Release Of Tumor Necrosis Factor Research Articles

Apoptosis modulation by activin A and follistatin in human endometrial stromal cells

Activin A is a growth factor that stimulates decidualization and is abundantly expressed in endometrial proliferative disorders. Nevertheless, whether it directly affects endometrial cell survival is still unknown. This study investigated the effects of activin A on total death and apoptosis rates and on tumor necrosis factor (TNF) release by human endometrial stromal cells (HESC). We performed a controlled prospective in vitro study using primary HESC cultures obtained from healthy reproductive age women (n = 11). Cells were treated with medium alone (control) or activin A (25 ng/mL) or activin A (25 ng/mL) and its antagonist follistatin (250 ng/mL). Apoptosis and total cell death were measured by flow cytometry, while TNF concentrations in culture media were quantified by ELISA. Activin A decreased the percentage of apoptotic/dead cells from 31% to 22% (p < 0.05, paired t-test) and reduced TNF levels in culture medium by 14%, but there was no linear correlation between TNF release and apoptotic rates. Both effects of activin A were reversed by follistatin. These findings indicate that activin A promotes HESC survival, possibly by a TNF-independent pathway. This mechanism may be critical to the actions of activin A upon stromal cell growth and differentiation in physiology and disease.

Combination of new multifunctional molecules for erythematotelangiectatic rosacea disorder.

BackgroundRosacea, a common chronic skin disorder, is currently managed by patient education, pharmacological drugs, medical devices (laser and light therapies), and use of proper skin cares. Unfortunately, none of these actual treatments used alone or in combination is curative, and so we proposed a dermocosmetic active ingredient to mitigate some aspects of the rosacea and particularly for erythematotelangiectatic rosacea.MethodsDermocosmetic active ingredient is composed of three glucosylated derivatives of natural plants hydroxybenzoic acid and hydroxycinnamic acids (rosmarinic acid, gallic acid, and caffeic acid). Anti-inflammatory, anti-angiogenesis, and anti-degranulation studies were done on cellular models (keratinocytes, mast cells, and endothelial cells). Efficiency of the active ingredient in comparison to placebo was assessed clinically on human volunteers having erythematotelangiectatic rosacea. The active and placebo were applied topically twice a day for 28 days. Biometrical analyses were done using a siascope tool.ResultsWe found that the active ingredient decreases inflammation (inhibition of interleukin-8 and tumor necrosis factor release), decreases degranulation of mast cells (inhibition of histamine release), and controls angiogenesis mechanism (inhibition of the production of vascular endothelial growth factor and neovessel formation) on cellular models. Study on human volunteers confirmed macroscopically the efficiency of this active ingredient, as we observed no neovessel formation and less visible vessels.ConclusionAlthough rosacea is a skin condition disorder that is difficult to heal, the studies have shown that this active ingredient could be a dermocosmetic support, especially for erythematotelangiectatic rosacea armamentarium. The active ingredient was topically applied on the face for 28 days and improved erythematotelangiectatic rosacea symptoms either by decreasing them (vessels are less visible) or by limiting their development (any neovessels). The active ingredient decreases inflammation (inhibition of interleukin-8 and tumor necrosis factor release), decreases degranulation of mast cells (inhibition of histamine release), and limits the angiogenesis process (inhibition of vascular endothelial growth factor production and neovessel formation).

Human whole-blood culture system for ex vivo characterization of designer-cell function.

Encapsulated designer cells implanted into mice are currently used to validate the efficacy of therapeutic gene networks for the diagnosis and treatment of various human diseases in preclinical research. Because many human conditions cannot be adequately replicated by animal models, complementary and alternative procedures to test future treatment strategies are required. Here we describe a novel approach utilizing an ex vivo human whole-blood culture system to validate synthetic biology-inspired designer cell-based treatment strategies. The viability and functionality of transgenic mammalian designer cells co-cultured with primary human immune cells were characterized. We demonstrated that transgenic mammalian designer cells required adequate insulation from the human blood microenvironment to maintain viability and functionality. The biomaterial alginate-(poly-l-lysine)-alginate used to encapsulate the transgenic designer cells did neither affect the viability of primary granulocytes and lymphocytes nor the functionality of lymphocytes. Additionally, alginate-encapsulated transgenic designer cells remained responsive to the release of the pro-inflammatory cytokine tumor necrosis factor (TNF) from the whole-blood culture upon exposure to bacterial lipopolysaccharide (LPS). TNF diffused into the alginate capsules, bound to the specific TNF receptors on the transgenic designer cells' surface and triggered the expression of the reporter gene SEAP (human placental secreted alkaline phosphatase) that was rewired to the TNF-specific signaling cascade. Human whole-blood culture systems can therefore be considered as valuable complementary assays to animal models for the validation of synthetic circuits in genetically modified mammalian cells and may speed up preclinical research in a world of personalized medicine.

Vpr Enhances Tumor Necrosis Factor Production by HIV-1-Infected T Cells.

The HIV-1 accessory protein Vpr displays different activities potentially impacting viral replication, including the arrest of the cell cycle in the G2 phase and the stimulation of apoptosis and DNA damage response pathways. Vpr also modulates cytokine production by infected cells, but this property remains partly characterized. Here, we investigated the effect of Vpr on the production of the proinflammatory cytokine tumor necrosis factor (TNF). We report that Vpr significantly increases TNF secretion by infected lymphocytes. De novo production of Vpr is required for this effect. Vpr mutants known to be defective for G2 cell cycle arrest induce lower levels of TNF secretion, suggesting a link between these two functions. Silencing experiments and the use of chemical inhibitors further implicated the cellular proteins DDB1 and TAK1 in this activity of Vpr. TNF secreted by HIV-1-infected cells triggers NF-κB activity in bystander cells and allows viral reactivation in a model of latently infected cells. Thus, the stimulation of the proinflammatory pathway by Vpr may impact HIV-1 replication in vivo. The role of the HIV-1 accessory protein Vpr remains only partially characterized. This protein is important for viral pathogenesis in infected individuals but is dispensable for viral replication in most cell culture systems. Some of the functions described for Vpr remain controversial. In particular, it remains unclear whether Vpr promotes or instead prevents proinflammatory and antiviral immune responses. In this report, we show that Vpr promotes the release of TNF, a proinflammatory cytokine associated with rapid disease progression. Using Vpr mutants or inhibiting selected cellular genes, we show that the cellular proteins DDB1 and TAK1 are involved in the release of TNF by HIV-infected cells. This report provides novel insights into how Vpr manipulates TNF production and helps clarify the role of Vpr in innate immune responses and inflammation.

Skin-Derived Mesenchymal Stem Cells Alleviate Atherosclerosis via Modulating Macrophage Function.

Mesenchymal stem cells (MSCs) exhibit immunosuppressive efficacy and significantly inhibit the formation of the atherosclerosis (AS) plaque in apolipoprotein E-knockout (apoE(-/-)) mice. Of note, the largest lymphoid organ, the skin, provides a readily accessible and ideal source of tissue for the isolation of MSCs: skin-derived MSCs (S-MSCs). However, the effect and the mechanism of the therapeutic properties of S-MSCs in the progression of AS are unclear. We therefore investigated a direct effect of S-MSC treatment in the formation of atherosclerotic plaque in apoE(-/-) mice. Fifty apoE(-/-) mice were divided into four groups: the control group (AS), the S-MSC treatment group (S-MSC treatment), the nuclear factor-κB (NF-κB)(-/-)-S-MSC treatment group (KO-S-MSC treatment), and the additional S-MSC migration group. Brachiocephalic artery ultrasound biomicroscope (UBM) analysis showed that S-MSC treatment significantly reduced lesion size compared with the control groups (p < .01). Histological studies demonstrated that the plaque area of the mouse aortic arch was significantly decreased after S-MSC treatment. All alterations were dependent on NF-κB activation. After tail-vein injection, S-MSCs were capable of migrating to atherosclerotic plaque and selectively taking up residence near macrophages. S-MSC treatment reduced the release of the proinflammatory cytokine tumor necrosis factor (TNF)-α and increased the expression of the anti-inflammatory factor interleukin (IL)-10 in the atherosclerotic plaque, which was also dependent on NF-κB activation. In vitro, we found lipopolysaccharide (LPS) induced NF-κB-dependent expression of cyclooxygenase-2 (COX-2) in S-MSCs. Prostaglandin E2 (PGE2) expression was markedly increased after LPS-stimulated S-MSCs were cocultured with macrophages. LPS-stimulated macrophages produced less TNF-α/IL-1β and more IL-10 when cultured with S-MSCs, and although both were dependent upon NF-κB, the release of IL-10 was diminished if the S-MSCs were pretreated with a COX-2 inhibitor or an EP2/EP4 antagonist. Our data demonstrated that S-MSCs inhibited the formation of the atherosclerotic plaque in apoE(-/-) mice by modulating the functionality of macrophages, suggesting that S-MSCs may potentially have a role in stem cell-based therapy for AS. A combination of in vitro and in vivo experiments showed that skin-derived mesenchymal stem cells (S-MSCs) can attenuate the plaque size of atherosclerosis. This is probably because S-MSCs beneficially modulate the response of macrophages through an increased release of prostaglandin E2 acting on the EP2 and EP4 receptors of the macrophages, stimulating the production and release of the anti-inflammatory cytokine interleukin-10, and decreasing the production of proinflammatory cytokine tumor necrosis factor-α. S-MSCs inhibited the formation of the atherosclerotic plaque in apolipoprotein E-knockout mice by modulating the functionality of macrophages, and the suppressive property of S-MSCs is dependent on NF-κB signaling. This study provides direct evidence that S-MSCs have a potent immunosuppressive effect in the development of atherosclerosis in mice, suggesting that S-MSCs can easily be cultured and have similar function to bone marrow-derived MSCs, a promising cell source for stem cell-based therapies of atherosclerosis, and possibly also in transplantation.

Ozone Induced Cell Death in HeLa cell Culture Mediated through Stimulation of TNF- Alpha

Ozone has been in used as a therapeutic agent for the treatment of infective and inflammatory diseases, including cancer. The relationship of ozone with cancer and mechanism underlying the therapeutic effect of ozone on cancer remain unclear. This is first attempt to study the effect of ozone on cancer cells and how ozone induced cell death is mediated by activation of tumor necrosis factor (TNF)-? by using in vitro cell culture method. In the present study, HeLa cell cultures were subjected to ozone treatment at different dosages (10, 20, 30 & 40 ?g/ml). HeLa cells exhibited an increasing cell death with increase in ozone doses studied. A substantial enhancement of cell death was observed after 18 hours of ozone treatment onwards. Treatment with 30 and 40 ?g/ml of ozone showed an elevated sensitivity to growth inhibition compared to the treatments 10 and 20?g/ml. Additionally cell detachment and cell floating were also faster and higher in ozone treatment with 30 and 40?g/ml. Tumor necrosis factor alpha (TNF-?), is a multifunctional cytokine playing a key role in apoptosis and cell survival as well as in inflammation and immunity. Hence we also studied the release of TNF-? in response to various dosages of ozone-treatments by using ELISA technique. We found that TNF-?, secretion was found to be enhanced with increase in cell death and at dose 40?g/ml ozone treatment, cell death was highest and TNF-? secretion was also highest. Our study suggests that ozone induced cell death in HeLa cell lines is mediated through the activation of TNF-? pathway and the role of ozone in inducing apoptosis cannot be ruled out.

Evaluation of the Wound Healing Properties of Hancornia speciosa Leaves.

The leaves of Hancornia speciosa Gomes (Apocynaceae), a medicinal species found in the Brazilian cerrado biome, are traditionally used to treat wounds and inflammatory disorders. The goal of the present study was to investigate the in vitro wound healing properties of ethanolic extract of H. speciosa leaves and its isolated compounds, using the scratch assay, and to evaluate their effects on the release of the pro-inflammatory cytokine tumor necrosis factor (TNF-α) by lipopolysaccharide (LPS)-stimulated human acute monocytic (THP-1) cells. H. speciosa ethanolic extract significantly increased (42.8% ± 5.4 at 25 µg/mL) cell migration and proliferation of fibroblasts compared with control cells, as well as the isolated compounds bornesitol (80.8% ± 5.1) and quinic acid (69.1% ± 6.2), both assayed at 50 μM. TNF-α release by LPS-stimulated THP-1 cells was significantly reduced by the ethanolic extract (62.9% ± 8.2, i.e. 1791.1 ± 394.7 pg/mL) at 10 µg/mL, bornesitol (48.9% ± 0.9, i.e. 2461.6 ± 43.1 pg/mL) at 50 μM, and quinic acid (90.2% ± 3.4, i.e. 473.5 ± 164.4 pg/mL) and rutin (82.4% ± 5.6, i.e. 847.0 ± 271.8 pg/mL) at 10 μM. These results provided evidences to support the traditional use of H. speciosa leaves to treat wounds and inflammatory disorders.

Panaxadiol Saponin and Dexamethasone Improve Renal Function in Lipopolysaccharide-Induced Mouse Model of Acute Kidney Injury.

BackgroundAcute kidney injury (AKI) is a serious complication of systemic inflammatory response syndrome (SIRS), which has a high mortality rate. Previous studies showed that panaxadiol saponin (PDS) and Dexamethasone have similar anti-inflammatory properties and protect cardiopulmonary function in lipopolysaccharide (LPS)-induced septic shock rats. In the present study, we investigated whether PDS or Dexamethasone has a similar role in improving kidney function in LPS-induced AKI mice.Methods and ResultsMice subjected to LPS (10 mg/kg) treatment exhibited AKI demonstrated by markedly increased blood urea nitrogen and creatinine levels compared with controls (P<0.01). However, PDS and Dexamethasone induce similar reverse effects on renal function, such as reduced serum creatinine and blood urea nitrogen levels compared with the LPS group (P<0.05). PDS decreased the production and release of tumor necrosis factor (TNF)-α and interleukin (IL)-6 by inhibiting the NF-κB signaling pathway, down-regulating inducible nitric oxide synthase protein expression levels and inhibiting oxidative stress. In most anti-AKI mechanisms, PDS and dexamethasone were similar, but PDS are better at inhibition of TNF production, promote SOD activity and inhibition of IKB phosphorylation. In addition, nuclear glucocorticoid receptor expression was markedly enhanced in PDS and Dexamethasone treatment groups. Further research is required to determine whether PDS can combine with the glucocorticoid receptor to enter the nucleus.ConclusionThis study demonstrated that PDS and dexamethasone have similar reverse amelioration for renal functions, and have potential application prospects in the treatment of sepsis-induced AKI.

Ecto-5'-Nucleotidase (CD73) Deficiency in Mycobacterium tuberculosis-Infected Mice Enhances Neutrophil Recruitment.

The immune system needs safeguards that prevent collateral tissue damage mediated by the immune system while enabling an effective response against a pathogen. The purinergic pathway is one such mechanism and finely modulates inflammation by sensing nucleotides in the environment. Extracellular ATP is considered to be a danger signal leading to a proinflammatory response, whereas adenosine is immunosuppressive. CD73, also called ecto-5'-nucleotidase, occupies a strategic position in this pathway, as it is the main enzyme responsible for the generation of adenosine from ATP. Here, we explore the role of CD73 during tuberculosis, a disease characterized by an immune response that is harmful to the host and unable to eradicate Mycobacterium tuberculosis. Using CD73 knockout (KO) mice, we found that CD73 regulates the response to M. tuberculosis infection in vitro and in vivo. Mycobacterium-infected murine macrophages derived from CD73 KO mice secrete more keratinocyte chemoattractant (KC), tumor necrosis factor alpha (TNF-α), and interleukin-6 (IL-6) and release less vascular endothelial growth factor (VEGF) upon ATP stimulation than do those derived from wild-type (WT) mice. In vivo, CD73 limits the early influx of neutrophils to the lungs without affecting bacterial growth and dissemination. Collectively, our results support the view that CD73 fine-tunes antimycobacterial immune responses.

Saikosaponin-D reduces cisplatin-induced nephrotoxicity by repressing ROS-mediated activation of MAPK and NF-κB signalling pathways

The nephrotoxicity induced by cisplatin (DDP) severely limits the clinical efficacy of this widely used anticancer agent. The observed nephrotoxicity may be the result of DDP-induced inflammation and apoptosis. Saikosaponin-D (SSD), a triterpenoid saponin, has numerous pharmacological properties. The goal of the present study was to investigate whether and how SSD protected against DDP-induced nephrotoxicity. Non-cytotoxic levels of SSD significantly increased the viability rate, improved the nuclear morphology, and attenuated the caspase-3 activation and programmed apoptosis of DDP-treated HK-2 cells. In addition, SSD treatment markedly inhibited the release of tumour necrosis factor (TNF)-α, interleukin-1β (IL-1β), and interleukin-6 (IL-6), as well as the production of nitric oxide and the expression of inducible nitric oxide synthase (iNOS) by these cells. More importantly, SSD effectively blocked the DDP-induced activation of NF-κB, P38, JNK, and MAPKs. Furthermore, we found that U0126 (a specific inhibitor of MAPKs) strongly inhibited the IKK/IκB/NF-κB-dependent release of pro-inflammatory cytokines and iNOS gene expression. Finally, we demonstrated that SSD decreased the level of reactive oxygen species (ROS) accumulation and that the specific ROS scavenger N-acetylcysteine (NAC) markedly inhibited the DDP-induced activation of MAPK and phosphorylation of the downstream signal NF-κB, which in turn reduced the levels of pro-inflammatory cytokine release and iNOS gene expression. Our results suggest that the SSD-mediated alleviation of DDP-induced nephrotoxicity was due to uncoupling of the ROS, P38, and JNK/NF-κB signalling pathways.

The molecular mechanism of polygalasaponin F-mediated decreases in TNFα: emphasizing the role of the TLR4-PI3K/AKT-NF-κB pathway

Polygalasaponin F (PS-F), an oleanane-type triterpenoid saponin extracted from Polygala japonica, decreases the release of the inflammatory cytokine tumor necrosis factor α (TNFα), but the precise molecular mechanisms by which this event occurs are not fully understood. To study the anti-neuroinflammatory mechanisms of PS-F, enzyme-linked immunosorbent assay was used to detect the secretion of TNFα from BV-2 microglial cells. Nuclear proteins extracted from BV-2 microglial cells stimulated by lipopolysaccharide (LPS) and pretreated with/without inhibitors were measured by Western blotting, and cell viability was evaluated by MTT analysis. The results indicated that inhibition of toll-like receptor (TLR) 4 (CLI-095 1 μg/ml), phosphatidylinositol 3-kinase (PI3K) (Ly294002 10 μM) or IκBα phosphorylation (Bay11-7082 10 μM) completely prevents the release of TNFα induced by LPS without affecting cell viability and attenuated the nuclear translocation of p65 stimulated by LPS. In addition, PS-F exhibited a similar trend regarding TNFα release, AKT phosphorylation and NF-κB translocation. These results suggest that PS-F reduces neuroinflammatory cytokine secretion through the regulation of the TLR4-PI3K/AKT-NF-κB signaling pathway.

Functional expression of TRPV channels in T cells and their implications in immune regulation.

The importance of Ca(2+) signalling and temperature in the context of T cell activation is well known. However, the molecular identities of key players involved in such critical regulations are still unknown. In this work we explored the endogenous expression of transient receptor potential vanilloid (TRPV) channels, a group of thermosensitive and non-selective cation channels, in T cells. Using flow cytometry and confocal microscopy, we demonstrate that members belonging to the TRPV subfamily are expressed endogenously in the human T cell line Jurkat, in primary human T cells and in primary murine splenic T cells. We also demonstrate that TRPV1- and TRPV4-specific agonists, namely resiniferatoxin and 4α-phorbol-12,13-didecanoate, can cause Ca(2+) influx in T cells. Moreover, our results show that expression of these channels can be upregulated in T cells during concanavalin A-driven mitogenic and anti-CD3/CD28 stimulated TCR activation of T cells. By specific blocking of TRPV1 and TRPV4 channels, we found that these TRPV inhibitors may regulate mitogenic and T cell receptor mediated T cell activation and effector cytokine(s) production by suppressing tumour necrosis factor, interleukin-2 and interferon-γ release. These results may have broad implications in the context of cell-mediated immunity, especially T cell responses and their regulations, neuro-immune interactions and molecular understanding of channelopathies.

Metabolite profiling and pharmacokinetic evaluation of hydrocortisone in a perfused three-dimensional human liver bioreactor.

Endotoxin lipopolysaccharide (LPS) is known to cause liver injury primarily involving inflammatory cells such as Kupffer cells, but few in vitro culture models are applicable for investigation of inflammatory effects on drug metabolism. We have developed a three-dimensional human microphysiological hepatocyte-Kupffer cell coculture system and evaluated the anti-inflammatory effect of glucocorticoids on liver cultures. LPS was introduced to the cultures to elicit an inflammatory response and was assessed by the release of proinflammatory cytokines, interleukin 6 and tumor necrosis factor α. A sensitive and specific reversed-phase-ultra high-performance liquid chromatography-quadrupole time of flight-mass spectrometry method was used to evaluate hydrocortisone disappearance and metabolism at near physiologic levels. For this, the systems were dosed with 100 nM hydrocortisone and circulated for 2 days; hydrocortisone was depleted to approximately 30 nM, with first-order kinetics. Phase I metabolites, including tetrahydrocortisone and dihydrocortisol, accounted for 8-10% of the loss, and 45-52% consisted of phase II metabolites, including glucuronides of tetrahydrocortisol and tetrahydrocortisone. Pharmacokinetic parameters, i.e., half-life, rate of elimination, clearance, and area under the curve, were 23.03 hours, 0.03 hour(-1), 6.6 × 10(-5) l⋅hour(-1), and 1.03 (mg/l)*h, respectively. The ability of the bioreactor to predict the in vivo clearance of hydrocortisone was characterized, and the obtained intrinsic clearance values correlated with human data. This system offers a physiologically relevant tool for investigating hepatic function in an inflamed liver.

TGFβ1 inhibits IFNγ-mediated microglia activation and protects mDA neurons from IFNγ-driven neurotoxicity.

Microglia-mediated neuroinflammation has been reported as a common feature of familial and sporadic forms of Parkinson's disease (PD), and a growing body of evidence indicates that onset and progression of PD correlates with the extent of neuroinflammatory responses involving Interferon γ (IFNγ). Transforming growth factor β1 (TGFβ1) has been shown to be a major player in the regulation of microglia activation states and functions and, thus, might be a potential therapeutic agent by shaping microglial activation phenotypes during the course of neurodegenerative diseases such as PD. In this study, we demonstrate that TGFβ1 is able to block IFNγ-induced microglia activation by attenuating STAT1 phosphorylation and IFNγRα expression. Moreover, we identified a set of genes involved in microglial IFNγ signaling transduction that were significantly down-regulated upon TGFβ1 treatment, resulting in decreased sensitivity of microglia toward IFNγ stimuli. Interestingly, genes mediating negative regulation of IFNγ signaling, such as SOCS2 and SOCS6, were up-regulated after TGFβ1 treatment. Finally, we demonstrate that TGFβ1 is capable of protecting midbrain dopaminergic (mDA) neurons from IFNγ-driven neurotoxicity in mixed neuron-glia cultures derived from embryonic day 14 (E14) midbrain tissue. Together, these data underline the importance of TGFβ1 as a key immunoregulatory factor for microglia by silencing IFNγ-mediated microglia activation and, thereby, rescuing mDA neurons from IFNγ-induced neurotoxicity. Interferon γ (IFNγ) is a potent pro-inflammatory factor that triggers the activation of microglia and the subsequent release of neurotoxic factors. Transforming growth factor β1 (TGFβ1) is able to inhibit the IFNγ-mediated activation of microglia, which is characterized by the release of nitric oxide (NO) and tumor necrosis factor α (TNFα). By decreasing the expression of IFNγ-induced genes as well as the signaling receptor IFNγR1, TGFβ1 reduces the responsiveness of microglia towards IFNγ. In mixed neuron-glia cultures, TGFβ1 protects midbrain dopaminergic (mDA) neurons from IFNγ-induced neurotoxicity.

Altered relation between lipopolysaccharide-induced inflammatory response and excitotoxicity in rat organotypic hippocampal slice cultures during ethanol withdrawal.

Ethanol (EtOH) causes neurotoxicity by several mechanisms including excitotoxicity and neuroinflammation, but little is known about the interaction between these mechanisms. Because neuroinflammation is known to enhance excitotoxicity, we hypothesized that neuroinflammation contributes to the enhanced excitotoxicity, which is associated with EtOH withdrawal (EWD). The aim of this study was to evaluate the lipopolysaccharide (LPS)-induced inflammatory response of cultured hippocampal tissue during EWD and its effects on the enhanced N-methyl-d-aspartate (NMDA) receptor-mediated excitotoxicity, which occurs at this time. Using a neonatal organotypic hippocampal slice culture (OHSC) model, we assessed the effects of NMDA and LPS (separately or combined) during EWD after 10days of EtOH exposure. Neurotoxicity was assessed using propidium iodide uptake, and the inflammatory response was evaluated by measuring the release of tumor necrosis factor (TNF)-alpha (quantified by enzyme-linked immunosorbent assay) and nitric oxide (NO; quantified by the Griess reaction) into culture media. Furthermore, we explored the potential role of the microglial cell type using immortalized BV2 microglia treated with EtOH for 10days and challenged with LPS during EWD. As predicted, NMDA-induced toxicity was potentiated by LPS under control conditions. However, during EWD, the reverse was observed and LPS inhibited peak NMDA-induced toxicity. Additionally, LPS-induced release of TNF-alpha and NO during EWD was reduced compared to control conditions. In BV2 microglia, following EtOH exposure, LPS-induced release of NO was reduced, whereas TNF-alpha release was potentiated. During EWD following chronic EtOH exposure, OHSC exhibited a desensitized inflammatory response to LPS and the effects of LPS on NMDA toxicity were reversed. This might be explained by a change in microglia to an anti-inflammatory and neuroprotective phenotype. In support, studies on BV2 microglia indicate that EtOH exposure and EWD do alter the response of these cells to LPS, but this cannot fully explain the changes observed in the OHSC. The data suggest that neuroinflammation and excitotoxicity do interact during EWD. However, the interaction is not as simple as we originally proposed. This in turn illustrates the need to assess the extent, importance, and relation of these mechanisms in models of EtOH exposure producing neurotoxicity.

Inhibition effect of glycyrrhizin in lipopolysaccharide-induced high-mobility group box 1 releasing and expression from RAW264.7 cells.

High-mobility group box 1 (HMGB1) is a therapeutic target for sepsis. Glycyrrhizin (GL) is the aglycone of glycyrrhizin derived from licorice. We clarified the anti-inflammatory effects of GL. We explored the anti-HMGB1 effect of GL and elucidated its molecular mechanism, which will be of benefit for sepsis treatment. We stimulated murine macrophage-like RAW 264.7 cells with lipopolysaccharide (LPS) and LPS + GL, then measured the expression and release of HMGB1. The expression of related signal transduction factors was detected. High-mobility group box 1 was distributed mainly in the nucleus with lower cytoplasmic levels in RAW 264.7 cells before LPS stimulation. After stimulation, cytoplasmic HMGB1 levels increased gradually, whereas in nuclear fluctuation a trend of HMGB1 expression was observed. Significant upregulation of HMGB1 mRNA occurred 12 h after LPS stimulation. Glycyrrhizin prevented the transfer of HMGB1 from the nucleus to the cytoplasm and inhibited upregulation of HMGB1 mRNA induced by LPS. Phospho-p38 mitogen-activated protein kinase and activated activating protein 1 increased significantly 8 h after LPS stimulation. Tumor necrosis factor α and interleukin 6 increased 4 h after LPS stimulation and peaked at 48 h, and HMGB1 increased at 8 h. The Toll-like receptor 4/MD2/nuclear factor κB signaling pathway was activated 4 h after LPS stimulation. Glycyrrhizin inhibited this pathway. Glycyrrhizin inhibited the expression and release of HMGB1 through blocking the p38 mitogen-activated protein kinase/activating protein 1 signaling pathway then inhibited the massive release of tumor necrosis factor α and interleukin 6.

Studying α7 nicotinic receptor anti‐inflammatory signaling

α7 nicotinic receptors (α7 AChRs) reportedly play anti‐inflammatory roles in innate immune function, in part by blocking the transcription factor NFkB. We tested whether heterologously expressed α7 AChRs directly interact with NFkB signaling in the rat pituitary cell line GH3. GH3 cells show significant 125I‐α‐bungarotoxin (BT) binding when transfected with rat α7 nAChR, but in our hands, commercially available α7 antibodies do not detect α7 AChR in these transfected cells. For instance, α7 AChR‐GFP chimeras transfected into cells show BT binding, and anti‐GFP staining, but not anti‐α7 AChR staining in Western blots. Upon transfection with an NFkB promoter driving secreted alkaline phosphatase (NFkB‐SEAP), GH3 cells show up to 30 fold increases in SEAP secretion following 1‐100 ng/ml rat tumor necrosis factor (TNF) treatment. Nicotine (100 ‐1000 μM) and the selective α7 AChR agonist PNU282987 (PNU, 50‐300 μM) blocked TNF‐stimulated SEAP secretion in GH3 cells transfected with NFkB‐SEAP alone, while GTS21 did not. This suggests some nicotinic compounds have direct effects on NFkB signaling in non‐immune cells, since transfection with α7 had no further effect. To understand whether reported α7 AChR anti‐inflammatory effects are cell‐type dependent, we investigated α7 AChR drug effects in macrophage‐like cells (RAW 264.7 cells) using PNU, GTS21 and nicotine following lipopolysaccharide (LPS) stimulation (10 ng/ml). GTS21 (50‐200 µM) blocked LPS‐induced TNF release from RAW cells, but nicotine and PNU did not. Our findings suggest that α7 AChR do not directly interact with and subsequently block NFKB signaling, and that additional factors are required in heterologous cells to mimic the reported α7 AChR‐mediated anti‐inflammatory effects in immune cells.

Interleukin 23 levels are increased in carotid atherosclerosis: possible role for the interleukin 23/interleukin 17 axis.

Interleukin (IL)-23 is a cytokine in the IL-12 family, mainly produced by antigen-presenting cells with a central role in inflammation. We hypothesize that IL-23 is also important in atherogenesis and investigate this in a population with carotid atherosclerosis. Plasma levels of IL-23 were measured in patients with carotid artery stenosis and in healthy controls. The mRNA levels of IL-23 and its receptor, IL-23R, were measured in atherosclerotic plaques, nonatherosclerotic vessels, peripheral blood mononuclear cells, and plasmacytoid dendritic cells. Our findings were as follows: (1) patients with carotid atherosclerosis (n=177) had significantly raised plasma levels of IL-23 when compared with healthy controls (n=24) with particularly high levels in those with the most recent symptoms. (2) mRNA levels of IL-23 and IL-23R were markedly increased in carotid plaques (n=68) when compared with nonatherosclerotic vessels (n=8-10). Immunostaining showed colocalization to plaque macrophages. (3) Patients with carotid atherosclerosis had increased mRNA levels of both IL-23 and IL-23R in plasmacytoid dendritic cells, but not in peripheral blood mononuclear cells. (4) IL-23 increased IL-17 release in monocytes and particularly in peripheral blood mononuclear cells from patients with carotid atherosclerosis, but not in cells from healthy controls. (5) IL-23 gave a prominent tumor necrosis factor release in monocytes from patients with carotid atherosclerosis but not in cells from healthy controls. (6) High plasma levels of IL-23 were associated with increased mortality during follow-up. We have shown an association between IL-23 and disease progression in patients with carotid atherosclerosis, potentially involving IL-17-related mechanisms.

Transforming growth factor β1-mediated anti-inflammation slows progression of midbrain dopaminergic neurodegeneration in Parkinson's disease?

Transforming growth factor β1-mediated anti-inflammation slows progression of midbrain dopaminergic neurodegeneration in Parkinson's disease?

A Comparative Study of the Effects upon LPS Induced Macrophage RAW264.7 Inflammation in vitro of the Lipids of Hippocampus trimaculatus Leach.

The present study attempts to investigate the anti-inflammatory potential of the isolated lipid extracts of three-spot seahorse which is rare marine bony fish. Petroleum ether (PE) extract was obtained from systematic solvent extraction after reflux extraction with 95% ethanol. FrIV was collected after silica gel column chromatography, and neutral lipids (NL), glycolipids (GL), phospholipids (PL) were separated from FrIV. Basic compositions were detected and analyzed via thin layer chromatography (TLC) and Fourier transform infrared spectroscopy (FTIR). Anti-inflammatory activities of total lipids (TL), isolated NL, GL, and PL were detected by secretion of pro-inflammatory cytokines induced by lipopolysaccharide (LPS) in murine monocyte macrophage RAW264.7 cells in vitro. The results revealed that lipids of seahorse showed a positive correlation with the in vitro suppression of the release of nitric oxide (NO), interleukin (IL)-6, IL-1β and tumor necrosis factor (TNF)-α potently in a dose dependent manner, and showed cell compatibility. Among the fractions, GL (50 μg/mL) showed the highest capacity to attenuate the generation of pro-inflammatory cytokines which was comparable to that of the positive drug dexamethasone (DX) (20 μg/mL). Collectively, our findings indicated that the lipids from seahorse may be effective in the management of inflammation.