Gene Expression Of NF-κB P65 Research Articles

Regulation of tight junction proteins and cell death by peroxisome proliferator-activated receptor γ agonist in brainstem of hypertensive rats.

The decrease in tight junction proteins and their adapter proteins in the hypertensive brain is remarkable. Here, we aimed to investigate tight junction proteins and peroxisome proliferator-activated receptor (PPARγ) activation as well as inflammation factors and cell death proteins in the brainstem of hypertension models, namely spontaneously hypertensive rats (SHR) and borderline hypertensive rats (BHR). At first, SHR and BHR groups were treated with PPARγ agonist, pioglitazone. Then, occludin, claudin-1, claudin-2, claudin-12, ZO-1, and NF-κB p65 gene expression levels; pIKKβ, NF-κB p65, TNF, IL-1β, caspase-3, caspase-9 levels, and PARP-1 cleavage were evaluated. Significantly lower pIKKβ, NF-κB p65, TNF, and IL-1β levels were measured in pioglitazone-treated SHR. Results from this study confirm higher occludin (1.35-fold), claudin-2 (7.45-fold), claudin-12 (1.12-fold), and NF-κB p65 subunit (4.76-fold) expressions in the BHR group when compared to the SHR group. Pioglitazone was found effective in terms of regulating gene expression in SHR. Pioglitazone significantly increased occludin (8.17-fold), claudin-2 (2.41-fold), and claudin-12 (1.85-fold) mRNA levels, which were accompanied by decreased cleaved caspase-3, caspase-9 levels, PARP-1 activation, and proinflammatory factor levels in SHR (p ˂ 0.05). Our work has led us to conclude that alterations in tight junction proteins, particularly occludin, and cell death parameters in the brainstem following PPARγ activation may contribute to neuroprotection in essential hypertension.

Studies to elucidate the effect and antiapoptotic mechanism of 2-ethyl-3-hydroxy-6- methylpyridine-n-acetyltaurinate in a rat model of retinal ischemia–reperfusion

Background: The study of possible ways of effective correction of retinal ischemia-reperfusion injury, which accompanies a number of eye diseases, is relevant today. The aim of the study: To study the retinoprotective effect and antiapoptotic mechanism of 2-Ethyl-3-hydroxy-6-methyl-pyridine-N-acetyltaurinate (EHMP-NAT) in a rat model of retinal ischemia–reperfusion (I/R). Materials and methods: A pathology model with an increase in intraocular pressure (IOP) to 110 mmHg was used. The retinoprotective effect of EHMP-NAT at a dose of 4.4 mg/kg/day, in comparison with emoxipine and taurine in equimolar doses, was estimated by the changes in the retinal microcirculation, electroretinograms (the b/a coefficient), and retinal caspase-3, NF-κB p65, p53 gene expressions in Wistar rats. Results: The use of EHMP-NAT led to an increase in the retinal microcirculation level to 756.5 (median) perfusion units in comparison with emoxipine (p = 0.045) and taurine (p = 0.00029). The b/a coefficient increased in comparison with the group with emoxipine (p = 0.0099) and with the group with taurine (p = 0.015). In the group with EHMP-NAT, the caspase-3 gene expression decreased reliably in comparison with emoxipine (p = 0.0002) and with taurine (p = 0.0028); the NF-κB p65 gene expression decreased in comparison with emoxipine (p = 0.0009) and with taurine (p = 0.0022); the p 53 gene expression decreased in comparison with emoxipine (p = 0.0022) and with taurine (p = 0.0009). Conclusion: Based on the data obtained, in correction of retinal I/R by EHMP-NAT, improvements in the retinal microcirculation, functional state, and caspase-3, NF-κB p65, p53 gene expressions were more pronounced than in monotherapy with emoxipine or taurine.

Long Term High Protein Diet Feeding Alters the Microbiome and Increases Intestinal Permeability, Systemic Inflammation and Kidney Injury in Mice.

This study evaluates the effects of a chronic high protein diet (HPD) on kidney injury, intestinal permeability and gut microbiota perturbations in a mouse model. Mice are fed a diet containing either 20% or 52% energy from protein for 24 weeks; protein displaced an equivalent amount of wheat starch. The HPD does not alter glycemic control or body weight. The HPD induces kidney injury as evidenced by increase in albuminuria, urinary kidney injury molecule-1, blood urea nitrogen, urinary isoprostanes and renal cortical NF-κB p65 gene expression. HPD decreases intestinal occludin gene expression, increases plasma endotoxin and plasma monocyte chemoattractant protein-1, indicating intestinal leakiness and systemic inflammation. Cecal microbial analysis reveals that HPD feeding does not alter alpha diversity; however, it does alter beta diversity, indicating an altered microbial community structure with HPD feeding. Predicted metagenome pathway analysis demonstrates a reduction in branched-chain amino acid synthesis and an increase of the urea cycle with consumption of a HPD. These results demonstrate that long term HPD consumption in mice causes albuminuria, systemic inflammation, increase in gastrointestinal permeability and is associated with gut microbiome remodeling with an increase in the urea cycle pathway, which may contribute to renal injury.

Novel complementary antitumour effects of celastrol and metformin by targeting IκBκB, apoptosis and NLRP3 inflammasome activation in diethylnitrosamine-induced murine hepatocarcinogenesis.

One promising strategy for minimizing chemotherapeutic resistance in hepatocellular carcinoma (HCC) is the use of effective chemosensitizers. We studied the complementary multi-targeted molecular mechanisms of metformin and celastrol in mice with diethylnitrosamine-induced HCC to investigate whether metformin could augment the sensitivity of HCC tissue to the effect of celastrol. Simultaneous administration of celastrol (2mg/kg) and metformin (200mg/kg) improved liver function, enhanced the histological picture and prolonged survival. Additionally, combination therapy exerted anti-inflammatory activity, as indicated by the decreased levels of TNF-α and IL-6. This protective role could be attributed to inhibition of inflammasome activation. Herein, our data revealed downregulated NLRP3 gene expression, suppressed caspase-1 activity and reduced levels of the active forms of IL-1β and IL-18. Under this condition, pyroptotic activity was suppressed. In contrast, in the celastrol and celastrol + metformin groups, the apoptotic potential was amplified, as revealed by the increase in the caspase-9 and caspase-3 levels and Bax:BCL-2 ratio. In addition to their repressive effect on the gene expression of NFκBp65, TNFR and TLR4, metformin and celastrol inhibited phosphorylation-induced activation of IκBκB and NFκBp65 and decreased IκBα degradation. Combination therapy with metformin and celastrol repressed markers of angiogenesis, metastasis and tumour proliferation, as revealed by the decreased hepatic levels of VEGF, MMP-2/9 and cyclin D1 mRNA, respectively. In conclusion, by inhibiting NLRP3 inflammasome and its prerequisite NFκB signalling, simultaneous administration of metformin and celastrol appears to have additive benefits in the treatment of HCC compared to cela monotherapy. This effect warrants further clinical investigation.

Trichostatin A, a Histone Deacetylase Inhibitor, Alleviates Eosinophilic Meningitis Induced by Angiostrongylus cantonensis Infection in Mice.

Histone deacetylase inhibitor (HDACi) has been used in the treatment of neurodegenerative or autoimmune diseases. Angiostrongyliasis cantonensis caused by Angiostrongylus cantonensis infection is an emerging zoonosis of human eosinophilic meningitis or meningoencephalitis. Progressive neuronal apoptosis is the pathological basis of behavioral dysfunctions in angiostrongyliasis cantonensis. Neurological defects after anthelmintic treatment for angiostrongyliasis cantonensis are still common. In this study, we examined the effects of trichostatin A (TSA), a HDACi, on eosinophilic meningitis induced by A. cantonensis in mice. Intragastric administration of TSA significantly ameliorated brain injury and decreased cognitive impairments in mice at 15 days post-infection. TSA administration effectively reduced the inflammatory factor levels of iNOS, TNF-α, IL-5, IL-6, and IL-13 in infected mice. TSA treatment counteracted apoptosis with reduced expression levels of cleaved caspase-3, -4, -6, and RIP3 in A. cantonensis infected mice. In addition, TSA administration reduced total HDAC activity and increased the acetylation of histone H3 and H4 in the brain tissue of infected mice. The underlying mechanism of TSA on eosinophilic meningitis might be associated with decreased NF-κB p65 nuclear accumulation by inhibiting IκB phosphorylation. Furthermore, a co-expressive network of NF-κB p65 with 22 other genes was constructed according to our previous transcriptomic data in infected mice. We identified the correlations in the gene expression of NF-κB p65 with Lrp10, Il12rb1, Nfkbia, Ube2n, and Ube2d1 in infected mice after TSA administration. Thus, TSA has a protective effect on the progression of eosinophilic meningitis induced by A. cantonensis in mice.

Effect of intensive insulin therapy on high mobility group box-1/nuclear factor-ΚB pathway in severe traumatic brain injury patient with stress hyperglycemia

To explore the effect of intensive insulin therapy (IIT) on high mobility group box-1/nuclear factor-ΚB (HMGB1/NF-ΚB) signaling pathway in severe traumatic brain injury (sTBI) patient with stress hyperglycemia. Sixty-one sTBI patients with stress hyperglycemia [Glasgow coma scale (GCS) ≤ 8, three times of random blood glucose levels > 11.1 mmoL/L, glycosylated hemoglobin (HbA1c) < 0.065] admitted to the Affiliated Huaian No.1 People's Hospital of Nanjing Medical University from July 2015 to October 2017 were enrolled. Patients were divided into IIT group (29 cases, keeping blood glucose at 4.4-7.8 mmol/L) and conventional glycemic therapy (CGT) group (32 cases, keeping blood glucose at 7.8-12.2 mmo/L) according to the random number table method. Before treatment and 1, 7 and 14 days after treatment, the levels of plasma HMGB1 and tumor necrosis factor-α (TNF-α) were measured by enzyme linked immunosorbent assay (ELISA); C-reactive protein (CRP) was determined by automatic biochemical analyzer, and NF-ΚB p65 gene expression in peripheral blood mononuclear cells was detected by real-time quantitative polymerase chain reaction (PCR). Nine patients were withdrawn from the observation because the 4 consecutive blood glucose monitoring did not reach the target value, combined with severe infection, or abandoned the treatment with serious brain damage. Finally, 52 patients were enrolled in the analysis, including 28 in CGT group and 24 in IIT group. The levels of plasma HMGB1, TNF-α, CRP and the expression of NF-ΚB gene in monocytes of the two groups at 1 day after treatment were significantly higher than those before treatment, and reached the peak value, then gradually decreased. After 7 days of treatment, they were significantly lower than 1 day. The levels of plasma CRP and TNF-α in the IIT group were significantly lower than those in the CGT group [CRP (mg/L): 36.7±4.4 vs. 45.1±6.1, TNF-α (ng/L): 42.4±9.7 vs. 53.2±9.1, both P < 0.05], the level of HMGB1 in plasma and the expression of NF-ΚB p65 in monocytes were significantly lower than those in the CGT group after 14 days of treatment [HMGB1 (μg/L): 60.1±8.7 vs. 80.5±9.1, NF-ΚB p65 (ΔCt): 35.8±8.5 vs. 53.5±7.3, both P < 0.05]. IIT inhibits the inflammatory response in sTBI patients with stress hyperglycemia through HMGB1/NF-ΚB pathway.

Activation of EMT in colorectal cancer by MTDH/NF-κB p65 pathway.

Epithelial-mesenchymal transition (EMT) leads to tumor dissemination and metastasis. Metadherin (MTDH) is an oncogene that plays an important role in metastasis regulation. This study tries to investigate the effect of MTDH gene up-regulation on the activation of EMT in colorectal cancer (CRC) cells and identify the role of NF-κB p65. The CaCO2 cells were divided into three groups: one control group of cultured CaCO2 cells (C1), and two groups of CaCO2 cells co-transfected using human MTDH expression plasmid with either siRNA targeting human NF-κB p65 or its negative control (C2 and C3 respectively). The gene modification was confirmed by qPCR and the effect of gene modification on CRC aggravation was studied. MTDH up-regulation significantly promoted CRC cell proliferation, activated anaerobic respiration (glucose consumption and lactate production), and increased gene expression of multidrug resistance gene (MDR1), Snail transcription factor and NF-κB p65, but decreased the gene expression of E-cadherin. Moreover, MTDH up-regulation led to a significant increase in the acquisition of surface markers of CRC stem cells. Interference with NF-κB p65 gene expression reversed the action of MTDH gene up-regulation on MDR1 and E-cadherin gene expression and anaerobic respiration. Moreover, NF-κB p65 interference significantly decreased MTDH-induced cell proliferation and acquisition of surface markers of CRC stem cells but didn't affect the Snail transcription factor. MTDH-dependent EMT in CRC is activated via NF-κB p65 and is mediated by up-regulation of Snail. These results identify a pathway by which MTDH regulates NF-κB p65 induced EMT during CRC cell metastasis.

Telmisartan Modulates the Oral Mucositis Induced by 5-Fluorouracil in Hamsters.

Oral mucositis (OM) is a common adverse effect resulting from cancer therapy. The OM it has implications that may compromise oncologic treatment and decrease the patient’s quality of life. The therapeutic options to prevent or treat the symptoms of OM are scarce; there is no effective therapy that improves the symptoms. Based on the need for further research for the treatment of OM, the present study objective was to evaluate the effect of telmisartan (TELM) on the OM induced by 5-fluorouracil (5-FU), using as animal model Golden Syrian hamsters. 5-FU followed by mechanical trauma on day 4 was used to induce OM in hamsters. Euthanasia occurred on the day 10. The experiments were constituted by the groups saline, mechanical trauma, 5-FU, and TELM in three doses (1, 5, or 10 mg/kg). Macroscopic, histopathological, and immunohistochemical analyses as well as immunofluorescence experiments were performed on the oral mucosa of the animals. The samples also were used for analysis enzyme-linked immunosorbent assays and quantitative real-time polymerase chain reactions (qPCR). TELM (5 or 10 mg/kg) was able to reduce the inflammatory ulceration and infiltration in the oral mucosa of the animals, decreasing the levels of the cytokines TNF-α and IL-1β. These treatments was minimize the immunostaining for cyclooxygenase-2, matrix metalloproteinase-9, transforming growth factor-β, and smad 2/3. The nuclear transcription factor kappa B (NFκB) p65 and inducible nitric oxide synthase were reduced in the oral mucosa. Finally, TELM (10 mg/kg) increased the PPARγ gene expression and reduced STAT1 and NFκB p65 gene expression relative to the 5-FU group. Therefore, TELM prevents the OM produced by 5-FU on animal model.

Advanced glycation end products evoke inflammatory reactions in proximal tubular cells via autocrine production of dipeptidyl peptidase-4

We have previously shown that albuminuria and renal levels of advanced glycation end products (AGEs), receptor for AGEs (RAGE), and oxidative stress are suppressed in dipeptidyl peptidase-4 (DPP-4)-deficient diabetic rats, thus suggesting the crosstalk between AGE-RAGE axis and DPP-4 in experimental diabetic nephropathy. Therefore, we examined here the role of DPP-4 in AGE-evoked inflammatory reactions in human proximal tubular cells. Proteins were extracted from proximal tubular cells, and conditioned medium was collected, both of which were subjected to western blot analysis using anti-DPP-4 antibody. RAGE-aptamer was prepared using a systemic evolution of ligands by exponential enrichment. NF-κB p65 and monocyte chemoattractant protein-1 (MCP-1) gene expression was analyzed by reverse transcription-polymerase chain reaction. AGEs significantly increased DPP-4 expression and soluble DPP-4 production by tubular cells, the latter of which was attenuated by RAGE-aptamer or an anti-oxidant, N-acetylcysteine. AGEs or DPP-4 up-regulated NF-κB p65 or MCP-1 mRNA levels in tubular cells, which were suppressed by linagliptin, an inhibitor of DPP-4. AGEs stimulated NF-κB p65 gene expression in tubular cells isolated from control rats, but not from DPP-4-deficient rats. Our present results suggest that the AGE-RAGE-mediated oxidative stress could evoke inflammatory reactions in proximal tubular cells via autocrine production of DPP-4.

Effect of polysaccharides from Vitis vinifera L. on NF-κB/IκB-α signal pathway and inflammatory factors in Alzheimer's model rats

ABSTRACTThe aim of this study was to investigate the mechanism of action underlying the effect of polysaccharide from Vitis vinifera L. (VTP) on the nuclear factor kappa B/inhibitor kappa B alpha (NF-κB/IκB-α) signaling pathway and related inflammatory factors in hippocampus of rats with Alzheimer's disease (AD). Amyloid-β 25–35 was injected into the hippocampus to establish AD model rats, and the rats were administered with donepezil and VTP. The levels of interleukin-1β, interleukin-6 and tumour necrosis factor-α in the serum were determined by enzyme-linked immunosorbent assay. The phosphorylation level, protein and gene expression of NF-κBp65 and IκB-α in hippocampus were detected by immunohistochemistry, western blot and real-time quantitative polymerase chain reaction (PCR), respectively. VTP effectively improved the learning and memory ability of AD rats. Transmission electron microscopy indicated that VTP reduced the toxic effects of amyloid-β 25–35 on neurons in AD rats. Enzyme-linked immunosorbent assay showed that VTP inhibited the expression of interleukin-1β, interleukin-6 and tumour necrosis factor-α in a dose-dependent manner. Compared with the model group, the expression levels of NF-κBp65 in the nucleus of the VTP group decreased, which was consistent with the Western blotting results. The expression of p-IκB-α and IκB-α, and, the mRNA level of NF-κBp65 and IκB-α in VTP group were significantly decreased compared to that in the model group, indicating that VTP has a therapeutic effect on AD. Its mechanism may be related to the inhibition of inflammatory response.

The in vivo action of chronic bisphenol F showing potential immune disturbance in juvenile common carp (Cyprinus carpio)

Bisphenol F (BPF) has been increasingly introduced into industrial applications as a replacement for bisphenol A (BPA), and has emerged as a ubiquitous environmental contaminant worldwide. Few studies have assessed the in vivo toxicities of BPF, particularly long-term exposure toxicities. In the present study, we examined whether long-term BPF exposure in vivo would evoke oxidative stress in the immune system of juvenile common carp. The results suggested that BPF exposure increased ROS content, oxidative stress indices, complement component 3, and immunoglobulin M contents, as well as the expression of inflammatory cytokine genes. Moreover, higher levels of nf-κb p65 gene expression were correlated with the induced ROS content and NF-κB pathway-associated genes, a strong indication that the mode of action of BPF is related to the NF-κB signaling pathway. We also provide evidence that the effects of BPF are comparable to those of BPA with regards to regulation of the immune response in teleosts, and therefore suggest that such chemical analogs should be thoroughly evaluated for their potential toxicity before they can be considered as “safer” replacements.

The pre-protective effect of vitamin A on LPS-induced oxidative stress of bovine mammary epithelial cells

All-trans retinoic acid (RA, derivative of vitamin A) has been demonstrated to have anti-oxidative activity and is needed for the proper function of the immune system. In this study, the protective effects of RA against lipopolysaccharide (LPS)-induced oxidative stress on bovine mammary epithelial cells (BMECs) have been investigated. BMECs were divided into four equal groups with six replicates. The first group was used as a control without RA or LPS. The three experimental groups were treated with RA (1 μg/mL) alone, LPS (1 μg/mL) alone and RA plus LPS, respectively. The cells were treated for 30 h in all. Our results showed that cells in LPS-treated group have a significantly higher inflammatory cytokines concentration and NF-κBp50 and NF-κBp65 gene expression than the control (p < .01). Furthermore, compared with control, selenoproteins’ activity of glutathione peroxidase (GPx), thioredoxin reductase (TrxR), the concentration of SelP and the gene expression of Nrf2 were significantly decreased while the activity of inducible nitric oxide synthase (iNOS), the production of intracellular reactive oxygen species (ROS) and malonaldehyde (MDA) were elevated dramatically in LPS-treated group (p < .01). However, RA in combination with LPS enhanced the antioxidant enzyme activities of selenoprotein compared to LPS-treated group. In conclusion, RA ameliorated the LPS-induced BMECs damage by improving levels of antioxidant markers and reducing inflammatory cytokines contents, indicating that RA has the potential to counter measure the immunosuppressive condition on BMECs oxidative damage and to improve antioxidative function. However, further studies are still required to elucidate the molecular mechanisms underlying the protective effects of RA as an antioxidant.

NF-κBp65 and Expression of Its Pro-Inflammatory Target Genes Are Upregulated in the Subcutaneous Adipose Tissue of Cachectic Cancer Patients.

Cancer cachexia, of which the most notable symptom is severe and rapid weight loss, is present in the majority of patients with advanced cancer. Inflammatory mediators play an important role in the development of cachexia, envisaged as a chronic inflammatory syndrome. The white adipose tissue (WAT) is one of the first compartments affected in cancer cachexia and suffers a high rate of lipolysis. It secretes several cytokines capable of directly regulating intermediate metabolism. A common pathway in the regulation of the expression of pro-inflammatory cytokines in WAT is the activation of the nuclear transcription factor kappa-B (NF-κB). We have examined the gene expression of the subunits NF-κBp65 and NF-κBp50, as well as NF-κBp65 and NF-κBp50 binding, the gene expression of pro-inflammatory mediators under NF-κB control (IL-1β, IL-6, INF-γ, TNF-α, MCP-1), and its inhibitory protein, nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκB-α). The observational study involved 35 patients (control group, n = 12 and cancer group, n = 23, further divided into cachectic and non-cachectic). NF-κBp65 and its target genes expression (TNF-α, IL-1β, MCP-1 and IκB-α) were significantly higher in cachectic cancer patients. Moreover, NF-κBp65 gene expression correlated positively with the expression of its target genes. The results strongly suggest that the NF-κB pathway plays a role in the promotion of WAT inflammation during cachexia.

Anti-Inflammatory and Anti-Mutagenic Effect of the YHK Phytocompound in Hepatocytes: In View of an Age-Management Liver-Protecting Approach

The receptor for advanced glycation end products (RAGE) regulates cellular proliferation in hepatocellular carcinoma (HCC). The aim of this study was to test the in vitro effect of Yo Jyo Hen Shi Ko (YHK), a nutraceutical with prior data suggesting its hepatocyte-protecting role, in regulating RAGE in the proliferation of the HCC cell line HuH7 as well checking also its potential modulation in the expression of the transcriptional factor nuclear factor-κB (NF-κB) p65. Our study showed that YHK significantly reduced cellular growth in the HuH7 cell line (p<0.05). Moreover, this phytocompound partly reduced gene expression of NF-κB p65 (by 35%, p<0.05). These data suggest that YHK has a potential role as a modulator of RAGE and RAGE ligands for potential healthy liver intervention in HCC prevention strategies.

Small interfering RNA-mediated knockdown of NF-κBp65 attenuates neuropathic pain following peripheral nerve injury in rats

Recent reports show that the nuclear factor-κB (NF-κB) can control numerous genes encoding inflammatory and nociceptive mediators and play an important role in the development of central pain sensitization. The aim of the present study is to assess the role of NF-κB signal pathway and its downstream pro-inflammatory cytokines in the modulation of neuropathic pain, by using small interfering RNAs (siRNAs) technique, which has been shown to result in potent, long-lasting post-transcriptional silencing of specific genes. We developed a highly efficient method of lentivirus-mediated delivery of short-hairpin RNA (shRNA) targeting NF-κBp65 for gene silencing. This method successfully transduced LV-shNF-κBp65 into cultured spinal cord neurons in vitro and spinal cord cells in vivo, inhibited the expression of NF-κBp65 and pro-inflammatory factors (TNF-α, IL-1β and IL-6) and alleviated mechanical allodynia and thermal hyperalgesia for more than 4weeks in chronic constriction injury (CCI) model of rats. Taken together, our results suggest that siRNA against NF-κBp65 is a potential strategy for analgesia. Furthermore, the lentiviral vector derived shRNA approach shows a great promise for the management of neuropathic pain and the study of functional NF-κBp65 gene expression.

Suppression of heart NF-kappaB p65 expression by jugular vein injection of RNAi in mice

The nuclear factor-kappaB (NF-κB) in cardiac vascular endothelial cells (type II VEC) is a key factor that activates delayed xenograft rejection (DXR), and therefore inhibition of NF-κB gene expression may alleviate post-transplant rejection. siRNA technology was used to inhibit NF-κB p65 gene expression in ICR mice. After jugular vein injection of siRNA/in vivo-jetPEI complex, fluorescence levels of FAM-labeled siRNA in hearts and lungs were much higher after jugular vein injection than tail vein injection, suggesting more efficient siRNA delivery to the heart through the jugular vein. The amount of FAM fluorescence of hearts increased to the highest level between 48 and 72 hours after injection, and decreased gradually 1 week after injection. A minimum dose of 6 nmol NF-κB p65 siRNA and a siRNA/in vivo-jetPEI ratio of 6 (N/P = 6) were required for in vivo siRNA-mediated gene silencing in the heart. Under these conditions, application of siRNA/in vivo-jetPEI complexes from the jugular vein successfully suppressed NF-κB p65 expression in the heart. The same strategy can be applied to heart transplant animal models to protect against NF-κB gene-related type II VEC activation and xenograft rejection.

Hyperglycemia Induces a Dynamic Cooperativity of Histone Methylase and Demethylase Enzymes Associated With Gene-Activating Epigenetic Marks That Coexist on the Lysine Tail

OBJECTIVEResults from the Diabetes Control Complications Trial (DCCT) and the subsequent Epidemiology of Diabetes Interventions and Complications (EDIC) Study and more recently from the U.K. Prospective Diabetes Study (UKPDS) have revealed that the deleterious end-organ effects that occurred in both conventional and more aggressively treated subjects continued to operate >5 years after the patients had returned to usual glycemic control and is interpreted as a legacy of past glycemia known as “hyperglycemic memory.” We have hypothesized that transient hyperglycemia mediates persistent gene-activating events attributed to changes in epigenetic information.RESEARCH DESIGN AND METHODSModels of transient hyperglycemia were used to link NFκB-p65 gene expression with H3K4 and H3K9 modifications mediated by the histone methyltransferases (Set7 and SuV39h1) and the lysine-specific demethylase (LSD1) by the immunopurification of soluble NFκB-p65 chromatin.RESULTSThe sustained upregulation of the NFκB-p65 gene as a result of ambient or prior hyperglycemia was associated with increased H3K4m1 but not H3K4m2 or H3K4m3. Furthermore, glucose was shown to have other epigenetic effects, including the suppression of H3K9m2 and H3K9m3 methylation on the p65 promoter. Finally, there was increased recruitment of the recently identified histone demethylase LSD1 to the p65 promoter as a result of prior hyperglycemia.CONCLUSIONSThese studies indicate that the active transcriptional state of the NFκB-p65 gene is linked with persisting epigenetic marks such as enhanced H3K4 and reduced H3K9 methylation, which appear to occur as a result of effects of the methyl-writing and methyl-erasing histone enzymes.

Lipid induced overexpression of NF-κB in skeletal muscle cells is linked to insulin resistance

Lipid induced NF-κB activation is known to be associated with insulin resistance and type2 diabetes. Here we show that incubation of L6 skeletal muscle cells with palmitate significantly increased NF-κB p65 and NF-κB p50 expression along with their phosphorylation. NF-κB p65 siRNA inhibited palmitate induced overexpression of NF-κB p65 indicating palmitate effect on transcriptional activation. RT-PCR and real time PCR experiments also showed a significant increase in NF-κB p65 gene expression due to palmitate. Overexpression of NF-κB p65 by palmitate was linked to impairment of insulin activity. Palmitate effect on NF-κB gene and protein expression was found to be mediated by phospho-PKCɛ as calphostin C (an inhibitor of PKC) and ɛV1 (PKCɛ translocation inhibitor) significantly reduced NF-κB expression. To understand the underlying mechanism, we purified NF-κB and pPKCɛ from palmitate incubated skeletal muscle cells and their interaction in cell free system demonstrated the transfer of phosphate from PKCɛ to NF-κB. This prompted us to transduct pPKCɛ to the skeletal muscle cells. These cells showed increased amount of pNF-κB and NF-κB. Excess of NF-κB p65 pool thus created in the cells made them insulin resistant. Addition of NF-κB p65 siRNA and SN50 inhibited palmitate induced NF-κB p65 expression indicating NF-κB regulation of its gene expression. Increase of NF-κB did not affect the activation of IKK/IκB indicating NF-κB p65 expression to be a distinct effect of palmitate. Since NF-κB p65 is linked to several diseases, including type2 diabetes, this report may be important in understanding the pathogenicity of these diseases.