Nuclear factor-kappaB Activation Research Articles

Gold nanoparticles induce serum amyloid A 1–Toll-like receptor 2 mediated NF-kB signaling in lung cells in vitro

Gold nanoparticles (AuNPs) have emerging applications in biomedicine and the industry. Exposure to AuNPs has previously been shown to alter the transcriptional activity of nuclear factor kappa B (NF-kB), which is known to mediate physiological and pathological processes. This study seeks to provide mechanistic insights into AuNP-induced NF-kB activation in Small Airway Epithelial Cells (SAECs) in vitro. Increased NF-kB transcriptional activity (quantified by the luciferase reporter assay) was observed in AuNP-treated SAECs. Transcriptomic analysis revealed differential expression of 42 genes, which regulate functional processes that include cellular response to stimulus, chemicals and stress as well as immune response. Notably, the gene expression of serum amyloid A1 (SAA1), an acute phase protein and Toll-like receptor 2 (TLR2) were found to be up-regulated. As TLR2 is known to be a functional receptor of SAA1, a co-immunoprecipitation assay was performed. SAA1 was observed to be co-immunoprecipitated with the TLR2 protein and this protein-protein interaction was further supported by in silico computer based protein modeling. The present study suggests that AuNPs may potentially induce SAA1-TLR2-mediated NF-kB transcription factor activation in lung epithelial cells, highlighting that nano-bio interactions could result in biological effects that may affect cells.

Osteoporosis Therapy With Denosumab in Organ Transplant Recipients.

Osteoporosis and fragility fractures represent serious complications for the solid organ transplant population. The recommended osteoporosis therapy for organ recipients involves supplementation with calcium and vitamin D and bisphosphonate administration. However, these options can prove limited for patients with impaired renal function. An alternative therapy option is offered by denosumab, a monoclonal antibody that targets receptor activator of nuclear factor kappa-B ligand. We evaluated 63 patients with osteoporosis (23 males and 40 females, age 56.4 ± 13.1 years) following solid organ transplantation (15 diabetic patients after simultaneous transplantation of the kidney and pancreas, 34 patients after kidney transplantation, and 14 patients with liver grafts). Osteoporosis was diagnosed according to standard DEXA examination using the Lunar Prodigy apparatus. Transplanted patients with impaired renal function were treated for osteoporosis of the lumbar spine (L-spine) and/or proximal femur with calcium and vitamin D supplementation and 60 mg of denosumab every 6 months between the years 2012 and 2017. The mean duration of the therapy was 1.65 ± 0.7 years. After denosumab therapy, L-spine T-scores improved across the whole group, ranging from -2.7 ± 0.09 to -1.8 ± 1.0 (p < 0.001). T-score values for the proximal femur increased from -2.5 ± 0.8 to -2.0 ± 0.7 after the therapy (p < 0.01). We observed only a mild, statistically insignificant improvement in distal forearm T-scores. The mean increase in L-spine bone mineral density (BMD) was 11.5 ± 6.2% in subjects with osteoporosis at this site and 10.4 ± 6.1% in the case of all patients. BMD of the proximal femur increased by 10.4 ± 8.3% in patients with osteoporosis and by 7.5 ± 7.3% in all patients. Denosumab therapy decreased the prevalence of osteoporosis in the L-spine from 75 to 27% (p < 0.001) and proximal femur osteoporosis from 54 to 36% (p < 0.05). Denosumab therapy reduced elevated levels of osteocalcin and beta-crosslaps (βCTX) in comparison with baseline levels (p < 0.001) across the whole group of graft recipients. Denosumab therapy was well-tolerated and improved bone density in our group of solid organ transplant recipients. The indications are that denosumab could be a viable therapeutic option for transplanted patients with osteoporosis, especially in those with renal function impairment or bisphosphonate intolerance.

Phosphosulindac is efficacious in an improved concanavalin A-based rabbit model of chronic dry eye disease

Dry eye disease (DED) currently has no satisfactory treatment partly because of the lack of informative animal models. We evaluated the anti-inflammatory phosphosulindac (PS) for the treatment of DED using a new rabbit model of DED based on the concanavalin A (Con A) acute DED model: we injected all lacrimal glands with Con A weekly under ultrasound guidance, which prolonged DED to >3 weeks, and thoroughly assessed efficacy with tear break-up time (TBUT), tear osmolarity, Schirmer test, and tear lactoferrin levels. Rabbits with DED (n = 8-10 eyes per group) were treated topically with PS or vehicle 3×/day for 21days. PS restored TBUT, tear osmolarity, and lactoferrin levels (P < 0.0001-0.04) to normal but did not significantly improve the results of the Schirmer test. PS showed no side effects and was much more efficacious than cyclosporine or lifitegrast. In the cornea, PS suppressed the activation of nuclear factor kappa-B, the levels of transforming growth factor beta, interleukin-1 beta, interleukin-6, and interleukin-8, and the levels of matrix metalloproteinase (MMP)-1 and MMP-9, and MMP activity. Levels of prostaglandin E2 (PGE2) in tears and cornea were preserved in PS-treated rabbits. Ketorolac and diclofenac, two ophthalmic NSAIDs causing corneal melt, nearly completely suppressed PGE2 levels but had no effect on MMPs. The effects of PS on PGE2 and MMPs likely account for its apparent ocular safety. Our results establish an animal model for acute and chronic DED suitable for drug efficacy studies and indicate that PS merits evaluation for DED.

Inhibition of STAT3 phosphorylation by curcumin partly reduces adhesion molecule expression in vascular endothelial cell

OBJECTIVESWe investigated whether curcumin, an ingredient of turmeric, can suppress the expression of intercellular adhesion molecule 1 (ICAM‐1) and vascular cell adhesion molecule 1 (VCAM‐1) in human umbilical vein endothelial cells (HUVEC) stimulated with lipopolysaccharide (LPS), and the underlying mechanisms.MATERIALS AND METHODOLOGYThe levels of adhesion molecules, activities of nuclear factor kappaB (NF‐kB) and signal transducer and activator of transcription 3 (STAT3) were determined using Western blot analysis. The effect of curcumin on monocyte binding to endothelial cells was determined using THP‐1 monocyte (human acute monocytic leukemia cell line) adhesion assay.RESULTSCurcumin prevented the LPS‐mediated increase in ICAM‐1 and VCAM‐1 expression in HUVEC in a dose‐dependent manner. Curcumin did not affect LPS induced degradation of IkappaBα (IkBα). On the other hand, curcumin reduced the LPS‐induced STAT3 phosphorylation. Curcumin significantly reduced LPS‐mediated THP‐1 monocyte adhesion to HUVEC. Furthermore, in C57BL/6 mice, curcumin prevented the LPS induced increase in aortic ICAM‐1 and VCAM‐1 expression.CONCLUSIONSCurcumin prevented LPS‐induced ICAM–1 and VCAM–1 expression on vascular endothelial cells associated with inhibition of STAT3 phosphorylation.Support or Funding InformationThis work was supported by the research grants of the Chungbuk National University in 2014.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

BONE REMODELING IN NORM AND IN PRIMARY OSTEOPOROSIS: THE SIGNIFICANCE OF BONE REMODELING MARKERS

Osteoporosis is a systemic skeletal disease is characterized by low bone mass and microarchitectural deterioration of bone tissues, leading to bone fragility and low-energy The incidence of osteoporosis has risen because the life expectancy of the population has been increasing . Osteoporosis is an extremely common disease: osteoporosis affects more than 200million people worldwide and causes more than 8.9 million fractures. In Russia, among people aged 50 years and older, osteoporosis is diagnosed in 34% of women and 27% of men. The social significance of osteoporosis is determined by its consequences — fractures of the bones of the peripheral skeleton and vertebral fractures, leading to high material costs and causing a high level of disability and mortality. The normal physiological process of bone remodeling involves a balance between bone resorption and bone formation. In osteoporosis, this process becomes unbalanced, resulting in gradual losses of bone mass and density due to enhanced bone resorption and/or inadequate bone formation. Several signaling pathways underlying primary osteoporosis have been identified, such as the osteoprotegerin/ receptor activator of nuclear factor kappa-B (RANK)/RANK ligand(RANKL), bone morphogenetic proteins, canonical wnt-signaling pathway. In addition, genetic disorders are involved in the development of the pathogenesis of osteoporosis. To identify osteoporosis, WHO recommends the use of dualenergy X-ray absorptive densitometry, which allows you to study the quantitative characteristics of bone tissue. Currently, there are various methods for evaluation of the quality of bone (microarchitectonics, the ability of bone tissue to be resistant to fracture), but these methods have limitations such as high cost and limited availability for their widespread using. The study of markers of bone remodeling in norm and in pathology helps to assess the quality of bone tissue indirectly, gives prospects in the selection of targeted therapy and improvement of early diagnosis of osteoporosis.

Regulation of the antiapoptotic protein cFLIP by the glucocorticoid Dexamethasone in ALL cells.

We recently reported that the Smac mimetic BV6 and glucocorticoids, e.g. Dexamethasone (Dexa), synergize to induce cell death in acute lymphoblastic leukemia (ALL) in vitro and in vivo. Here, we discover that this synergism involves Dexa-stimulated downregulation of cellular FLICE-like inhibitory protein (cFLIP) in ALL cells. Dexa rapidly decreases cFLIPL protein levels, which is further enhanced by addition of BV6. While attenuating the activation of non-canonical nuclear factor-kappaB (NF-κB) signaling by BV6, Dexa suppresses cFLIPL protein but not mRNA levels pointing to a transcription-independent downregulation of cFLIPL by Dexa. Analysis of protein degradation pathways indicates that Dexa causes cFLIPL depletion independently of proteasomal, lysosomal or caspase pathways, as inhibitors of the proteasome, lysosomal enzymes or caspases all failed to protect from Dexa-mediated loss of cFLIPL protein. Also, Dexa alone or in combination with BV6 does not affect overall activity of the proteasome. Importantly, overexpression of cFLIPL to an extent that is no longer subject to Dexa-imposed downregulation rescues Dexa/BV6-mediated cell death. Vice versa, knockdown of cFLIP increases BV6-mediated cell death, thus mimicking the effect of Dexa. Altogether, these data demonstrate that Dexa-mediated downregulation of cFLIPL protein promotes Dexa/BV6-mediated cell death, thereby providing novel insights into the synergistic antitumor activity of this combination treatment.

Bipolar disorder in youth is associated with increased levels of vitamin D-binding protein

Genetic, dietary, and inflammatory factors contribute to the etiology of major mood disorders (MMD), thus impeding the identification of specific biomarkers to assist in diagnosis and treatment. We tested association of vitamin D and inflammatory markers in 36 adolescents with bipolar disorder (BD) and major depressive disorder (MDD) forms of MMD and without MMD (non-mood control). We also assessed the overall level of inflammation using a cell-based reporter assay for nuclear factor kappa-B (NFκB) activation and measuring antibodies to oxidized LDL. We found that these factors were similar between non-mood and MMD youth. To identify potential biomarkers, we developed a screening immunoprecipitation-sequencing approach based on inflammatory brain glia maturation factor beta (GMFβ). We discovered that a homolog of GMFβ in human plasma is vitamin D-binding protein (DBP) and validated this finding using immunoprecipitation with anti-DBP antibodies and mass spectrometry/sequencing analysis. We quantified DBP levels in participants by western blot. DBP levels in BD participants were significantly higher (136%) than in participants without MMD (100%). The increase in DBP levels in MDD participants (121.1%) was not statistically different from these groups. The DBP responds early to cellular damage by binding of structural proteins and activating inflammatory cells. A product of enzymatic cleavage of DBP has been described as macrophage-activating factor. Circulating DBP is comprised of heterogenous high and low molecular fractions that are only partially recognized by mono- and polyclonal ELISA and are not suitable for the quantitative comparison of DBP in non-mood and MDD participants. Our data suggest DBP as a marker candidate of BD warranting its validation in a larger cohort of adolescent and adult MMD patients.

The protective effects of metformin in an in vitro model of aging 3T3 fibroblast under the high glucose conditions.

Metformin is the most widely used anti-diabetic drug in the world. It reduces advanced glycation end product (AGEs)-induced ROS generation in high glucose condition. Protein glycation contributes to skin aging as it deteriorates the existing collagen by crosslinking. The progressive increase of AGE during aging not only causes oxidative damage to cellular macromolecules but also modulates the activation of transcription factors nuclear factor kappa-B(NF-kB). However, it is still unclear whether metformin can change collagen production and NF-kB activity induced by high glucose conditions in 3T3 fibroblast. The effects of metformin on proliferation, apoptosis, and collagen levels and NF-kB activity of in vitro cell aging model of 3T3 fibroblast cells in high glucose conditions. At first, we investigated the effects of 50mM high glucose concentration, with or without metformin, on 3T3 fibroblast proliferation, by BrdU immunostaining for cell proliferation. Apoptotic levels were analyzed by flow cytometric assay. NF-kB(p65) activity was measured by transcription factor assay kit and collagen I and III levels by Collagen Estimation Assay through ELISA. We observed that metformin exposure leads to decreased apoptosis levels and increased proliferation of 3T3 fibroblast in high glucose media. We also determined that metformin exposure leads to increased production of collagen I-III and decreased activation of NF-kB(p65) activity. The data are consistent with the observation that metformin has a protective effect in this in vitro model of aging 3T3 fibroblasts under high glucose conditions inducing cell proliferation, collagen I and III production, protection from apoptosis, and reducing NF-kB(p65) activity.

Magnesium Lithospermate B Protects against Lipopolysaccharide-Induced Bone Loss by Inhibiting RANKL/RANK Pathway.

Lipopolysaccharide (LPS) can induce bone loss by stimulating bone resorption. Natural compounds have great potential for the treatment of osteolytic bone diseases. Magnesium lithospermate B (MLB) plays an important role in protecting against oxidative damage and also has potential anti-inflammatory pharmacological properties. However, its role in LPS-induced bone loss is still unknown. In the present study, we observed the effects of MLB on LPS-induced bone damage and investigated the possible mechanisms. The bone loss models were established by LPS administration in male Sprague–Dawley rats. MLB (200 mg/kg body weight) was given by subcutaneous injection. MicroCT analysis, biomarker assay, histological examination and immunohistochemical staining were performed at the 8th weeks. In addition, RAW264.7 cells were treated with LPS in the presence or absence of MLB. The osteoclast formation, resorption activity and differentiation-related genes [(receptor activator of nuclear factor kappa-B (RANK), Traf6, Fra-1, and c-src)] expression were evaluated. LPS induced bone loss shown as the decrease in bone volume fraction and trabecular number, and increase in trabecular separation. LPS also markedly enhanced the osteoclast formation and resorption activity compared with the control. MLB significantly abolished the LPS-induced bone microstructure damage (p < 0.05) and osteoclast formation. MLB also inhibited the increases of serum tartrate-resistant acid phosphatase 5b, RANK ligand (RANKL) and TNF-α level enhanced by LPS (p < 0.05). Immunohistochemical staining indicated that MLB attenuated the high expression of RANKL and RANK stimulated by LPS. In addition, MLB significantly abolished the LPS-enhanced osteoclast formation, resorption activity, RANK, Traf6, Fra-1, and c-src expression in vitro. Our data demonstrate that MLB can suppress LPS-induced bone loss via inhibiting RANKL/RANK related osteoclast formation.

Using Surface Enhanced Raman Spectroscopy to Probe Single-Cell NF-kB Dynamics during TNF-α Stimulation

Surface-enhanced Raman spectroscopy (SERS) is an optical device that is complicated but fast, that deals with high precision cells, noninvasive and label-free. Tumor necrosis factor-alpha (TNF-α) is primarily produced by macrophages an inflammatory cytokine. When TNF-α binds to TNFR, it activates nuclear factor kappa B (NF-kB) transcript factors. This will lead to oscillation in nuclear NF-kB and steering gene expression by negative feedback loops. We applied biotin anti-human TNF-α antibody to bind with TNF-α proceeded by chemical bonding for streptavidin- Phycoerythrin (PE) to detect the space structure between TNF-α and TNFR. The intracellular p65 was labeled with GFP for NF-kB tracking. We indicated the probing of spatiotemporal interaction between binding of TNF-α to TNF-receptor. PE interacted with TNF-α on a ratio of 1:1 and 1:4. Quantum dots (QDs) with TNF-α at 1:1. The signal strength had decreased in the ratio 1:4. There was low bright field in QD interaction to TNF-α. We used real-time imaging for single TNF-α molecule on the living NIH-3T3 cell surface. The result showed the ligand-induced receptor dimerization and trimerization. The bioconjugation and diameter of fluorescent probes both affected the spatial binding of TNF-α to its receptor. Using real-time imaging of GFP-P65, we successfully visualized the intracellular NF-kB nuclear translocation. Our goal is to compare the results from fluorescence imaging to SERS experiment. SERS has the potential to analyze NF-kB. With this image, label-free SERS space detection and with potentially patentable novel gold nano-particles (Au NPs) it can be seen NF-kB activation in two hours. In addition, we can conduct a 96 or 384 test in parallel and increase the throughput by two or three orders by 100-1000 faults. This will be the ultimate goal to move this device into clinical application.

Lactation induces increases in the RANK/RANKL/OPG system in maxillary bone.

The underlying causes of maxillary bone loss during lactation remain poorly understood. We evaluated the impact of lactation on physiological and mechanically-induced alveolar bone remodeling. Nulliparous non-lactating (N-LAC) and 21-day lactating (LAC) mice underwent mechanically-induced bone remodeling by orthodontic tooth movement (OTM). Micro-computed tomography (microCT) was performed in the maxilla, femur and vertebra. Tartrate-resistant-acid phosphatase (TRAP) and Masson's trichrome labelling was performed in the maxillary bone and gene expression was determined in the periodontal ligament. The effect of prolactin on osteoclast (OCL) and osteoblast (OBL) differentiation was also investigated in N-LAC and LAC mice. Lactation increased alveolar bone loss in the maxilla, femur and vertebra, while OTM was enhanced. The number of OCL and OBL was higher in the maxilla of LAC mice. OTM increased OCL in both groups; while OBL was increased only in N-LAC but not in LAC mice, in which cell numbers were already elevated. The alveolar bone loss during lactation was associated with increased expression of receptor activator of nuclear factor-KappaB (RANK), RANK ligand (RANKL), and osteoprotegerin (OPG) in the maxilla. OTM induced the same responses in N-LAC mice, whereas it had no further effect in LAC mice. Lactation enhanced differentiation of OCL and OBL from bone marrow cells, and prolactin recapitulated OCL differentiation in N-LAC mice. Thus, lactation increases physiological maxillary bone remodeling and OTM, and both require activation of RANK/RANKL/OPG system. These findings expand our knowledge of lactation-induced osteopenia and have possible impact on clinical practice regarding orthodontic treatments and dental implants in lactating women.

Amentoflavone Inhibits Osteoclastogenesis and Wear Debris-Induced Osteolysis via Suppressing NF-κB and MAPKs Signaling Pathways.

Wear debris-induced osteolysis is one of the major reasons for subsequent aseptic loosening after cementless hip arthroplasty. Increasing evidence suggests that receptor activator of nuclear factor kappa-B (NF-κB) ligand-mediated osteoclastogenesis and osteolysis are responsible for wear debris-induced aseptic loosening. In the present study, we explored the effect of amentoflavone (AMF) on inhibiting osteoclast generation and wear debris-induced osteolysis in vitro and in vivo. Twenty-four male C57BL/J6 mice were randomly divided into four groups: a sham group and groups with titanium wear debris treatment followed by intraperitoneal injection of various concentrations of AMF (0, 20, and 40 mg/kg/day). The micro computed tomography scanning and histological analysis were performed. Bone marrow-derived macrophages were cultured to investigate the effect of AMF on osteoclast generation and function. The results showed that AMF suppressed osteoclastogenesis, F-actin ring formation, and bone absorption without cytotoxicity. AMF prevented titanium wear debris-induced osteolysis in mice. AMF suppressed the relative proteins of NF-κB and mitogen-activated protein kinase (MAPKs) signaling pathways. Thus, the present study suggests that AMF derived from plants could inhibit osteoclastogenesis and titanium wear debris-induced osteolysis via suppressing NF-κB and MAPKs signaling pathways.

Anti-Inflammatory Effects of Vardenafil Against Cholestatic Liver Damage in Mice: a Mechanistic Study

Background/Aims: Phosphodiesterase-5 inhibitors have beneficial effects in multiple liver diseases possibly through the reduction of oxidative stress and inflammatory response. However, these effects have not yet been examined in cholestatic liver dysfunction. Hence, this study aimed to explore the ability of vardenafil, a known phosphodiesterase-5 inhibitor, to repress lithocholic acid (LCA)-induced cholestatic liver injury and investigate the possible molecular pathways. Methods: Male Swiss albino mice were treated with LCA (0.125 mg/g) twice daily for 7 days to induce cholestatic liver damage. Vardenafil was administered 3 days before and throughout the administration of LCA. Serum markers of hepatotoxicity and hepatic nitro-oxidative stress along with antioxidant parameters were measured, and the histopathology of liver tissues was assessed. The expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and its target genes was examined using PCR. The activation of nuclear factor kappa-B (NF-κB) and the levels of inflammatory cytokines were determined. NLRP3 inflammasome and its components were studied by PCR and western blot. Results: LCA induced marked cholestatic liver damage as demonstrated by increased serum transaminases, alkaline phosphatase (ALP), lactate dehydrogenase (LDH), bilirubin, and bile acids. Examination of liver specimens confirmed the biochemical results. Nitro-oxidative stress parameters were significantly elevated along with reduced antioxidant capacity in hepatic tissue following LCA administration. LCA suppressed Nrf2 and its target genes and decreased the mRNA expression and binding capacity of Nrf2 as well as the mRNA expression of GCLm, GCLc, Nqo1, and HO-1. Additionally, LCA enhanced the activation of NF-κB, which was accompanied by elevations of inflammatory cytokines. Importantly, LCA induced the activation of NLRP3 inflammasome. LCA increased the expression of NLRP3, ASC, caspase-1, and IL-1β genes and proteins in hepatic tissue. The activities of IL-1β and caspase-1 were increased in the LCA group. Interestingly, vardenafil ameliorated LCA-induced hepatic injury and alleviated all biochemical, histopathological, and inflammatory parameters. Conclusions: These data elucidated the effects of Nrf2 inhibition and NLRP3 inflammasome activation in LCA-induced liver injury. The hepatoprotective activity of vardenafil in LCA-induced cholestatic damage may result from the drug’s ability to activate Nrf2 signaling and prevent the activation of NLRP3, which could suppress the inflammatory responses in hepatic tissue. Thus, vardenafil can be considered a novel anti-inflammatory remedy for cholestatic liver damage.

Casein glycomacropeptide hydrolysates inhibit PGE2 production and COX2 expression in LPS-stimulated RAW 264.7 macrophage cells via Akt mediated NF-κB and MAPK pathways.

A casein glycomacropeptide hydrolysate (GMPH) was found to possess inhibitory activity against lipopolysaccharide (LPS)-induced inflammatory response in our previous study. In the current study, the inhibitory effect and the underlying molecular mechanism of GMPH on inflammatory response in LPS-stimulated RAW264.7 macrophages were further investigated. Results showed that GMPH significantly suppressed LPS-induced intracellular reactive oxygen species (ROS) and malondialdehyde (MDA) production. GMPH reduced the production of prostaglandin E2 (PEG2) and the expression of cyclooxygenase-2 (COX-2) and cytosolic phospholipase A2 (cPLA2) in LPS-stimulated macrophages. GMPH also attenuated LPS-induced phosphorylation of MAPK (c-Jun NH2-terminal kinase (JNK), extracellular signal-regulated kinase (ERK) and p38) and protein kinase B (Akt). Furthermore, GMPH inhibited nuclear transcription factor kappa-B (NF-κB) activation by suppressing the nuclear translocation of NF-κB p65, which was markedly reversed by LY294002, an Akt inhibitor. These results demonstrated that GMPH exerts anti-inflammatory functions through the inactivation of MAPK and Akt in LPS-stimulated RAW264.7 macrophages, therefore may hold potential to ameliorate inflammation-related metabolic disorders.

Evaluation of Aculeatin and Toddaculin Isolated from Toddalia asiatica as Anti-inflammatory Agents in LPS-Stimulated RAW264 Macrophages.

Anti-inflammatory activity of aculeatin and toddaculin, which are coumarins with a similar structure isolated from Toddalia asiatica (L.) LAM., was evaluated using lipopolysaccharide (LPS)-stimulated RAW264 mouse macrophage cells. Both aculeatin and toddaculin significantly inhibited mRNA expression of inflammatory mediators and nitric oxide production. Furthermore, Toddaculin suppressed LPS-induced phosphorylation of p38 and extracellular signal-regulated kinase (ERK)1/2 and inhibited LPS-induced activation of nuclear factor-kappaB (NF-κB). However, aculeatin did not exhibit such effects, suggesting that aculeatin and toddaculin suppress LPS-induced inflammation of RAW264 cells via different mechanisms. The cellular uptake of these compounds was also evaluated. Toddaculin was detected in RAW264 cells after 4 and 24 h. However, aculeatin levels were not observed in RAW264 cells at all incubation intervals. These results indicate that de-epoxidation of a prenyl group can increase hydrophobicity of molecule and is thought to accelerate cellular uptake and/or interactions with the phospholipid bilayers of cell membranes.

Key Triggers of Osteoclast-Related Diseases and Available Strategies for Targeted Therapies: A Review.

Osteoclasts, the only cells with bone resorption functions in vivo, maintain the balance of bone metabolism by cooperating with osteoblasts, which are responsible for bone formation. Excessive activity of osteoclasts causes many diseases such as osteoporosis, periprosthetic osteolysis, bone tumors, and Paget’s disease. In contrast, osteopetrosis results from osteoclast deficiency. Available strategies for combating over-activated osteoclasts and the subsequently induced diseases can be categorized into three approaches: facilitating osteoclast apoptosis, inhibiting osteoclastogenesis, and impairing bone resorption. Bisphosphonates are representative molecules that function by triggering osteoclast apoptosis. New drugs, such as tumor necrosis factor and receptor activator of nuclear factor kappa-B ligand (RANKL) inhibitors (e.g., denosumab) have been developed for targeting the receptor activator of nuclear factor kappa-B /RANKL/osteoprotegerin system or CSF-1/CSF-1R axis, which play critical roles in osteoclast formation. Furthermore, vacuolar (H+)-ATPase inhibitors, cathepsin K inhibitors, and glucagon-like peptide 2 impair different stages of the bone resorption process. Recently, significant achievements have been made in this field. The aim of this review is to provide an updated summary of the current progress in research involving osteoclast-related diseases and of the development of targeted inhibitors of osteoclast formation.

Anticancer activity of Astragalus polysaccharide in human non-small cell lung cancer cells

BackgroundWe have reported that Chinese herbs Astragalus polysaccharide (APS) can inhibit nuclear factor kappaB (NF-κB) activity during the development of diabetic nephropathy in mice. NF-κB plays important roles in genesis, growth, development and metastasis of cancer. NF-κB is also involved in the development of treatment resistance in tumors. Here we investigated the antitumor activity of APS in human non-small cell lung cells (A549 and NCI-H358) and the related mechanisms of action.MethodsThe dose–effect and time-effect of antitumor of APS were determined in human lung cancer cell line A549 and NCI-H358. The inhibition effect of APS on the P65 mRNA and protein was detected by reverse transcriptase-PCR (RT-PCR) and Western blot in A549 cells respectively. The inhibition effect of APS on the p50, CyclinD1 and Bcl-xL protein was detected by Western blot in A549 cells respectively. The effect of APS on NF-κB transcription activity was measured with NF-κB luciferase detection. Finally, the nude mice A549 xenograft was introduced to confirm the antitumor activity of APS in vivo.ResultsCell viability detection results indicated that APS can inhibit the proliferation of human lung cancer cell line A549 and NCI-H358 in the concentration of 20 and 40 mg/mL. NF-κB activator Phorbol 12-myristate13-acetate (PMA) can attenuate the antitumor activity of APS in both cell lines, but NF-κB inhibitor BAY 11-7082 (Bay) can enhance the effect of APS in both cell lines. In vivo APS can delay the growth of A549 xenograft in BALB/C nude mice. APS can down-regulate the expression of P65 mRNA and protein of A549 cells and decrease the expression of p50, CyclinD1 and Bcl-xL protein. The luciferase detection showed that the APS could reduce the P65 transcription activity in A549 cells. PMA can partially alleviate the inhibition activity of P65 transcription activity of APS in A549 cells, and Bay can enhance the down-regulation of the P65 transcription activity induced by APS in A549 cells.ConclusionAPS has a significant antitumor activity in human lung cancer cells A549 and NCI-H358. NF-κB inhibition may mediate the antitumor effect.

AT1 receptor blockage impairs NF-κB activation mediated by thyroid hormone in cardiomyocytes.

We have previously demonstrated that calcium-binding protein S100A8 and myeloid differentiation factor-88 (MyD88) are important mediators of nuclear transcription factor kappa-B (NF-κB) activation in cardiomyocytes and that signalling molecules are involved in the hypertrophic response that is stimulated by thyroid hormones (TH). Angiotensin II (Ang II), the main active peptide of the renin-angiotensin system (RAS), binds to type 1 Ang II receptor (AT1R) and subsequently promotes cardiac hypertrophy and the inflammatory response with NF-κB activation underlying the cardiovascular effects. Considering the amount of evidence that RAS is an important mediator of TH actions on the cardiovascular system, we aimed to investigate whether cardiac expression of NF-κB and upstream associated molecules could be altered in hyperthyroidism, as well as whether AT1R could mediate the effects of TH on cardiac tissue and in cardiomyocytes in culture. Wistar rats were subjected to hyperthyroidism with or without the AT1R blocker losartan. The TH serum levels, haemodynamic parameters and cardiac mass were assessed to confirm the hyperthyroid status. The S100A8, MyD88 and nuclear NF-κB expression levels were increased in the hearts of the hyperthyroid rats, and the losartan treatment attenuated these TH effects. In addition, the cultured cardiomyocytes that had been stimulated with losartan exhibited blunted S100A8 upregulation and NF-κB activation compared with the TH-treated cells. Together, our results suggest that AT1R participates in TH-induced cardiac hypertrophy partly by mediating S100A8, MyD88 and NF-κB activation via TH. These findings indicate the important crosstalk between TH and RAS, highlighting the participation of AT1R in the triggered mechanisms of TH that contribute to the cardiac hypertrophy response.

Effect of vitamin D supplementation on inflammation and nuclear factor kappa-B activity in overweight/obese adults: a randomized placebo-controlled trial

In-vitro studies suggest that vitamin D reduces inflammation by inhibiting nuclear factor kappa-B (NFκB) activity. Yet, no trials have examined the effects of vitamin D supplementation on NFκB activity in-vivo in humans. We conducted a double-blind randomized trial (RCT) examining effects of vitamin D supplementation on inflammatory markers and NFκB activity in peripheral blood mononuclear cells (PBMCs). Sixty-five overweight/obese, vitamin D-deficient (25-hydroxyvitamin D [25(OH)D] ≤ 50 nmol/L) adults were randomized to a single 100,000 IU bolus followed by 4,000 IU daily cholecalciferol or matching placebo for 16 weeks. We measured BMI, % body fat, serum 25(OH)D, high-sensitivity C-reactive protein (hsCRP), tumour necrosis factor (TNF), monocyte chemoattractant protein-1 (MCP-1), interferon-gamma (IFN-γ), several interleukins, and NFκB activity in PBMCs. Fifty-four participants completed the study. Serum 25(OH)D concentrations increased with vitamin D supplementation compared to placebo (p < 0.001). Vitamin D and placebo groups did not differ in any inflammatory markers or NFκB activity (all p > 0.05). Results remained non-significant after adjustment for age, sex, and % body fat, and after further adjustment for sun exposure, physical activity, and dietary vitamin D intake. Although in-vitro studies report anti-inflammatory effects of vitamin D, our RCT data show no effect of vitamin D supplementation on inflammatory markers or NFκB activity in-vivo in humans.

Zinc Potentiates Lipopolysaccharide-induced Nitric Oxide Production in Cultured Primary Rat Astrocytes.

Zn2+ plays a crucial role in the CNS where it accumulates in synaptic vesicles and is released during neurotransmission. Synaptically released Zn2+ is taken up by neurons and astrocytes. The majority of previous work has focused on neuronal damage caused by excess Zn2+. However, its effect on astrocyte function is not well understood. We examined the effect of extracellularly applied Zn2+ on nitric oxide (NO) production in primary cultured rat astrocytes, which were experimentally activated by lipopolysaccharide (LPS). Zn2+, at a concentration up to 125μM, augmented LPS-induced NO production without affecting cell viability. LPS induced expression of both mRNA and protein of inducible NO synthase; this expression was enhanced by 125µM Zn2+. Zn2+ also increased LPS-induced production of intracellular reactive oxygen species. Zn2+ enhanced the phosphorylation of p38-mitogen-activated protein kinase (MAPK) at 1-6h after LPS treatment. The LPS-induced nuclear factor-kappaB (NFκB) activation was sustained for 6h by Zn2+. Intracellular Zn2+ chelation with N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) or inhibition of p38-MAPK diminished the Zn2+ enhancement of LPS-induced NO production. These findings suggest that activation of MAPK and NFκB is important for mediating Zn2+enhancement of LPS-induced NO production in astrocytes. Such changes may exacerbate glial and neuronal damage during neuroinflammation.