Anxiolytic effects of Formononetin in an inflammatory pain mouse model.

Chronic pain is commonly accompanied with anxiety disorder, which complicates treatment. In this study, we investigated the analgesic and anxiolytic effects of Formononetin (FMNT), an active component of traditional Chinese medicine red clover (Trifolium pratense L.) that is capable of protecting neurons from N-methyl-D-aspartate (NMDA)-evoked excitotoxic injury, on mice suffering from complete Freund's adjuvant (CFA)-induced chronic inflammatory pain. The results show that FMNT administration significantly reduces anxiety-like behavior but does not affect the nociceptive threshold in CFA-injected mice. The treatment reverses the upregulation of NMDA, GluA1, and GABAA receptors, as well as PSD95 and CREB in the basolateral amygdala (BLA). The effects of FMNT on NMDA receptors and CREB binding protein (CBP) were further confirmed by the potential structure combination between these compounds, which was analyzed by in silico docking technology. FMNT also inhibits the activation of the NF-κB signaling pathway and microglia in the BLA of mice suffering from chronic inflammatory pain. Therefore, the anxiolytic effects of FMNT are partially due to the attenuation of inflammation and neuronal hyperexcitability through the inhibition of NMDA receptor and CBP in the BLA.

Short-term high-fat diet affects macrophages inflammatory response, early signs of a long-term problem

Obesity is a chronic inflammatory disease that affects millions of people worldwide. Most studies observe the effects of a high-fat diet (HFD) in 10-12 weeks. This work investigated the effects induced by a HFD administered for 6 weeks on the nutritional status of mice and some aspects of the inflammatory response in mouse peritoneal macrophages. Male Swiss Webster mice, 2-3 months of age, were fed a control diet or HFD for 6 weeks. After this period, the mice were euthanized, and peritoneal macrophages were collected for immunoassays and assessment of biochemical parameters. A HFD was associated with increased cholesterol, insulin resistance, C-reactive protein (CRP), leptin, and serum resistin levels. Lipopolysaccharide (LPS)- stimulated adipocyte cultures of animals subjected to a HFD showed increased production of proinflammatory cytokines such as tumor necrosis factor alpha (TNF-α), interleukin-1 (IL-1), and interleukin-6 (IL-6). However, peritoneal macrophages of the HFD group showed no changes in the levels of these cytokines. LPS-stimulated peritoneal macrophages from HFD-treated animals showed a reduction in mRNA expression of TNF-α and IL-6, as well as a decrease in expression of the transcription factor nuclear factor-kappa B (NF-kB). In conclusion, HFD treatment for 6 weeks induces similar signs to metabolic syndrome and decreases the capacity of peritoneal macrophages to develop an appropriate inflammatory response to a bacterial component

Hyaluronan synthase 3 promotes plaque inflammation and atheroprogression.

OBJECTIVE: Hyaluronan (HA) is a prominent component of the provisional extracellular matrix (ECM) present in the neointima of atherosclerotic plaques. Here the role of HA synthase 3 (HAS3) in atheroprogression was studied. APPROACH AND RESULTS: It is demonstrated here that HAS isoenzymes 1, -2 and -3 are expressed in human atherosclerotic plaques of the carotid artery. In Apolipoprotein E (Apoe)-deficient mice Has3 expression is increased early during lesion formation when macrophages enter atherosclerotic plaques. Importantly, HAS3 expression in vascular smooth muscle cells (VSMC) was found to be regulated by interleukin 1 ß (IL-1ß) in an NFkB dependent manner and blocking antibodies to IL-1ß abrogate Has3 expression in VSMC by activated macrophages. Has3/Apoe double deficient mice developed less atherosclerosis characterized by decreased Th1-cell responses, decreased IL-12 release, and decreased macrophage-driven inflammation. CONCLUSIONS: Inhibition of HAS3-dependent synthesis of HA dampens systemic Th1 cell polarization and reduces plaque inflammation. These data suggest that HAS3 might be a promising therapeutic target in atherosclerosis. Moreover, because HAS3 is regulated by IL-1ß, our results suggest that therapeutic anti-IL-1ß antibodies, recently tested in human clinical trials (CANTOS), may exert their beneficial effects on inflammation in post-myocardial infarction patients in part via effects on HAS3. TOC categorybasic study TOC subcategoryarteriosclerosis.

A signature of microRNA-155 in the pathogenesis of diabetic complications

The current study was designed to explore the potential involvement of miR-155 in the pathogenesis of diabetes complications. Male rats were divided into control and diabetic groups (n = 6). Type 2 diabetes was induced by a single-dose injection of nicotinamide (110 mg/kg; intraperitoneal (i.p.)), 15 min before injection of streptozotocin (STZ; 50 mg/kg; i.p.) in 12-h fasted rats. Two months after induction of diabetes, the rats were sacrificed for subsequent measurements. The nuclear factor kappa B (NF-κB) activity was higher in diabetic peripheral blood mononuclear cells (PBMCs), aorta, heart, kidney, liver, and sciatic nerve, than the control counterparts. Also, apoptosis rate was increased in these tissues, except the aorta. NF-κB messenger RNA (mRNA) expression level was higher in the kidney, heart, PBMCs, and sciatic nerve of diabetic rats than their control counterparts. Except the liver, the miR-155 expression level was significantly decreased in diabetic kidney, heart, aorta, PBMCs, and sciatic nerve versus the controls. Moreover, the expression of miR-155 was negatively correlated with NF-κB activity and apoptosis rate. These results suggest that changes in the expression of miR-155 may participate in the pathogenesis of diabetes-related complications, but causal relationship between miR-155 dysregulation and diabetic complications is unknown

Targeting N-acetylglucosamine-bearing polymer-coated liposomes to vascular smooth muscle cells.

The targeted delivery of anti-inflammatory agents has great therapeutic potential for treating restenosis following percutaneous coronary intervention. To develop a drug delivery system targeted to injured blood vessels, we examined whether N-acetylglucosamine (GlcNAc)-bearing polymer-coated liposomes (GlcNAc-Ls) are specifically taken up by vascular smooth muscle cells (VSMCs). Flow cytometric analysis revealed that GlcNAc-Ls were taken up by VSMCs in vitro. Furthermore, GlcNAc-Ls were intravenously administered to mice that had undergone wire-mediated vascular injury. GlcNAc-Ls markedly accumulated at the intramural site of the injured vessel walls but not at the contralateral (uninjured) vessel walls. These results demonstrated that GlcNAc-Ls can be specifically taken up by VSMCs both in vitro and in vivo. We propose a novel strategy of using GlcNAc-Ls that has potential for application in drug delivery targeted to injured blood vessels.

Differential activation of NF-κB and nitric oxide in lymphocytes regulates in vitro and in vivo radiosensitivity.

Lymphocytes are more sensitive to radiation in vivo than in vitro. However, the mechanism of this differential response is poorly understood. In the present study, it was found that the lipid peroxidation and cell death were significantly higher in lymphocytes following whole body irradiation (WBI) as compared to lymphocytes exposed to radiation in vitro. EL-4 cells transplanted in mice were also more sensitive to radiation than EL-4 cells irradiated in vitro. DNA repair, as assessed by comet assay, was significantly faster in lymphocytes exposed to 4Gy radiation in vitro as compared to that in lymphocytes obtained from whole body irradiated mice exposed to the same dose of radiation. This was associated with increased NF-κB activation in response to genotoxic stress and lesser activation of caspase in lymphocytes in vitro compared to in vivo. To explain the differential radiosensitivity, we postulated a role of nitric oxide, an extrinsic diffusible mediator of radiosensitivity that has also been implicated in DNA repair inhibition. Nitric oxide levels were significantly elevated in the plasma of whole body irradiated mice but not in the supernatant of cells irradiated in vitro. Addition of sodium nitroprusside (SNP), a nitric oxide donor to cells irradiated in vitro inhibited the repair of DNA damage and enhanced apoptosis (increased Bax to Bcl-2 ratio). Administration of l-NAME, a nitric oxide synthase inhibitor, to mice significantly protected lymphocytes against WBI-induced DNA damage and inhibited in vivo radiation-induced production of nitric oxide. These results confirm that the observed differential radiosensitivity of lymphocytes was due to slow repair of DNA due to nitric oxide production in vivo.

Anti-inflammatory properties of dietary flavonoids / Propiedades antiinflamatorias de los flavonoides de la dieta

Flavonoids are a group of natural substances that are located in sources of vegetal origin. More than 4,000 varieties of flavonoids have been identified. All of them are phenyl-benzopyrones of low molecular weight with a basic structure formed by two benzene rings united through a heterocyclic pyrane or pyrone. Besides their relevance in plants, flavonoids are important for human health. Their antioxidant capacity confers a therapeutic potential in cardiovascular diseases, gastric or duodenal ulcers, cancer or hepatic pathologies. Also important are their antiviral and anti-allergic actions, as well as their anti-thrombotic and anti-inflammatory properties. Prostaglandins and nitric oxide biosynthesis is involved in inflammation, and isoforms of inducible nitric oxide synthase (iNOS) and of cyclooxygenase (COX-2) are responsible for the production of a great amount of these mediators. It has been demonstrated that flavonoids are able to inhibit both enzymes, as well as other mediators of the inflammatory process such as reactive C protein or adhesion molecules. Modulation of the cascade of molecular events leading to the overexpression of those mediators include inhibition of transcription factors such as nuclear factor kappa B and AP-1, through the inhibition of protein kinases involved in signal transduction. Increased antioxidant defenses through activation of the NF-E2 related factor 2 (Nrf2) also contribute to the anti-inflammatory capacity of flavonoids

Los flavonoides son un grupo de las sustancias naturales que se encuentran en fuentes de origen vegetal, existiendo más de 4.000 variedades. Todos son fenilbenzopironas de peso molecular bajo con una estructura básica formada por dos anillos heterocíclicos de benceno unidos a través de un pirano o de una pirona. Además de su función en las plantas, los flavonoides son importantes para la salud humana. Su capacidad antioxidante confiere un potencial terapéutico en enfermedades cardiovasculares, úlceras gástricas o duodenales, cáncer o patologías hepáticas. También son importantes sus acciones antivirales y antialérgicas, así como sus características antitrombóticas y antiinflamatorias. La síntesis de prostaglandinas y de óxido nítrico está implicada en la inflamación, e isoformas de la óxido nítrico sintetasa (iNOS) y de la ciclooxigenasa (COX-2) son responsables de la producción de una gran cantidad de estos mediadores. Se ha demostrado que los flavonoides pueden inhibir ambas enzimas, así como otros mediadores del proceso inflamatorio tales como la proteína C reactiva o diversas moléculas de adhesión. La modulación de la cascada de los acontecimientos moleculares que conducen al aumento en la expresión de estos mediadores incluye la inhibición de factores de transcripción tales como el factor nuclear kappaB y el factor AP-1, a través de la inhibición de diferentes proteína quinasas. Otros factores, tales como el incremento de las defensas antioxidantes a través del factor Nrf2 pueden también contribuir a las propiedades antiinflamatorias de los flavonoides

Folate-linked lipid-based nanoparticles deliver a NFkappaB decoy into activated murine macrophage-like RAW264.7 cells.

Activated macrophages are the key effector cells in rheumatoid arthritis (RA) and secrete multiple mediators of inflammation including proinflammatory cytokines. We investigated delivery of a nuclear factor kappa B (NFkappaB) decoy by folate-linked lipid-based nanoparticles (NP-F) into murine macrophages. The expression of folate receptor (FR) in RAW264.7 cells activated by lipopolysaccaride was confirmed by strong expression of FR mRNA, and association of FITC-labeled folate-BSA conjugate. When transfected via NP-F, the NFkappaB decoy was strongly detected in the cytoplasm, and an inhibitory effect on the translocation of NFkappaB into the nucleus was observed at 0.03 microM of the decoy, suggesting that NP-F effectively delivered the NFkappaB decoy into the cytoplasm. This information is of value for the design of NFkappaB decoy carrier systems targeting FR in activated macrophages in gene therapy for autoimmune diseases such as RA.

Activation of nuclear factor-kappaB in human prostate carcinogenesis and association to biochemical relapse.

Nuclear factor (NF)-kappaB/p65 regulates the transcription of a wide variety of genes involved in cell survival, invasion and metastasis. We characterised by immunohistochemistry the expression of NF-kappaB/p65 protein in six histologically normal prostate, 13 high-grade prostatic intraepithelial neoplasia (PIN) and 86 prostate adenocarcinoma specimens. Nuclear localisation of p65 was used as a measure of NF-kappaB active state. Nuclear localisation of NF-kappaB was only seen in scattered basal cells in normal prostate glands. Prostatic intraepithelial neoplasias exhibited diffuse and strong cytoplasmic staining but no nuclear staining. In prostate adenocarcinomas, cytoplasmic NF-kappaB was detected in 57 (66.3%) specimens, and nuclear NF-kappaB (activated) in 47 (54.7%). Nuclear and cytoplasmic NF-kappaB staining was not correlated (P=0.19). By univariate analysis, nuclear localisation of NF-kappaB was associated with biochemical relapse (P=0.0009; log-rank test) while cytoplasmic expression did not. On multivariate analysis, serum preoperative prostate specific antigen (P=0.02), Gleason score (P=0.03) and nuclear NF-kappaB (P=0.002) were independent predictors of biochemical relapse. These results provide novel evidence for NF-kappaB/p65 nuclear translocation in the transition from PIN to prostate cancer. Our findings also indicate that nuclear localisation of NF-kappaB is an independent prognostic factor of biochemical relapse in prostate cancer.

Down-regulation of brain nuclear factor-kappa B pathway in the cyclooxygenase-2 knockout mouse.

Cyclooxygenase (COX) is the rate-limiting enzyme in the synthesis of prostaglandins (PGs) from arachidonic acid. Evidence suggests that neuronal COX-2 gene expression is mainly regulated by the transcription factor nuclear factor kappa-B (NF-kappaB). The present study was undertaken to determine whether there is a shared regulation of NF-kappaB or of nuclear factor of activated T-cells cytoplasmic (NFATc) with COX-2 and whether these transcription factors known to regulate COX-2 expression are altered in brain from COX-2 knockout (COX-2-/-) mice compared to wild type. We found a decrease in NF-kappaB DNA-protein binding activity, which was accompanied by a reduction of the phosphorylation state of both I-kappaBalpha and p65 proteins in the COX-2-/- mice. The mRNA and protein levels of p65 were also reduced in COX-2-/- mice, whereas total cytoplasmic I-kappaB protein level was not significantly changed. Taken together, these changes may be responsible for the observed decrease in NF-kappaB DNA binding activity. NF-kappaB DNA binding activity was selectively affected in the COX-2-/- mice compared to the wild type as there was no significant change in NFATc DNA binding activity. Overall, our data indicate that constitutive brain NF-kappaB activity is decreased in COX-2 deficient mice and suggest a reciprocal coupling between NF-kappaB and COX-2.

The structure of PEG-modified poly(ethylene imines) influences biodistribution and pharmacokinetics of their complexes with NF-kappaB decoy in mice.

PURPOSE: To study the relationship between structure of poly(ethylene imine-co-ethylene glycol), PEI-PEG, copolymers and physicochemical properties as well as in vivo behavior of their complexes with NF-kappaB decoy. METHODS: A variety of copolymers of PEG grafted onto PEI as well as PEI grafted onto PEG were synthesized and their complexes with a double stranded 20mer oligonucleotide were examined regarding size, surface charge, biodistribution and pharmacokinetics. RESULTS: Polyplexes of copolymers were smaller compared to polyplexes formed by non-PEGylated PEI 25 kDa (58 - 334 nm vs. 437 nm for a nitrogen/phosphate ratio of 3.5 and 85 - 308 nm vs. 408 nm for N/P 6.0) and showed reduced zeta potential (-2.5 - 6.4 mV vs. 14.5 mV for N/P 6.0). IV injection into mice revealed liver (35-76% of injected dose), kidney (3 - 22%) and spleen (2 - 16%) to be the main target organs for all injected complexes. Complexes formed by copolymers with few PEG blocks of higher molecular weight (5 kDa and 20 kDa) grafted onto PEI 25 kDa did not show different blood levels from PEI 25 kDa. In contrast, a copolymer with more short PEG blocks (550 Da) grafted onto PEI showed elevated blood levels with an increase in AUC of 62 %. CONCLUSIONS: A sufficiently high density of PEG molecules is necessary to effectively prevent opsonization and thereby rapid clearance from blood stream.