Reversal of LPS induced endothelial cell TNF synthesis and increased permeability with microencapsulated antisense oligomers to NF-κB

Endothelial cells form the barrier between the circulation and interstitial space. Changes in permeability of endothelial cells allow penetration of inflammatory cells such as polymorphonuclear cells and macrophages to respond to infections and other inflammatory stimuli. Endothelial cells have also been shown to be phagocytic and produce pro-inflammatory cytokines such as TNF. It is the purpose of this study to evaluate endothelial cell phagocytosis of albumin microspheres containing antisense oligonucluetide to NF-κB (MASO), the effect of MASO on TNF synthesis after LPS stimulation and the effect of TNF inhibition on the permeability of endothelial cells in vitro. Results were (1) endothelial cells avidly phagocytozed albumin miocrospheres 1.0 and 1.7?µm in size, (2) phagocytosis of microspheres was potentiated by LPS, (3) TNF is synthesized by endothelial cells in cell culture with the peak concentrations occurring 4?h after stimulation with LPS, (4) MASO results in high intracellular concentration of oligomer, (5) MASO inhibits TNF synthesis to a greater extent than equivalent amounts of NF-κB antisense in solution and (6) the inhibition of TNF by MASO significantly decreases the permeability of albumin through endothelial cells in vitro.     

The effect of intracellular delivery of catalase and antisense oligonucleotides to NF-κB using albumin microcapsules in the endotoxic shock model

Microencapsulated (MC) catalase has been shown to inhibit H₂O₂ and tumor necrosis factor (TNF) in vitro after endotoxin stimulation. It is the purpose of this study to determine whether MC catalase improves pro-inflammatory cytokine inhibition and mortality in an endotoxic shock model in vivo. We also examined whether MC catalase and antisense oligonucleotides (ASO) to nuclear factor κB (NF-κB) together improved survival by inhibiting pro-inflammatory cytokines using different mechanisms. Methods: Albumin microcapsules containing catalase and ASO to NF-κB were prepared 2–7 μm in size by using a Büchi spray dryer. Progressively increasing doses of MC catalase, MC ASO to NF-κB, and the combination were given to rats before the administration of Escherichia coli endotoxin. Results demonstrated 60% survival in rats given 15?mg/kg MC catalase, 70% survival with 20?mg/kg MC ASO NF-κB, and 80% survival with the combination. TNF was inhibited by 53% in the MC catalase group 4?h after endotoxin administration, 43% in the ASO NF-κB group, and 78% in the combination group compared to controls. In conclusion, this study demonstrates the effectiveness of MC intracellular delivery of the naturally occurring antioxidant catalase in improving animal survival. The addition of ASO to NF-κB improved both cytokine inhibition and animal survival in endotoxic shock.     

Formulation and in vitro characterization of spray-dried antisense oligonucleotide to NF-κB encapsulated albumin microspheres

The aim of this study was to formulate and characterize microspheres containing antisense oligonucleotide to NF-κB using bovine serum albumin as the polymer matrix. Microspheres were prepared by spray-drying technique with 5, 10 and 15% drug loading. Glutaraldehyde was used as a cross-linking agent. The particle sizes ranged from 3–5 µm. Microspheres were smooth and spherical in shape, as determined by scanning electron microscopy (SEM). The yield of microspheres ranged from 70–75% and the encapsulation efficiencies were found to be in the range of 59–60%, as determined by a novel HPLC method. Zeta potential of the microspheres ranged between ?39 to ?53 mV, thus indicating good suspension stability in water. In-vitro release studies performed using phosphate buffer saline demonstrated extended drug release up to 72 h. Kinetic model fitting showed high correlation with the Higuchi model, suggesting that the drug release was primarily diffusion controlled.     

Celastrol-induced apoptosis in human HaCaT keratinocytes involves the inhibition of NF-κB activity

Psoriasis is a chronic inflammatory skin disease affecting 1-3% of the world's population. Traditional Chinese medicines have been extensively used for treating psoriasis with promising clinical results. Celastrol, a triterpenoid isolated from a Chinese herb Celastrus orbiculatus caulis, has been known to have diverse pharmacological effects such as anti-inflammatory, anti-cancer and antioxidant activities. The present study aimed at evaluating the anti-proliferative action of celastrol on cultured HaCaT cells and elucidating the mechanisms of action involved. Celastrol was shown to inhibit HaCaT cells growth with an IC?? value of 1.1 μM as measured by MTT assay. The ability of celastrol to induce apoptosis was studied by flow cytometric and western blot analyses. Celastrol was found to be capable of inducing apoptosis in HaCaT cells as characterized by phosphatidyl-serine (PS) externalization, depolarization of mitochondrial membrane potential and activation of caspase-3. The apoptosis induced by celastrol could be suppressed by Z-IETD-FMK and Z-LEHD-FMK, the respective caspase-8 and caspase-9 inhibitor. In addition, western blot analysis revealed a significant augmentation in the protein expression of Bax and attenuation in Bcl-2, suggesting that the celastrol-induced apoptosis acts through both death receptor and mitochondrial pathways. Moreover, western blot analysis on the expression of Rel/NF-κB demonstrated that the celastrol-mediated apoptosis on HaCaT cells was associated with the inhibition of the NF-κB pathway. Taken together, the present project has for the first time identified celastrol as a naturally occurring compound with potent apoptogenic action on cultured human keratinocytes, rendering it a promising candidate for further development into an anti-psoriatic agent.     

Formula PSORI-CM01 inhibits the inflammatory cytokine and chemokine release in keratinocytes via NF-κB expression

Psoriasis is a common chronic inflammatory disease in which T-helper 1(Th1) and T-helper 17(Th17) cells play an important role in its pathology. Formula PSORI-CM01 was a novel formulated Chinese medicine used for psoriasis therapy. It had been demonstrated previously that PSORI-CM01 and serum contained Formula PSORI-CM01 (PCM01CS) could improve psoriasis by inhibiting the epithelial hyperplasia, how PSORI-CM01 affects inflammatory cytokine and chemokine in dermis is still unknown. In this study we found PSORI-CM01 pre-treated 3 days before IMQ painting could ameliorated IMQ-induced mice skin lesion as PASI score was apparently reduced. Th1 related cytokine IFN-γ and Th17 related cytokine IL-17/IL-22 was used to induce inflammatory models on human keratinocyte cell line HaCaT in vitro, respectively. PCM01CS significantly reduced IFN-γ induced mRNA expression of IL-6, IL-12 and CXCL-10, reduced IL-6 and CXCL-10 release into HaCaT supernatant. 20 ng/ml IL-17/IL-22 co-stimulation significantly upregulated expression of IL-6, IL-8 and CCL20 mRNA expression in HaCaT cells, PCM01CS significantly inhibit these cytokines expression both in mRNA and in protein levels. Finally, PCM01CS could obviously inhibit nuclear NF-κB p65 expression which activated by IFN-γ and IL-17/IL-22 stimulation. Thus, our new findings reveal that Formula PSORI-CM01 may possess therapeutic action on psoriasis by inhibiting inflammatory within skin environments.     

Tanshinone IIA prevents uric acid nephropathy in rats through NF-κB inhibition

The purpose of this research is to investigate the effect of tanshinone IIA, an extract of the Chinese medicine Que Xie Hua Yu Tang, on uric acid nephropathy (UAN) and to elucidate the underlying mechanisms. UAN rat model was established. Fifty UAN rats were randomly allocated into 5 groups: adenine-treated group, allopurinol-treated group, and low/middle/high dose of tanshinone IIA-treated groups. Meanwhile, another 10 rats were used as normal controls. Serum uric acid (UA), blood urea nitrogen (BUN), serum creatinine (Scr), MCP-1, and IL-1β levels were measured. Histological staining was performed. Comparison between the adenine group and treatment (allopurinol and tanshinone IIA) groups showed compound treatment could attenuate the inflammation status of the kidneys and decrease serum UA levels. Among different kinds of medicine, tanshinone IIA had similar effects as allopurinol and exerted anti-inflammatory and renal protective effect in a dose-dependent manner. Furthermore, we found tanshinone IIA alone could also inhibit urate-induced MCP-1 and IL-1β overexpression both in vivo and in vitro, accompanied with inhibition of NF-κB translocation from cytosome to nucleus. Tanshinone IIA could protect rats from uric acid-induced kidney damage, probably by attenuating renal inflammatory status.     

Soluble Siglec-9 alleviates intestinal inflammation through inhibition of the NF-κB pathway

BackgroundSialic acid-binding immunoglobulin-like lectins (Siglecs) are a superfamily of immunoreceptors recognizing sialic acid. Siglec-9 has been shown to mediate inhibitory immune responses. The aim of this study was to evaluate the effect of a soluble form of Siglec-9 (sSiglec-9) on inflamed intestinal epithelial cells (IECs), murine macrophages, and experimental murine colitis models.MethodsCOLO 205 human IECs and RAW 264.7 murine macrophages were pretreated with sSiglec-9 and then stimulated with TNF-α or lipopolysaccharides, respectively. The expression of proinflammatory cytokines such as IL-8 and TNF-α was measured using real-time RT-PCR and ELISA. To demonstrate the inhibitory effects of sSiglec-9 on the NF-κB pathway, IκBα phosphorylation/degradation was determined using western blotting and the DNA binding activity of NF-κB was evaluated using an electrophoretic mobility shift assay. Further, mouse models with dextran sulfate sodium-induced acute colitis and piroxicam-induced IL-10^-/- chronic colitis were generated. Intraperitoneal injections of sSiglec-9 were performed, and body weight, colon length, and histopathologic findings were examined.ResultssSiglec-9 suppressed IL-8 and TNF-α gene expression in stimulated COLO 205 and RAW 264.7 cells. sSiglec-9 inhibited IκBα phosphorylation/degradation and the DNA binding activity of NF-κB. sSiglec-9 injections significantly ameliorated weight loss, colon shortening, and the severity of intestinal inflammation in acute and chronic colitis mouse models.ConclusionsSiglec-9 may inhibit NF-κB activation in IECs and macrophages and alleviate experimental colitis in mice, suggesting that sSiglec-9 is a potential therapeutic agent for the treatment of inflammatory bowel disease.     

Bovine glycomacropeptide induces cytokine production in human monocytes through the stimulation of the MAPK and the NF-κB signal transduction pathways

Background and purpose:

Bovine glycomacropeptide (BGMP) is a natural milk peptide that is produced naturally in the gastrointestinal tract during digestion. Glycomacropepide has intestinal anti-inflammatory activity, but the mechanism of action is unknown. Here we have characterized the effects of BGMP on monocytes.

Experimental approach:

We have used human THP-1 cells as an in vitro monocyte model. The effect of BGMP on the secretion of tumour necrosis factor (TNF), interleukin (IL)-1β and IL-8 was assessed, as well as the involvement of the NF-κB and MAP kinase signalling pathways. The stimulatory effect of BGMP was also tested in human peripheral blood monocytes.

Key results:

BGMP up-regulated the secretion of TNF, IL-1β and IL-8 in a concentration-dependent fashion. The biological activity was exerted by the intact peptide, because cytokine secretion was not affected by protease inhibitors. The secretion of IL-8 and specially TNF and IL-1β was blocked by PD98059, SP600125, SB203580 and Bay11-7082, suggesting the involvement of the MAP kinases p38, c-Jun N-terminal kinase and ERK and particularly the NF-κB pathway, although IL-8 secretion was independent of p38. BGMP was shown to elicit the phosphorylation of IκB-α and the nuclear translocation of the NF-κB subunits p50 and p65. The effect of BGMP on cytokine secretion was validated in human primary blood monocytes.

Conclusions and implications:

BGMP stimulates human monocytes, operating via MAP kinase and NF-κB pathways. BGMP may exert an indirect intestinal anti-inflammatory effect by potentiating host defences against invading microorganisms.     

The effect of antisense to NF-κB in an albumin microsphere formulation on the progression of left-ventricular remodeling associated with chronic volume overload in rats

Abstract

Background: Increased NF-κB levels play a crucial role in the pathophysiology of heart failure and are known to cause ventricular remodeling. Antisense therapy can be used for blocking the expression of NF-κB and subsequently avoiding heart failure. However, as with most biotechnology products, molecular instability and overall cost are often the major issues and concerns limiting the advancement of most antisense drugs to the market. Therefore, a cost-efficient biodegradable sustained release particle drug delivery system to transport and target NF-kB antisense to its intended site of action would be ideal.

Purpose: To evaluate the in vivo performance of a sustained release spray-dried albumin microsphere formulation for effective delivery and treatment of left ventricular remodeling with antisense to NF-κB.

Methods: Albumin-based microspheres encapsulating antisense to NF-kB were prepared by spray drying and studied in a rat model to treat congestive heart failure.

Results: The NF-κB activation and TNF-α release seen in treated animals were significantly lower than control animals. Ventricular remodeling was controlled in animals with antisense-treated AV fistulas as ΔV_0–25 and ΔV₀ were significantly lower compared to animals with untreated AV fistulas.

Conclusion: This treatment was successful in curbing ventricular remodeling by suppressing NF-κB activation.     

Pinocembrin attenuates allergic airway inflammation via inhibition of NF-κB pathway in mice

Pinocembrin, one of the primary flavonoids in propolis, possesses many biological activities, including anti-inflammation, anti-oxidation and immunoregulation. This study aimed to evaluate whether pinocembrin could attenuate ovalbumin (OVA)-induced allergic airway inflammation in mice and to explore the possible mechanism. BALB/c mice sensitized and challenged with OVA were administered intraperitoneally with pinocembrin. Airway inflammation and airway hyperresponsiveness were examined. T-helper type (Th) 2 cytokines in bronchoalveolar lavage fluid (BALF) and OVA-specific immunoglobulin E (IgE) in serum were determined. The activation of nuclear factor kappa B (NF-κB) p65 were also measured. Our results showed that pinocembrin resulted in significant inhibition of pathophysiological signs of allergic asthma, including increased pulmonary eosinophilia infiltration, mucus hypersecretion and airway hyperresponsiveness (AHR). Treatment with pinocembrin significantly reduced Th2 cytokines interleukin (IL)-4, IL-5 and IL-13 in BALF, and OVA-specific IgE in serum. Moreover, pinocembrin treatment suppressed phosphorylation of inhibitor-κBα (IκBα) and NF-κB subunit p65 activation in lung tissue of OVA-sensitized mice. These data suggest that pinocembrin may inhibit allergic airway inflammation, and providing potential benefits in the treatment of inflammatory disease.     

Silver nanoparticles induced changes in the expression of NF-κB related genes are cell type specific and related to the basal activity of NF-κB

Silver nanoparticles (AgNPs) are widely used in industry and medicine but the recent evidence for their cytotoxicity rise a concern about the safety of their use. We have previously shown that human A549 cells are resistant to AgNPs cytotoxicity, as compared with similarly treated HepG2 cells. In order to check for the role of the NF-κB signaling pathway in response of A549 and HepG2 cell lines to the treatment with 20 nm and 200 nm AgNps, we analyzed the expression of 84 key genes related to the functionality of the NF-κB signaling pathway. We observed considerable alternations in gene expression in HepG2 cells treated with 20 nm AgNPs, and minor changes when exposed to 200 nm AgNPs. Surprisingly, no changes in gene expression were observed in A549 cells treated with both size AgNPs. Using the NF-κB luciferase reporter system, we further tested the basal activity and inducibility of the NF-κB pathway in both cell lines and found that the inducibility of NF-κB signaling in A549 cells is approximately 5 times lower than this of HepG2 cells, but the basal activity is approximately 3.5 times higher. In accordance, the NF-κB activation after AgNPs treatment was observed in HepG2 but not in A549. Altogether indicate that NF-kB mediated cellular response to AgNPs is cell type specific and related to the basal activity of NF-κB.     

Damnacanthal-induced anti-inflammation is associated with inhibition of NF-κB activity

Morinda citrifolia L. (Rubiaceae), commonly called noni, is a traditional folk medicinal plant with a long history of use for several diseases. Its anti-inflammation activity has been proposed, but detailed knowledge of this antiinflammation mechanism remains unclear. Here, we investigated the effects of noni extract and its major bioactive component damnacanthal on anti-inflammation in vivo as well as in vitro. Our data demonstrate that noni extract and its bioactive component damnacanthal exhibit suppression of inflammation as evidenced by the suppression of paw and ear edema in rats and mice, and down-regulation of lipopolysaccharide-induced nuclear factor-κB (NF-κB) activity, respectively. As a result, the expression of pro-cytokines, cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS) were suppressed in the presence of damnacanthal. These results provide a potential use of damnacanthal in the treatment of inflammatory-related diseases.     

FKBP51 and the NF-κB regulatory pathway in cancer

Constitutive activation of NF-κB occurs in a significant percentage of human cancers. Genetic abnormalities of tumors often enhance normal NF-κB signaling. Chronic inflammation is also associated with constitutive NF-κB activation and increases the risk of cancer. Aberrant NF-κB activation favors cellular transformation, sustains cancer survival, and contributes to tumor invasion. Strategies to inhibit NF-κB represent a promising therapeutic option against cancer. In the last decade, several studies point to the large immunophilin FKBP51 as an important element for the control of NF-κB activation in human neoplasia. This article is an overview of the causes of aberrant NF-κB regulation in cancer and highlights recent papers that implicate FKBP51 in such deregulation.