDA) content, as well as decrease in hepatic glutathione (GSH) content. In addition, elevation in hepatic hemeoxygenase-1 (HO-1) and NADPH oxidase expression was observed accompanied with a significant reduction in glutathione synthase and cystathionine-beta-synthase (CβS) expression. Furthermore, nuclear factor kappa-B (NF-κB) activity was enhanced in APAP-treated rats. Administration of the standard APAP antidote; N-acetylcysteine (NAC, 1200mg/kg) or α-LA (20mg/kg), simultaneously or 1.5h after APAP, ameliorated APAP-induced alterations in liver function, oxidant and inflammatory markers. Importantly, simultaneous administration of NAC or α-LA (20mg/kg) was more protective than their later administration. However, the beneficial effect of α-LA was lost at higher dose level (100mg/kg). Taken together, the beneficial effects of α-lipoic acid (20mg/kg) were comparable to those of NAC which provides a new possible treatment for APAP-induced hepatotoxicity in patients who cannot tolerate NAC. However, careful dose selection is warranted since the beneficial effects of α-LA were lost at higher doses.

The enhanced antitumor effect of combined triptolide and paclitaxel on pancreatic cancer cell lines.

335 Background: Pancreatic cancer is a disease with dismal prognosis and its treatment options are limited. Recently albumin-bound paclitaxel (nab-paclitaxel) was approved by FDA for advanced pancreatic cancer patients. This study aims to explore the enhancement of therapeutic effect on human pancreatic cancer cell lines (AsPC-1 and PANC-1) via the combination of triptolide, derived from the herb Tripterygium wilfordii, and paclitaxel. Methods: Pancreatic cells were treated with paclitaxel or triptolide alone, and the combination of them. Cell proliferation was measured by sulforhodamine B (SRB). Apoptotic cells were assessed by flow cytometry and western blot for cleaved caspase-3, 8, 9 and PARP. To explore the role of nuclear factor kappaB (NF-kB) activity in pancreatic cancer cell lines, AsPC-1/IkBaM and PANC-1/IkBaM cells (NF-kB activity of cells was silenced by IkB-a mutation) were treated with triptolide plus paclitaxel and NF-kB activity were analyzed. Results: Triptolide demonstrated cytotoxicity on pancreatic cancer cell lines with the IC50 of 25-40nM. The combination of triptolide (IC30 concentration) and paclitaxel enhanced the cytotoxicity significantly . Combination index (CI) indicated that the effect of triptolide plus paclitaxel was synergistic. Furthermore, pancreatic cancer cells treated with triptolide plus paclitaxel exhibited increased apoptosis, as evidenced by stronger Annexin V/ PI staining, higher levels of pro-apoptotic proteins including cleaved caspases and activated PARP compared to cells treated with paclitaxel or gemcitabine alone. In the mechanism study, the combination of triptolide and paclitaxel did not demonstrate synergistic effect on AsPC-1/IkBaM and PANC-1/IkBaM cells. It indicated that the enhanced effect of triptolide plus paclitaxel was correlated with NF-kB activity. Conclusions: (1) the combination of triptolide and paclitaxel enhances the apoptosis in vitro, low concentration of triptolide showed synergistic effect with paclitaxel; (2) the antitumor effect of triptolide plus paclitaxel on pancreatic cancer is mediated by the induction of apoptosis and triptolide enhances paclitaxel-mediated apoptosis via suppression of NF-kB activity.

NOD2 Expression is Regulated by microRNAs in Colonic Epithelial HCT116 Cells

Crohn's disease (CD) is associated with defective sensing of pathogens in genetically susceptible individuals. Nucleotide-binding oligomerization domain containing 2 (NOD2) mutations in coding regions are strongly linked to CD pathogenesis. Our laboratory has reported that microRNAs (miRNAs) are differentially expressed in CD. However, miRNA regulation of NOD2 remains unknown. This study was designed to determine whether miRNAs regulate NOD2 expression as well as downstream nuclear factor kappaB activation and inflammatory responses in colonic epithelial HCT116 cells. NOD2 and miRNA expression in stimulated HCT116 cells were assessed by quantitative reverse transcription-polymerase chain reaction. Regulation of NOD2 expression by miRNAs was determined by luciferase reporter construct assays and transfection of specific miRNA mimics. Regulation of NOD2 signaling and immune response by miRNAs was assessed by transfection of mimics followed by muramyl dipeptide stimulation. Muramyl dipeptide-induced increases in NOD2, interleukin-8, and CXCL3 expression were inversely associated with miRNA expression. Overexpression of miR-192, miR-495, miR-512, and miR-671 suppressed NOD2 expression, muramyl dipeptide-mediated NF-κB activation, and messenger RNA expressions of interleukin-8 and CXCL3 in HCT116 cells. A single-nucleotide polymorphism (rs3135500) located in the NOD2 3'-untranslated region significantly reduced miR-192 effects on NOD2 gene expression. To our knowledge, this is the first report demonstrating that miRNAs regulate NOD2 and its signaling pathway. Four miRNAs downregulate NOD2 expression, suppress NF-κB activity, and inhibit interleukin-8 and CXCL3 messenger RNA expression. Treatment of CD with miRNAs may represent a potential anti-inflammatory therapeutic strategy in CD patients with and without NOD2 gene mutations.

Cordycepin prevented IL-β-induced expression of inflammatory mediators in human osteoarthritis chondrocytes

Cordycepin, a nucleoside derivative isolated from Cordyceps, has been reported to exert anti-inflammatory, antitumor, antidiabetic and renoprotective effects. Osteoarthritis (OA) is a degenerative joint disease with an inflammatory component that drives the degradation of cartilage extracellular matrix. This study aimed to assess the effects of cordycepin on human OA chondrocytes. In this study, human OA chondrocytes were pretreated with cordycepin at 10, 50 or 100 μM and subsequently stimulated with interleukin-1β (IL-1β) (5 ng/ml) for 24 h. Production of prostaglandin E2 (PGE2) and nitric oxide (NO) were evaluated by the Griess reaction and an enzyme-linked immunosorbent assay (ELISA). Gene expression of matrix metalloproteinase (MMP)-13, IL-6, inducible nitric oxide synthase (iNOS) and cyclo-oxygenase (COX-2) was measured by real-time polymerase chain reaction (PCR). MMP-13 and IL-6 proteins in culture medium were determined using cytokine-specific ELISA. Western immunoblotting was used to analyse the iNOS and COX-2 protein production in culture medium. Nuclear factor kappa-B (NF-κB) activity regulation was explored using Western immunoblotting. Pretreatment with cordycepin significantly inhibited the production of PGE2 and NO induced by IL-1β. Cordycepin also significantly decreased the IL-1β-stimulated gene expression and production of MMP-13, IL-6, iNOS and COX-2 in OA chondrocytes. Pretreatment with cordycepin attenuated IL-1β-induced activation of NF-κB by suppressing degradation of its inhibitory protein nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκB-α) in the cytoplasm. We show for the first time the anti-inflammatory activity of cordycepin in human OA chondrocytes. Thus, with this unique profile of actions, cordycepin may prove to be a potentially attractive and new therapeutic/preventive agent for OA.

Ursolic Acid Provides Kidney Protection in Diabetic Rats

BackgroundDiabetic nephropathy (DN) is one of the most serious microvascular complications of diabetes and the leading cause of end-stage renal failure. However, the treatment of DN is still a problem in the world. Inflammatory process plays a critical role in the development of DN. Therefore, anti-inflammatory treatment of DN is worth exploring now and in the future. ObjectiveThe study aimed to evaluate the impact of ursolic acid (UA) on renal function in streptozotocin-induced diabetes. MethodsRats with streptozotocin-induced diabetes were treated with UA for 16 weeks. After 16 weeks, urine albumin excretion, serum creatinine, and blood urea nitrogen were measured. In addition, renal oxidative stress level, nuclear factor kappa-B (NF-κB) activity, P-selectin expression, and kidney histopathologic changes were evaluated. ResultsSixteen weeks following streptozotocin injection, the rats produced significant alteration in renal function and increased oxidative stress, NF-κB activity, and P-selectin expression in the kidneys. Interestingly, UA significantly prevented biochemical and histopathologic changes in the kidneys associated with diabetes. Compared with untreated diabetic rats, UA treatment lowered urine albumin excretion, renal oxidative stress level, NF-κB activity, and P-selectin expression. Moreover, UA treatment also improved renal histopathologic changes in rats with diabetes. ConclusionsUA treatment exhibited a protective effect on kidneys in diabetic rats, implying that UA could be a potential treatment for diabetic nephropathy.