NF-κB Decoy Research Articles

Potential use of iontophoresis for transdermal delivery of NF-κB decoy oligonucleotides

Topical application of nuclear factor-κB (NF-κB) decoy appears to provide a novel therapeutic potency in the treatment of inflammation and atopic dermatitis. However, it is difficult to deliver NF-κB decoy oligonucleotides (ODN) into the skin by conventional methods based on passive diffusion because of its hydrophilicity and high molecular weight. In this study, we evaluated the in vitro transdermal delivery of fluorescein isothiocyanate (FITC)-NF-κB decoy ODN using a pulse depolarization (PDP) iontophoresis. In vitro iontophoretic experiments were performed on isolated C57BL/6 mice skin using a horizontal diffusion cell. The apparent flux values of FITC-NF-κB decoy ODN were enhanced with increasing the current density and NF-κB decoy ODN concentration by iontophoresis. Accumulation of FITC-NF-κB decoy ODN was observed at the epidermis and upper dermis by iontophoresis. In mouse model of skin inflammation, iontophoretic delivery of NF-κB decoy ODN significantly reduced the increase in ear thickness caused by phorbol ester as well as the protein and mRNA expression levels of tumor necrosis factor-α (TNF-α) in the mice ears. These results suggest that iontophoresis is a useful and promising enhancement technique for transdermal delivery of NF-κB decoy ODN.

Oxidized Ultrashort Nanotubes as Carbon Scaffolds for the Construction of Cell-Penetrating NF-κB Decoy Molecules

Oligonucleotide (ODN) decoys are synthetic ODNs containing the DNA binding sequence of a transcription factor. When delivered to cells, these molecules can compete with endogenous sequences for binding the transcription factor, thus inhibiting its ability to activate the expression of target genes. Modulation of gene expression by decoy ODNs against nuclear factor-kappaB (NF-kappaB), a transcription factor regulating many genes involved in immunity, has been achieved in a variety of immune/inflammatory disorders. However, the successful use of transcription factor decoys depends on an efficient means to bring the synthetic DNA to target cells. It is known that single-walled carbon nanotubes (SWCNTs), under certain conditions, are able to cross the cell membrane. Thus, we have evaluated the possibility to functionalize SWCNTs with decoy ODNs against NF-kappaB in order to improve their intracellular delivery. To couple ODNs to CNTs, we have exploited the carbodiimide chemistry which allows covalent binding of amino-modified ODNs to carboxyl groups introduced onto SWCNTs through oxidation. The effective binding of ODNs to nanotubes has been demonstrated by a combination of microscopic, spectroscopic, and electrophoretic techniques. The uptake and subcellular distribution of ODN decoys bound to SWCNTs was analyzed by fluorescence microscopy. ODNs were internalized into macrophages and accumulated in the cytosol. Moreover, no cytotoxicity associated with SWCNT administration was observed. Finally, NF-kappaB-dependent gene expression was significantly reduced in cells receiving nanomolar concentrations of SWCNT-NF-kappaB decoys compared to cells receiving SWCNTs or SWCNTs functionalized with a nonspecific ODN sequence, demonstrating both efficacy and specificity of the approach.

Inhibitory effect of NF-κB decoy on insulin resistance in adipocytes of patients with type 2 diabetes mellitus

Objective To investigate the relationship of abnormal insulin signal transduction with NF-κB activation in the adipose tissue of patients with type 2 diabetes mellitus(T2DM) ,and to study the effect of NF-κB decoy on insulin resistance in vitro. Methods Decoy targeting NF-κB was designed and synthesized. Adipocytes from abdominal subcutaneous adipose tissues were obtained from T2DM patients and non-diabetic patients. The phosphorylation of insulin signal transduction molecules IRS-1 tyrosine and Akt-Ser473 of adipocytes were examined by immunoprecipitation and Western blotting analysis after stimulation with insulin. The activity of NF-κB in the adipocytes was analyzed by electrophoretic mobility shift assay (EMSA). The NF-κB decoy was transfected into the adipocytes of T2DM patients via liposome. Results The phosphorylation levels of insulin signal transduction molecule IRS-1 tyrosine and Akt-Ser473 in adipocytes T2 DM patients were significantly less than that in adipocytes from non-diabetes mellitus controls( P 0. 05). The activity of NF-κB in adipocytes of T2 DM patients was significantly higher than that in the controls( P 0. 01). After transfection of NF-κB decoy,the activity of NF-κB was decreased compared with that before transfection( P 0. 05) ,and the phosphorylation levels of IRS-1 tyrosine and Akt-Ser473 in adipocytes of T2 DM patients were significantly increased compared with those before transfection( P 0. 05). Conclusion Insulin resistance and excessive activation of NF-κB exist in the adipocytes of patients with T2DM; NF-κB decoy can partly ameliorate insulin resistance.

Effect of nuclear factor-kappa B decoy oligodeoxynncleotides on respiratory function and cytokine expression after severe lung contusion in rabbits

Objective To explore the effect of nuclear factor-kappa B （ NF-κB ） decoy oligodeoxynueleotides （ODN） on respiratory function and expressions of IL-1β and IL-13 in serum following severe lung contusion in rabbits. Methods A total of 40 New-Zealand rabbits were randomly divided into four groups, ie, severe lung contusion group （Group A, n = 12）, severe lung contusion with NF-κB scrambled decoy ODN intervention group （ Group B, n = 12） , severe lung contusion with sense NF- B decoy ODN intervention group （Group C, n = 12） and nomlal control group （Group D, n =4）. After the contusion model was set up, the sense and scrambled NF-κB decoy ODN were infused into the rabbits via the jugular veins in different groups, with 20 g per experimental rabbit. After contusion, respiratory frequency, tidal volume, airway pressure, respiration flow rate curve and end expiration nitric oxide concentration were detected at 1, 2, 3 and 4 hours. The expressions of IL-1β and IL-13 in serum were observed by means of ELISA. Results After sense NF-κB decoy ODN intervention, alveolar ventilation, arterial PO2 and pulmonary compliance were improved, compared with Group A and Group B, with statistical difference （ P 〈 0. 01 ）. The expression of IL-1β was decreased and that of IL-13 increased after sense NF-κB decoy ODN intervention to the severe lung contusion, compared with Groups A and B, with statistical difference （P 〈0.01 ）. The expression of IL-1β was increased to peak level at 1 hour after contusion, which continued to the end of the experiment. While expression of IL-13 was decreased at 1 hour after contusion and reached the minimum level at 4 hours. With intervention with sense decoy ODN, the increased expression of IL-1β was down-regulated, but expression of IL-13 remained at high level, with statistical difference compared with Group A and Group B （ P 〈 0. 01 ）. Conclusions Intervention with sense NF-κB decoy ODN can significantly protect the respiratory function, reduce the expression of IL-1β, and increase expression of IL-13 after severe lung contusion. Key words: Thoracic injuries ; Nuclear factor-kappa B ; Decoy oligodeoxynucleotides ; Respiratory mechanics; Pulmonary ventilation; Pulmonary gas exchange; Inflammation mediators

Intratracheally instilled mannosylated cationic liposome/NFκB decoy complexes for effective prevention of LPS-induced lung inflammation

The nuclear factor kappa B (NFκB) signaling pathway is a key mechanism in the pathophysiology of lung inflammation. NFκB is critically responsible for the expression of pro-inflammatory mediators following activation. The specific inhibition of NFκB by a NFκB decoy via inhalation appears to improve therapeutic effects. However, administration of naked NFκB decoy limits the efficacy of the decoy strategy due to low targeting ability to immune cells such as alveolar macrophages. In this study, we have assessed the effect of alveolar macrophage-targeted NFκB decoy by mannosylated (Man) cationic liposomes in a LPS-induced lung inflammation model after intratracheal administration. The complex of Man-cationic liposome/NFκB decoy was physically stable during spraying. Man-cationic liposome/NFκB decoy complex was selectively delivered to alveolar macrophages for subsequent localization of NFκB decoy in the cytoplasm and to a lesser extent in the nucleus. In the LPS-induced lung inflammation model, pre-treatment with Man-cationic liposome/50 μg NFκB decoy complex significantly inhibited the release of TNF-α, IL-1β and CINC-1, neutrophil infiltration and NFκB activation compared with naked NFκB decoy, cationic liposome/NFκB decoy complex and Man-cationic liposome/scrambled decoy complex treatments. This study demonstrates the sufficient targeting of NFκB decoy using Man-cationic liposomes in a novel effective anti-inflammatory therapy for lung inflammation.

Encapsulation of NF-κB decoy oligonucleotides within echogenic liposomes and ultrasound-triggered release

Echogenic liposomes (ELIP) have additional promise, beyond diagnostic agents, as vehicles for delivering oligonucleotides (ODN), especially if the release of the agent can be triggered and its uptake can be enhanced by ultrasound application at a specific site. The purpose of this study was to co-encapsulate air and NF-κB decoy ODN within ELIP allowing ultrasound to release encapsulated ODN from ELIP, and to accurately quantify release of encapsulated ODN from ELIP upon ultrasound application. FITC-labeled sense ODN (2 mM) was incorporated within ELIP using freeze/thaw method. Encapsulation efficiency of FITC–ODN was spectrofluorometrically analyzed by quenching fluorescence of unencapsulated FITC–ODN using a complementary strand tagged with Iowa Black FQ–ODN. Quenching of FITC–ODN (0.05 μM) with Iowa Black FQ–ODN (0.1 µM) was found to be efficient (92.4 ± 0.2%), allowing accurate determination of encapsulated ODN. Encapsulation efficiency of ODN was 14.2 ± 2.5% in DPPC/DOPC/DPPG/CH liposomes and 29.6 ± 1.5% in DPPC/DOPE/DPPG/CH liposomes. Application of ultrasound (1 MHz continuous wave, 0.26 MPa peak-to-peak pressure amplitude, 60 s.) to the latter formulation triggered 41.6 ± 4.3% release of ODN from ODN–containing ELIP. We have thus demonstrated that ODN can be encapsulated into ELIP and released efficiently upon ultrasound application. These findings suggest potential applications to gene therapy for atherosclerosis as well as a variety of other diseases.

New Treatment of Periodontal Diseases by Using NF-κB Decoy OligodeoxynucleotidesviaPrevention of Bone Resorption and Promotion of Wound Healing

Nuclear factor-kappa B (NF-kappaB) is involved in osteoclast differentiation and activation. Thus, the blockade of the NF-kappaB pathway might be a novel therapeutic strategy for treating bone metabolic diseases. Periodontitis is subgingival inflammation caused by bacterial infection; this disease also is thought to be a chronic focal point responsible for systemic diseases. In this study, NF-kappaB decoy oligodeoxynucleotides (ODNs) were topically applied for experimental periodontitis in a debris-accumulation model and wound healing in a bone-defect model of beagle dogs to investigate the effect of decoy ODN on bone metabolism. Application of NF-kappaB decoy ODN significantly reduced interleukin-6 activity in crevicular fluid and improved alveolar bone loss in the analysis of dental radiographs and DEXA. Direct measurement of exposed root that lost alveolar bone support revealed that NF-kappaB decoy treatment dramatically protected bone from loss. In a bone-defect model, NF-kappaB decoy ODN promoted the healing process as compared with control scrambled decoy in micro-CT analysis. Overall, inhibition of NF-kappaB by decoy strategy prevented the progression of bone loss in periodontitis and promoted the wound healing in bone defects through the inhibition of osteoclastic bone resorption. Targeting of NF-kappaB might be a potential therapy in various bone metabolic diseases.

Lipopolysaccharide (LPS) increases the invasive ability of pancreatic cancer cells through the TLR4/MyD88 signaling pathway

Inflammation plays a multifaceted role in cancer progression, and NF-kappaB is one of the key factors connecting inflammation with cancer progression. We have shown that lipopolysaccharide (LPS) promotes NF-kappaB activation in colon cancer cells and pancreatic cancer cells. However, it is unclear why inflammatory stimuli can induce NF-kappaB activation in cancer cells. We used two human pancreatic cancer cells, Panc-1 and AsPC-1, as target cells. LPS was used as an inflammatory stimulus. To confirm the participation of TLR4/NF-kappaB signaling pathway, we used three different NF-kappaB inhibitors (PDTC, IkappaBalpha mutant, and NF-kappaB decoy ODN) and siRNAs (against TLR4, MyD88, and MMP-9). Effect of LPS on pancreatic cancer cell invasive ability was determined by Matrigel invasion assay. LPS increased the invasive ability of pancreatic cancer cells, while blockade of NF-kappaB pathway decreased the LPS-dependent increased invasive ability. Blockade of TLR4 or MyD88 by siRNA also decreased the LPS-dependent increased invasive ability. These results suggest that TLR/MyD88/NF-kappaB signaling pathway plays a significant role in connecting inflammation and cancer invasion and progression.

Effects of intratracheal administration of nuclear factor-kappaB decoy oligodeoxynucleotides on long-term cigarette smoke-induced lung inflammation and pathology in mice

To determine if nuclear factor-κB (NF-κB) activation may be a key factor in lung inflammation and respiratory dysfunction, we investigated whether NF-κB can be blocked by intratracheal administration of NF-κB decoy oligodeoxynucleotides (ODNs), and whether decoy ODN-mediated NF-κB inhibition can prevent smoke-induced lung inflammation, respiratory dysfunction, and improve pathological alteration in the small airways and lung parenchyma in the long-term smoke-induced mouse model system. We also detected changes in transcriptional factors. In vivo, the transfection efficiency of NF-κB decoy ODNs to alveolar macrophages in BALF was measured by fluorescein isothiocyanate (FITC)-labeled NF-κB decoy ODNs and flow cytometry post intratracheal ODN administration. Pulmonary function was measured by pressure sensors, and pathological changes were assessed using histology and the pathological Mias software. NF-κB and activator protein 1(AP-1) activity was detected by the electrophoretic motility shift assay (EMSA). Mouse cytokine and chemokine pulmonary expression profiles were investigated by enzyme-linked immunosorbent assay (ELISA) in bronchoalveolar lavage fluid (BALF) and lung tissue homogenates, respectively, after repeated exposure to cigarette smoke. After 24 h, the percentage of transfected alveolar macrophages was 30.00 ± 3.30%. Analysis of respiratory function indicated that transfection of NF-κB decoy ODNs significantly impacted peak expiratory flow (PEF), and bronchoalveolar lavage cytology displayed evidence of decreased macrophage infiltration in airways compared to normal saline-treated or scramble NF-κB decoy ODNs smoke exposed mice. NF-κB decoy ODNs inhibited significantly level of macrophage inflammatory protein (MIP) 1α and monocyte chemoattractant protein 1(MCP-1) in lung homogenates compared to normal saline-treated smoke exposed mice. In contrast, these NF-κB decoy ODNs-treated mice showed significant increase in the level of tumor necrosis factor-α(TNF-α) and pro-MMP-9(pro-matrix metalloproteinase-9) in mice BALF. Further measurement revealed administration of NF-κB decoy ODNs did not prevent pathological changes. These findings indicate that NF-κB activation play an important role on the recruitment of macrophages and pulmonary dysfunction in smoke-induced chronic lung inflammation, and with the exception of NF-κB pathway, there might be complex mechanism governing molecular dynamics of pro-inflammatory cytokines expression and structural changes in small airways and pulmonary parenchyma in vivo.

NFκB decoy delivery using dendritic poly( l-lysine) for treatment of endotoxin-induced hepatitis in mice

Mono-dispersed, 6th generation dendritic poly( l-lysine) (KG6) forms a stable complex with plasmid DNA and this complex can circulate in vivo for extended times before the DNA finally accumulates in the liver. In this study, we attempted to use KG6 as a carrier of NFκB decoy oligonucleotide to the liver to treat hepatitis, induced by lipopolysaccharide and d-galactosamine. KG6 formed a complex with the NFκB decoy. Serum aspartate aminotransferase and alanine aminotransferase were dramatically suppressed in the hepatitis mouse model after intravenous injection of KG6/NFκB decoy complexes. Expression levels of several cytokines and proteins related to the inflammatory reaction were also suppressed by intravenous administration of KG6/NFκB decoy complexes. Because [ 32P] NFκB decoy was found in non-parenchymal cells after intravenous injection, KG6 has been shown to be a promising carrier molecule of various oligonucleotides to non-parenchymal liver cells, including Kupffer cells.

Reduced Collagen Biosynthesis Is the Hallmark of Cerebral Aneurysm

Reduced extracellular matrix is a prominent feature of cerebral aneurysms (CAs). We previously reported excessive ECM degradation in CA walls. In the present study, we examined collagen biosynthesis in CA walls and the molecular mechanisms underlying it in CA progression. RT-PCR and immunohistochemistry showed reduced expression of procollagen type I, III, and lysyl oxidase (LOX) in CA walls. Treatment with the LOX inhibitor beta-aminopropionitrile resulted in enhanced progression of CA. Expression of procollagen type I, III, and LOX was inhibited by interleukin-1beta (IL-1beta) in cultured rat aortic smooth muscle cells (RASMCs) in vitro. Nuclear factor kappa-B (NF-kappaB) was activated in IL-1beta-stimulated RASMCs, and treatment with NF-kappaB decoy oligodeoxynucleotides (ODN) restored reduced expression of procollagen type I, III, and LOX in vitro. NF-kappaB decoy ODNs ameliorated the expression of procollagen type I, III, and LOX in CA walls in vivo. Collagen biosynthesis was significantly inhibited at the transcriptional level and in the posttranscriptional enzymatic modification in CA walls through upregulated expression of IL-1beta and the NF-kappaB pathway. Reduced collagen biosynthesis may contribute to CA progression, and inhibition of this process may lead to the prevention of the progression and rupture of CAs.

Nanoparticle-Mediated Delivery of Nuclear Factor κB Decoy Into Lungs Ameliorates Monocrotaline-Induced Pulmonary Arterial Hypertension

Pulmonary arterial hypertension (PAH) is an intractable disease of the small pulmonary artery that involves multiple inflammatory factors. We hypothesized that a redox-sensitive transcription factor, nuclear factor kappaB (NF-kappaB), which regulates important inflammatory cytokines, plays a pivotal role in PAH. We investigated the activity of NF-kappaB in explanted lungs from patients with PAH and in a rat model of PAH. We also examined a nanotechnology-based therapeutic intervention in the rat model. Immunohistochemistry results indicated that the activity of NF-kappaB increased in small pulmonary arterial lesions and alveolar macrophages in lungs from patients with PAH compared with lungs from control patients. In a rat model of monocrotaline-induced PAH, single intratracheal instillation of polymeric nanoparticles (NPs) resulted in delivery of NPs into lungs for <or=14 days postinstillation. The NP-mediated NF-kappaB decoy delivery into lungs prevented monocrotaline-induced NF-kappaB activation. Blockade of NF-kappaB by NP-mediated delivery of the NF-kappaB decoy attenuated inflammation and proliferation and, thus, attenuated the development of PAH and pulmonary arterial remodeling induced by monocrotaline. Treatment with the NF-kappaB decoy NP 3 weeks after monocrotaline injection improved the survival rate as compared with vehicle administration. In conclusion, these data suggest that NF-kappaB plays a primary role in the pathogenesis of PAH and, thus, represent a new target for therapeutic intervention in PAH. This nanotechnology platform may be developed as a novel molecular approach for treatment of PAH in the future.

Nanoparticle-Mediated Drug Delivery and Pulmonary Hypertension

Nanotechnology offers great potential benefits for drug delivery and therapy of respiratory and systemic diseases. Nanoparticles (NPs) have been of significant interest for some time because they can be designed to simultaneously carry a drug payload, specifically target features of diseased tissues, and carry an imaging molecule to track drug accumulation and clearance in tissues. Moreover, they can be engineered to tailor drug delivery and improve pharmacokinetics. A variety of NPs have been investigated in experimental animal models as tools to improve the delivery and therapeutic efficacy of drugs or genes delivered to the lung or other organ systems.1 The nanotechnology platform for drug delivery contains a number of very different types of nanostructures with widely varying properties. Examples of these NPs include dendrimers, fullerenes, carbon nanotubes, and polymeric NPs. In this issue of Hypertension , Kimura et al2 report that nuclear factor κB (NF-κB) decoy oligodeoxynucleotides (ODNs) encapsulated in poly-(ethylene glycol)- block -lactide/glycolide copolymer NPs and delivered to the lungs of rats by intratracheal instillation reduced pulmonary arterial hypertension (PAH) induced by monocrotaline (MCT). They showed that NP-encapsulated NF-κB decoy, visualized by fluorescein isothiocyanate labeling, reached the distal regions of the lungs and was present in alveolar macrophages and small pulmonary arteries for ≤14 days after a single instillation. The small pulmonary arteries were also found to be a site of NF-κB activation and NF-κB–dependent inflammatory cytokine production (monocyte chemoattractant protein 1, interleukin 1, and tumor necrosis factor α) in patients with PAH and in rats with MCT-induced PAH. The decoy ODNs, unlike antisense ODNs, which bind specific regions in mRNA, bind directly to the transcription factor and inhibit transcription factor binding to target DNA and initiation of gene transcription (Figure). It was speculated by the authors that cellular uptake of the NPs might slowly release encapsulated …

The Effects of Nuclear Factor-κB Decoy Oligodeoxynucleotide on Lipopolysaccharide-Induced Direct Acute Lung Injury

Background: The pathophysiologic mechanisms of early acute lung injury (ALI) differ according to the type of primary insult. It is important to differentiate between direct and indirect pathophysiologic pathways, and this may influence the approach to treatment strategies. NF-κB decoy oligodeoxynucleotide (ODN) is a useful tool for the blockade of the expression of NF-κB-dependent proinflammatory mediators and has been reported to be effective in indirect ALI. The purpose of this study was to investigate the effect of NF-κB decoy ODN in the lipopolysaccharide (LPS)-induced direct ALI model. Methods: Five-week-old specific pathogen-free male BALB/c mice were used for the experiment. In the preliminary studies, tumor necrosis factor (TNF)-α, interleukine (IL)-6 and NF-κB activity peaked at 6 hours after LPS administration. Myeloperoxidase (MPO) activity and ALI score were highest at 36 and 48 hours, respectively. Therefore, it was decided to measure each parameter at the time of its highest level. The study mice were randomly divided into three experimental groups: (1) control group which was administered 50 μL of saline and treated with intratracheal administration of 200 μL DW containing only hemagglutinating virus of Japan (HVJ) vector (n=24); (2) LPS group in which LPS-induced ALI mice were treated with intratracheal administration of 200 μL DW containing only HVJ vector (n=24); (3) LPS＋ODN group in which LPS-induced ALI mice were treated with intratracheal administration of 200 μL DW containing 160 μg of NF-κB decoy ODN and HVJ vector (n=24). Each group was subdivided into four experimental subgroups: (1) tissue subgroup for histopathological examination for ALI at 48 hours (n=6); (2) 6-hour bronchoalveolar lavage (BAL) subgroup for measurement of TNF-α and IL-6 in BAL fluid (BALF) (n=6); (3) 36-hour BAL subgroup for MPO activity assays in BALF (n=6); and (4) tissue homogenate subgroup for measurement of NF-κB activity in lung tissue homogenates at 6 hours (n=6). Results: NF-κB decoy ODN treatment significantly decreased NF-κB activity in lung tissues. However, it failed to improve the parameters of LPS-induced direct ALI, including the concentrations of tumor necrosis factor-α and interleukin-6 in BALF, myeloperoxidase activity in BALF and histopathologic changes measured by the ALI score. Conclusion: NF-κB decoy ODN, which has been proven to be effective in indirect models, had no effect in the direct ALI model.

Oligonuleotide Delivery with Dendritic Poly(L-lysine) for Treatments of the Liver Disorders

AbstractA cationic peptide dendrier, dendritic poly(L-lysine), forms complexes with oligonucleotides and can deliver them to liver after intravenous injection. Here, we tried to deliver apolipoprotein B-specific siRNA for the treatment of hypercholesterolemia and NFκB decoy for the hepatitis treatment. Significant therapeutic effects in those disease model mice were observed after intravenous injection of the oligonucleotides complexes with dendritic poly(L-lysine).

Transcriptional regulation of NF-κB by ring type decoy oligodeoxynucleotide in an animal model of nephropathy

Inflammation of the tubulointerstitial compartment, leading to fibrosis, is a major factor in the progressive loss of renal function in a wide variety of kidney diseases. In order to develop a therapeutic approach for nephropathy, we examined the simultaneous inhibition of transcription factor nuclear factor-κB (NF-κB), which is responsible for a wide range of cellular processes, especially inflammation, in a mouse model of unilateral ureteral obstruction. In this study, we employed a ring-type NF-κB (R-NF-κB) decoy oligodeoxynucleotide (ODN), containing consensus promoter sequences of NF-κB. This R-NF-κB decoy ODN is more highly resistant to degradation by nucleases than is the current phosphothiolated double stranded NF-κB decoy ODN. The inhibitory effect of R-NF-κB decoy ODN on nephropathy was confirmed by molecular and histological examinations. In addition, treatment with R-NF-κB decoy ODN reduced the activities of inflammatory cytokines, such as tumor necrosis factor-α and interleukin-1β. Interestingly, the treatment with R-NF-κB decoy ODN also suppressed the gene expression of transforming growth factor-β1 and fibronectin, resulting in the inhibition of fibrotic changes. These results suggest that the inhibition of NF-κB using R-NF-κB decoy ODN has potential therapeutic application in the prevention of renal fibrosis.

Tumor necrosis factor receptor 1 induces interleukin-6 upregulation through NF-kappaB in a rat neuropathic pain model

Peripheral nerve injury resulting in neuropathic pain induces the upregulation of interleukin (IL)-6 and tumor necrosis factor-α, which binds to tumor necrosis factor receptor 1 (TNFR1) and induces NF-κB and p38 MAPK activation in the spinal cord and dorsal root ganglia (DRG). We here investigated whether TNFR1 regulates IL-6 expression through NF-κB or p38 MAPK activations in the spinal cord and DRG in rats with chronic constriction injury (CCI) of the sciatic nerve. Intrathecal treatment with a TNFR1 antisense oligonucleotide (ASO) significantly inhibited CCI-elevated IKKs phosphorylation, IkB-α degradation, the nuclear translocation, phosphorylation, and DNA-binding activity of NF-κB, p38 MAPK activation, and IL-6 mRNA and protein expression in the spinal cord and DRG. Interestingly, CCI remarkably elevated IKKα and p65 phosphorylations in the spinal cord rather than in the DRG. In addition, NF-κB decoy, but not p38 MAPK inhibitor, SB203580 reduced CCI-elevated IL-6 expression in the spinal cord and DRG. Therefore, these data suggest that TNFR1 induces IL-6 upregulation and neuropathic pain through NF-κB, but not p38 MAPK activation in the spinal cord and DRG and that the NF-κB/IL-6 pathways in the DRG may be less dependent on TNFR1 than the spinal cord pathway.

Amelioration of murine dextran sulfate sodium-induced colitis by nuclear factor-κb decoy oligonucleotides

Background Activation of nuclear factor (NF)-κB has been shown to play a critical role in the pathogenesis of ulcerative colitis (UC). The purpose of the current study was to investigate the effects of NF-κB decoy oligonucleotides (ODNs) on an experimental model of UC. Methods NF-κB decoy ODNs were administered in experimental colitis induced by dextran sulfate sodium (DSS). The disease activity index (DAI) and histological score were observed. NF-κB DNA binding activity was assessed by electrophoretic mobility shift assay (EMSA). The expression of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) were measured by reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). Results A significant improvement was observed in DAI and histological score in mice with NF-κB decoy ODNs, and the increase in NF-κB DNA binding activity, myeloperoxidase (MPO) activity, IL-1β, and TNF-a in mice with DSS-induced colitis was significantly reduced following administration of NF-κB decoy ODNs. Conclusions The administration of NF-κB decoy ODNs leads to an amelioration of DSS-induced colitis, suggesting administration of NF-κB decoy ODNs may provide a therapeutic approach for UC.

Suppressive effects of sugar-modified cationic liposome/NF-κB decoy complexes on adenovirus vector-induced innate immune responses

To date, no countermeasures have been developed to overcome adenovirus (Ad) vector-induced innate immune responses in gene therapies that utilize these vectors. In the present study, we attempted to suppress Ad vector-induced innate immune responses and hepatotoxicity by delivering an NF-κB decoy to splenic macrophages and dendritic cells, as well as to Kupffer cells in the liver, using sugar-modified cationic liposomes. Pre-injection of the sugar-modified cationic liposome/NF-κB decoy complexes reduced serum levels of interleukin (IL)-12, which is a typical inflammatory cytokine induced by Ad vectors, and also reduced hepatotoxicity following Ad vector injection. Electrophoretic mobility shift assay demonstrated a reduction of Ad vector-induced NF-κB activation by pre-injection of the sugar-modified cationic liposome/NF-κB decoy complexes. Two types of sugar-modified cationic liposomes, fucosylated cationic liposomes (Fuc-liposomes) and mannosylated cationic liposomes (Man-liposomes), were tested in this study. Man-liposomes, which are considered to have greater affinity than Fuc-liposomes for splenocytes, appeared to have superior suppressive effects to those of Fuc-liposomes. Pre-injection of the sugar-modified cationic liposome/NF-κB decoy complexes did not inhibit Ad vector-mediated transduction in the organs. This study provides important findings for overcoming Ad vector-induced innate immune responses.

Inhibition of Tissue Factor Expression in Brain Microvascular Endothelial Cells by Nanoparticles Loading NF-κB Decoy Oligonucleotides

To investigate a nuclear factor-kappa B decoy oligonucleotides strategy on the inhibition of tissue factor (TF) expression in cultured rat brain microvascular endothelial cells (BMECs) by polylactic acid (PLA) nanoparticles delivery system and to evaluate this new vector for in vitro gene therapy. Nanoparticles were formulated using poly D,L-polylactic acid with surface modifying by polysorbates 80. 3-[4,5-Dimethylthiazol-2,5-diphenyl-2H-tetrazolium bromide] (MTT) assays showed that PLA nanoparticles were not toxic to the cultured BMECs.The decoy oligonuceotides (ODNs) loaded within nanoparticles was 6 μg/mg, encapsulation efficacy was (60.5±1.5)%. It was observed by flow cytometry that the cellular uptake of nanoparticles depended on the time of incubation and the concentration of nanoparticles in the medium. And confocal microscopy demonstrated that nanoparticles localized mostly in the BMECs cytoplasm. The released decoy oligonuceotides (ODNs) uptaked by BMECs retained their biologic activity and led to reduced level of tissue factor expression as compared to control cultures. These findings offer a potential therapeutic strategy in the control of TF expression in BMECs in vitro and suggest that PLA nanoparticles may be appropriate as delivery vehicles for decoy strategy in the gene therapy of cerebral thrombosis.