Inhibition Of Pro-inflammatory Gene Expression Research Articles

Protective role of Bu-Zhong-Yi-Qi decoction on aristolochic acid-intoxicated zebrafish


 
 
 
 Purpose: To investigate the potential nephroprotective effects of a traditional Chinese medical prescription, Bu-Zhong-Yi-Qi Decoction (BZYQD) in an aristolochic acid (AA)-intoxicated zebrafish model.
 Methods: A green fluorescent zebrafish line Tg (wt1b:EGFP) was used, and different exposure protocols were applied. Once a suitable protective concentration of BZYQD was found, antibody staining and real-time PCR methods were applied and the pro-inflammatory gene expressions were determined.
 Results: The results showed that low dose (10 ppm) of BZYQD attenuates the AA-induced malformed kidney phenotype. This finding was further substantiated by an examination of the integrity of pronephric tubes in the treated embryos. Pre-treatment with BZYQD suppressed the elevated expressions of pro- inflammatory genes, including tumor necrosis factor-α and myeloperoxidase, induced by AA exposure (1.6 ~ 2.3-fold). This indicates that the nephroprotective effect of BZYQD may be mediated by suppression of pro-inflammatory gene expression. On the other hand, a high dose (500 ppm) of BZYQD caused nephrotoxic effect.
 Conclusion: An efficient model to identify AA-protective compounds using zebrafish embryos has been successfully established. Using this strategy, it has been found that BZYQD is nephroprotective in zebrafish embryos, and might have the potential to be applied in humans.
 
 
 

Parasympathetic neural activity and the reciprocal regulation of innate antiviral and inflammatory genes in the human immune system

The vagus nerve mediates parasympathetic nervous system control of peripheral physiological processes including cardiovascular activity and immune response. In mice, tonic vagal activation down-regulates inflammation via nicotinic acetylcholine receptor-mediated inhibition of the pro-inflammatory transcription factor NF-κB in monocyte/macrophages. Because Type I interferon and pro-inflammatory genes are regulated reciprocally at the level of transcription factor activation and cell differentiation, we hypothesized that vagal activity would up-regulate Type I interferon response genes concurrently with inflammatory downregulation in human immune cells. We mapped empirical individual differences in the circulating leukocyte transcriptome and vagal activity indexed by high frequency (0.15–0.40 Hz) heart rate variability (HF-HRV) in 380 participants in the Midlife in the US study. Here we show that promoter-based bioinformatics analyses linked greater HF-HRV to reduced NF-κB activity and increased activity of IRF transcription factors involved in Type I interferon response (independent of β-antagonists, BMI, smoking, heavy alcohol consumption, and demographic factors). Transcript origin analyses implicated myeloid lineage immune cells as targets, representing per-cell alterations in gene transcription as HF-HRV was not associated with differential prevalence of leukocyte subsets. These findings support the concept of parasympathetic inhibition of pro-inflammatory gene expression in humans and up-regulation of Type I interferons that could augment host defense against viral infections.

Identification and therapeutic modulation of a pro-inflammatory subset of disease-associated-microglia in Alzheimer\u2019s disease

BackgroundDisease-associated-microglia (DAM) represent transcriptionally-distinct and neurodegeneration-specific microglial profiles with unclear significance in Alzheimer’s disease (AD). An understanding of heterogeneity within DAM and their key regulators may guide pre-clinical experimentation and drug discovery.MethodsWeighted co-expression network analysis (WGCNA) was applied to existing microglial transcriptomic datasets from neuroinflammatory and neurodegenerative disease mouse models to identify modules of highly co-expressed genes. These modules were contrasted with known signatures of homeostatic microglia and DAM to reveal novel molecular heterogeneity within DAM. Flow cytometric validation studies were performed to confirm existence of distinct DAM sub-populations in AD mouse models predicted by WGCNA. Gene ontology analyses coupled with bioinformatics approaches revealed drug targets and transcriptional regulators of microglial modules predicted to favorably modulate neuroinflammation in AD. These guided in-vivo and in-vitro studies in mouse models of neuroinflammation and neurodegeneration (5xFAD) to determine whether inhibition of pro-inflammatory gene expression and promotion of amyloid clearance was feasible. We determined the human relevance of these findings by integrating our results with AD genome-wide association studies and human AD and non-disease post-mortem brain proteomes.ResultsWGCNA applied to microglial gene expression data revealed a transcriptomic framework of microglial activation that predicted distinct pro-inflammatory and anti-inflammatory phenotypes within DAM, which we confirmed in AD and aging models by flow cytometry. Pro-inflammatory DAM emerged earlier in mouse models of AD and were characterized by pro-inflammatory genes (Tlr2, Ptgs2, Il12b, Il1b), surface marker CD44, potassium channel Kv1.3 and regulators (NFkb, Stat1, RelA) while anti-inflammatory DAM expressed phagocytic genes (Igf1, Apoe, Myo1e), surface marker CXCR4 with distinct regulators (LXRα/β, Atf1). As neuro-immunomodulatory strategies, we validated LXRα/β agonism and Kv1.3 blockade by ShK-223 peptide that promoted anti-inflammatory DAM, inhibited pro-inflammatory DAM and augmented Aβ clearance in AD models. Human AD-risk genes were highly represented within homeostatic microglia suggesting causal roles for early microglial dysregulation in AD. Pro-inflammatory DAM proteins were positively associated with neuropathology and preceded cognitive decline confirming the therapeutic relevance of inhibiting pro-inflammatory DAM in AD.ConclusionsWe provide a predictive transcriptomic framework of microglial activation in neurodegeneration that can guide pre-clinical studies to characterize and therapeutically modulate neuroinflammation in AD.

5 - Beneficial Effect of Resolvins in Ethanol and LPS-Induced Liver Injury via Inhibition of Pro-Inflammatory Gene Expression and Inflammasome Activation

5 - Beneficial Effect of Resolvins in Ethanol and LPS-Induced Liver Injury via Inhibition of Pro-Inflammatory Gene Expression and Inflammasome Activation

DRES-07. MODULATION OF INFLAMMATION PROFILES AND AHR SIGNALING IN HUMAN GLIOBLASTOMA MULTIFORME CELL LINES BY INDIRUBIN DERIVATIVES E804 AND 7BIO

Glioblastoma Multiforme (GBM) is a highly invasive brain tumor that affects approximately 18,000 people annually in the US alone. The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that binds endogenous, indole-derived ligands as well as many planar xenobiotics. In addition to regulating drug and xenobiotic metabolism, AHR signaling is involved in hematopoiesis, immune function, cell cycling, and inflammation. As new treatment options for GBM are developed, inflammation is emerging as a concern in the success of these new modalities. Indirubin and many of its derivatives bind AHR with different affinities based on their structure activity relationships (SAR). Depending on the SAR, AHR activation has a range of effects on cell physiology. We examined the effects of two indirubin derivatives: Indirubin-3’-(2,3 dihydroxypropyl)-oximether (E804) and 7-Bromoindirubin-3’-oxime (7BIO) on AHR activity and inflammation profiles in LN-18 and T98G human glioma cell lines. CYP1B1 gene expression, a marker for AHR activation, is induced in both cell lines, and most potently by E804. Using a commercial cancer-inflammation qPCR array, we showed that gene expression profiles indicative of pro-inflammation are highly suppressed by E804, with 7BIO having little effect. Of note, this suppression of pro-inflammatory gene expression occurred in LN-18 cells, but not T98G cells. IL6 protein secretion, an iconic inflammatory cytokine, was decreased by E804 as compared to the control at two time points and in both cell lines. Furthermore, in LN-18 cells, 7BIO reduced the expression of indoleamine dioxygenase (IDO1), a key enzyme for glioma survival. In contrast, however, IDO1 expression was induced in 7BIO-treated T98G cells. Moreover, expression of the regulatory transcription factor STAT3 was reduced by E804, but not by 7BIO in both cell lines. Thus, E804 is both an AHR ligand and inhibitor of STAT3, and may have potential in future treatments for GBM.

Compound A influences gene regulation of the Dexamethasone-activated glucocorticoid receptor by alternative cofactor recruitment

The glucocorticoid receptor (GR) is a transcription factor of which the underlying gene regulatory mechanisms are complex and incompletely understood. The non-steroidal anti-inflammatory Compound A (CpdA), a selective GR modulating compound in various cell models, has been shown to favour GR-mediated gene repression but not GR-mediated gene activation. Shifting balances towards only a particular subset of GR gene regulatory events may be of benefit in the treatment of inflammatory diseases. We present evidence to support that the combination of CpdA with Dexamethasone (DEX), a classic steroidal GR ligand, can shape GR function towards a unique gene regulatory profile in a cell type-dependent manner. The molecular basis hereof is a changed GR phosphorylation status concomitant with a change in the GR cofactor recruitment profile. We subsequently identified and confirmed the orphan nuclear receptor SHP as a coregulator that is specifically enriched at GR when CpdA and DEX are combined. Combining CpdA with DEX not only leads to stronger suppression of pro-inflammatory gene expression, but also enhanced anti-inflammatory GR target gene expression in epithelial cells, making ligand combination strategies in future a potentially attractive alternative manner of skewing and fine-tuning GR effects towards an improved therapeutic benefit.

P26 Tamaractam, a natural lactam, induces apoptosis and inhibits inflammation in rheumatoid arthritis fibroblast-like synoviocytes

Tamaractam is a novel lactam isolated from Tamarix ramosissima Ledeb., a traditional herbal medicine used for rheumatoid arthritis (RA) treatment in northwest China. The present study investigated the pro-apoptotic and anti-inflammatory effects of tamaractam on fibroblast-like synoviocytes (FLS) from RA patients. Apoptosis was assessed by TUNEL assay and caspase-3 activity was assessed by a colorimetric assay. The expression levels of apoptosis-related proteins including Bcl-2, Bax, p-Akt and p53 were determined by western blot analyses. The levels of IL-1β, IL-6, MMP-9 and COX-2 were measured using ELISA, and the mRNA expressions of these pro-inflammatory mediators were measured with quantitative real-time PCR. Tamaractam (0.1 and 1 μM) treatment induced cellular apoptosis of RA-FLS and also resulted in a significant increase in caspase-3 activity. It was found that tamaractam could regulate the protein expression of Bcl-2, Bax, p53 and pAkt. The concentrations and mRNA expression levels of IL-1β, IL-6, MMP-9 and COX-2 from RA-FLS were suppressed by tamaractam treatment in a dose-dependent manner. In conclusion, tamaractam treatment was found to induce apoptosis of RA-FLS through regulation of apoptosis related protein expression and to reduce the level of pro-inflammatory factors through inhibition of pro-inflammatory gene expression in RA-FLS. These results suggest that the pro-apoptotic and anti-inflammatory activities of tamaractam may be used as a possible therapeutic option for RA.

Regulation of inflammatory responses by neuregulin-1 in brain ischemia and microglial cells in vitro involves the NF-kappa B pathway.

BackgroundWe previously demonstrated that neuregulin-1 (NRG-1) was neuroprotective in rats following ischemic stroke. Neuroprotection by NRG-1 was associated with the suppression of pro-inflammatory gene expression in brain tissues. Over-activation of brain microglia can induce pro-inflammatory gene expression by activation of transcriptional regulators following stroke. Here, we examined how NRG-1 transcriptionally regulates inflammatory gene expression by computational bioinformatics and in vitro using microglial cells.MethodsTo identify transcriptional regulators involved in ischemia-induced inflammatory gene expression, rats were sacrificed 24 h after middle cerebral artery occlusion (MCAO) and NRG-1 treatment. Gene expression profiles of brain tissues following ischemia and NRG-1 treatment were examined by microarray technology. The Conserved Transcription Factor-Binding Site Finder (CONFAC) bioinformatics software package was used to predict transcription factors associated with inflammatory genes induced following stroke and suppressed by NRG-1 treatment. NF-kappa B (NF-kB) was identified as a potential transcriptional regulator of NRG-1-suppressed genes following ischemia. The involvement of specific NF-kB subunits in NRG-1-mediated inflammatory responses was examined using N9 microglial cells pre-treated with NRG-1 (100 ng/ml) followed by lipopolysaccharide (LPS; 10 μg/ml) stimulation. The effects of NRG-1 on cytokine production were investigated using Luminex technology. The levels of the p65, p52, and RelB subunits of NF-kB and IkB-α were determined by western blot analysis and ELISA. Phosphorylation of IkB-α was investigated by ELISA.ResultsCONFAC identified 12 statistically over-represented transcription factor-binding sites (TFBS) in our dataset, including NF-kBP65. Using N9 microglial cells, we observed that NRG-1 significantly inhibited LPS-induced TNFα and IL-6 release. LPS increased the phosphorylation and degradation of IkB-α which was blocked by NRG-1. NRG-1 also prevented the nuclear translocation of the NF-kB p65 subunit following LPS administration. However, NRG-1 increased production of the neuroprotective cytokine granulocyte colony-stimulating factor (G-CSF) and the nuclear translocation of the NF-kB p52 subunit, which is associated with the induction of anti-apoptotic and suppression of pro-inflammatory gene expression.ConclusionsNeuroprotective and anti-inflammatory effects of NRG-1 are associated with the differential regulation of NF-kB signaling pathways in microglia. Taken together, these findings suggest that NRG-1 may be a potential therapeutic treatment for treating stroke and other neuroinflammatory disorders.

Design of a novel thiophene inhibitor of 15-lipoxygenase-1 with both anti-inflammatory and neuroprotective properties

The enzyme 15-lipoxygenase-1 (15-LOX-1) plays a dual role in diseases with an inflammatory component. On one hand 15-LOX-1 plays a role in pro-inflammatory gene expression and on the other hand it has been shown to be involved in central nervous system (CNS) disorders by its ability to mediate oxidative stress and damage of mitochondrial membranes under hypoxic conditions. In order to further explore applications in the CNS, novel 15-LOX-1 inhibitors with favorable physicochemical properties need to be developed. Here, we present Substitution Oriented Screening (SOS) in combination with Multi Component Chemistry (MCR) as an effective strategy to identify a diversely substituted small heterocyclic inhibitors for 15-LOX-1, denoted ThioLox, with physicochemical properties superior to previously identified inhibitors. Ex vivo biological evaluation in precision-cut lung slices (PCLS) showed inhibition of pro-inflammatory gene expression and in vitro studies on neuronal HT-22 cells showed a strong protection against glutamate toxicity for this 15-LOX-1 inhibitor. This provides a novel approach to identify novel small with favorable physicochemical properties for exploring 15-LOX-1 as a drug target in inflammatory diseases and neurodegeneration.

Biflorin, Isolated from the Flower Buds of Syzygium aromaticum L., Suppresses LPS-Induced Inflammatory Mediators via STAT1 Inactivation in Macrophages and Protects Mice from Endotoxin Shock.

Two chromone C-glucosides, biflorin (1) and isobiflorin (2), were isolated from the flower buds of Syzygium aromaticum L. (Myrtaceae). Here, inhibitory effects of 1 and 2 on lipopolysaccharide (LPS)-induced production of nitric oxide (NO) and prostaglandin E2 (PGE2) in RAW 264.7 macrophages were evaluated, and 1 (IC50 = 51.7 and 37.1 μM, respectively) was more potent than 2 (IC50 > 60 and 46.0 μM). The suppression of NO and PGE2 production by 1 correlated with inhibition of iNOS and COX-2 protein expression. Compound 1 reduced inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) mRNA expression via inhibition of their promoter activities. Compound 1 inhibited the LPS-induced production and mRNA expression of tumor necrosis factor-α (TNF-α) and interleukin (IL)-6. Furthermore, 1 reduced p-STAT1 and p-p38 expression but did not affect the activity of nuclear factor κ light-chain enhancer of activated B cells (NF-κB) or activator protein 1 (AP-1). In a mouse model of LPS-induced endotoxemia, 1 reduced the mRNA levels of iNOS, COX-2, and TNF-α, and the phosphorylation-mediated activation of the signal transducer and activator of transcription 1 (STAT1), consequently improving the survival rates of mice. Compound 1 showed a significant anti-inflammatory effect on carrageenan-induced paw edema and croton-oil-induced ear edema in rats. The collective data indicate that the suppression of pro-inflammatory gene expression via p38 mitogen-activated protein kinase and STAT1 inactivation may be a mechanism for the anti-inflammatory activity of 1.

Mapping the Interaction of B Cell Leukemia 3 (BCL-3) and Nuclear Factor κB (NF-κB) p50 Identifies a BCL-3-mimetic Anti-inflammatory Peptide

The NF-κB transcriptional response is tightly regulated by a number of processes including the phosphorylation, ubiquitination, and subsequent proteasomal degradation of NF-κB subunits. The IκB family protein BCL-3 stabilizes a NF-κB p50 homodimer·DNA complex through inhibition of p50 ubiquitination. This complex inhibits the binding of the transcriptionally active NF-κB subunits p65 and c-Rel on the promoters of NF-κB target genes and functions to suppress inflammatory gene expression. We have previously shown that the direct interaction between p50 and BCL-3 is required for BCL-3-mediated inhibition of pro-inflammatory gene expression. In this study we have used immobilized peptide array technology to define regions of BCl-3 that mediate interaction with p50 homodimers. Our data show that BCL-3 makes extensive contacts with p50 homodimers and in particular with ankyrin repeats (ANK) 1, 6, and 7, and the N-terminal region of Bcl-3. Using these data we have designed a BCL-3 mimetic peptide based on a region of the ANK1 of BCL-3 that interacts with p50 and shares low sequence similarity with other IκB proteins. When fused to a cargo carrying peptide sequence this BCL-3-derived peptide, but not a mutated peptide, inhibited Toll-like receptor-induced cytokine expression in vitro. The BCL-3 mimetic peptide was also effective in preventing inflammation in vivo in the carrageenan-induced paw edema mouse model. This study demonstrates that therapeutic strategies aimed at mimicking the functional activity of BCL-3 may be effective in the treatment of inflammatory disease.

Role of hydrogen sulfide in paramyxovirus infections.

Hydrogen sulfide (H2S) is an endogenous gaseous mediator that has gained increasing recognition as an important player in modulating acute and chronic inflammatory diseases. However, its role in virus-induced lung inflammation is currently unknown. Respiratory syncytial virus (RSV) is a major cause of upper and lower respiratory tract infections in children for which no vaccine or effective treatment is available. Using the slow-releasing H2S donor GYY4137 and propargylglycin (PAG), an inhibitor of cystathionine-γ-lyase (CSE), a key enzyme that produces intracellular H2S, we found that RSV infection led to a reduced ability to generate and maintain intracellular H2S levels in airway epithelial cells (AECs). Inhibition of CSE with PAG resulted in increased viral replication and chemokine secretion. On the other hand, treatment of AECs with the H2S donor GYY4137 reduced proinflammatory mediator production and significantly reduced viral replication, even when administered several hours after viral absorption. GYY4137 also significantly reduced replication and inflammatory chemokine production induced by human metapneumovirus (hMPV) and Nipah virus (NiV), suggesting a broad inhibitory effect of H2S on paramyxovirus infections. GYY4137 treatment had no effect on RSV genome replication or viral mRNA and protein synthesis, but it inhibited syncytium formation and virus assembly/release. GYY4137 inhibition of proinflammatory gene expression occurred by modulation of the activation of the key transcription factors nuclear factor κB (NF-κB) and interferon regulatory factor 3 (IRF-3) at a step subsequent to their nuclear translocation. H2S antiviral and immunoregulatory properties could represent a novel treatment strategy for paramyxovirus infections. RSV is a global health concern, causing significant morbidity and economic losses as well as mortality in developing countries. After decades of intensive research, no vaccine or effective treatment, with the exception of immunoprophylaxis, is available for this infection as well as for other important respiratory mucosal viruses. This study identifies hydrogen sulfide as a novel cellular mediator that can modulate viral replication and proinflammatory gene expression, both important determinants of lung injury in respiratory viral infections, with potential for rapid translation of such findings into novel therapeutic approaches for viral bronchiolitis and pneumonia.

Mesenchymal stem cells inhibit lipopolysaccharide-induced inflammatory responses of BV2 microglial cells through TSG-6.

Microglia are the primary immunocompetent cells in brain tissue and microglia-mediated inflammation is associated with the pathogenesis of various neuronal disorders. Recently, many studies have shown that mesenchymal stem cells (MSCs) display a remarkable ability to modulate inflammatory and immune responses through the release of a variety of bioactive molecules, thereby protecting the central nervous system. Previously, we reported that MSCs have the ability to modulate inflammatory responses in a traumatic brain injury model and that the potential mechanisms may be partially attributed to upregulated TNF-α stimulated gene/protein 6 (TSG-6) expression. However, whether TSG-6 exerts an anti-inflammatory effect by affecting microglia is not fully understood. In this study, we investigated the anti-inflammatory effects of MSCs and TSG-6 in an in vitro lipopolysaccharide (LPS)-induced BV2 microglial activation model. We found that MSCs and TSG-6 significantly inhibited the expression of pro-inflammatory mediators in activated microglia. However, MSC effects on microglia were attenuated when TSG-6 expression was silenced. In addition, we found that the activation of nuclear factor (NF)-κB and mitogen-activated protein kinase (MAPK) pathways in LPS-stimulated BV2 microglial cells was significantly inhibited by TSG-6. Furthermore, we found that the presence of CD44 in BV2 microglial cells was essential for MSC- and TSG-6-mediated inhibition of pro-inflammatory gene expression and of NF-κB and MAPK activation in BV2 microglial cells. The results of this study suggest that MSCs can modulate microglia activation through TSG-6 and that TSG-6 attenuates the inflammatory cascade in activated microglia. Our study indicates that novel mechanisms are responsible for the immunomodulatory effect of MSCs on microglia and that MSCs, as well as TSG-6, might be promising therapeutic agents for the treatment of neurotraumatic injuries or neuroinflammatory diseases associated with microglial activation.

The SIRT1 Activator SRT1720 Extends Lifespan and Improves Health of Mice Fed a Standard Diet

The prevention or delay of the onset of age-related diseases prolongs survival and improves quality of life while reducing the burden on the health care system. Activation of sirtuin 1 (SIRT1), an NAD(+)-dependent deacetylase, improves metabolism and confers protection against physiological and cognitive disturbances in old age. SRT1720 is a specific SIRT1 activator that has health and lifespan benefits in adult mice fed a high-fat diet. We found extension in lifespan, delayed onset of age-related metabolic diseases, and improved general health in mice fed a standard diet after SRT1720 supplementation. Inhibition of proinflammatory gene expression in both liver and muscle of SRT1720-treated animals was noted. SRT1720 lowered the phosphorylation of NF-κB pathway regulators in vitro only when SIRT1 was functionally present. Combined with our previous work, the current study further supports the beneficial effects of SRT1720 on health across the lifespan in mice.

Human mesenchymal stem cell transplantation changes proinflammatory gene expression through a nuclear factor‐κB‐dependent pathway in a rat focal cerebral ischemic model

Previous studies have demonstrated the immunomodulatory functions of mesenchymal stem cells (MSCs) in cerebral ischemic rats. However, the underlying mechanisms are unclear. The purpose of this study is to investigate the effects of MSC transplantation on transcriptional regulations of proinflammatory genes in cerebral ischemia. Transient ischemia was induced by middle cerebral artery occlusion (MCAO) in adult male Sprague-Dawley rats. After 24 hr, vehicle (PBS) or a human MSC line (B10) was transplanted intravenously. The neurological deficits, infarct volume, cellular accumulations, and gene expression changes were monitored by means of behavior tests, MRI, immunohistochemistry, Western blotting, laser capture microdissection, and real-time PCR. In the core area of the B10 transplantation group, the number of ED1-positive macrophage/microglia was decreased compared with the PBS group. In the core, nuclear factor-κB (NF-κB) was decreased, although CCAAT/enhancer-binding protein β was not changed; both were expressed mainly in ED1-positive macrophage/microglia. Likewise, mRNAs of NF-κB-dependent genes including interleukin-1β, MCP-1, and inducible nitric oxide synthase were decreased in ED1-positive and Iba-1-positive macrophage/microglia in the B10 transplantation group. Moreover, upstream receptors of the NF-κB pathway, including CD40 and Toll-like receptor 2 (TLR2), were decreased. Immunofluorescence results showed that, in the B10 transplantation group, the percentages of NF-κB-positive, CD40-positive, and TLR2-positive cells were decreased in ED1-positive macrophage/microglia. Furthermore, NF-κB-positive cells in the CD40- or TLR2-expressing cell population were decreased in the B10 transplantation group. This study demonstrates that B10 transplantation inhibits NF-κB activation, possibly through inhibition of CD40 and TLR2, which might be responsible for the inhibition of proinflammatory gene expression in macrophage/microglia in the infarct lesion.

Trichostatin A Modulates Thiazolidinedione-Mediated Suppression of Tumor Necrosis Factor α-Induced Lipolysis in 3T3-L1 Adipocytes

In obesity, high levels of tumor necrosis factor α (TNFα) stimulate lipolysis in adipocytes, leading to hyperlipidemia and insulin resistance. Thiazolidinediones (TZDs), the insulin-sensitizing drugs, antagonize TNFα-induced lipolysis in adipocytes, thereby increasing insulin sensitivity in diabetes patients. The cellular target of TZDs is peroxisome proliferator-activated receptor γ (PPARγ), a nuclear receptor that controls many adipocyte functions. As a transcription factor, PPARγ is closely modulated by coregulators, which include coactivators and corepressors. Previous studies have revealed that in macrophages, the insulin-sensitizing effect of PPARγ may involve suppression of proinflammatory gene expression by recruiting the corepressor complex that contains corepressors and histone deacetylases (HDACs). Therefore, we investigated whether the corepressor complex is involved in TZD-mediated suppression of TNFα-induced lipolysis in 3T3-L1 adipocytes. Trichostatin A (TSA), a pan HDAC inhibitor (HDACI) that inhibits class I and II HDACs, was used to examine the involvement of HDACs in the actions of TZDs. TSA alone increased basal lipolysis and attenuated TZD-mediated suppression of TNFα-induced lipolysis. Increased basal lipolysis may in part result from class I HDAC inhibition because selective class I HDACI treatment had similar results. However, attenuation of TZD-mediated TNFα antagonism may be specific to TSA and related hydroxamate-based HDACI rather than to HDAC inhibition. Consistently, corepressor depletion did not affect TZD-mediated suppression. Interestingly, TSA treatment greatly reduced PPARγ levels in differentiated adipocytes. Finally, extracellular signal-related kinase 1/2 (ERK1/2) mediated TNFα-induced lipolysis, and TZDs suppressed TNFα-induced ERK phosphorylation. We determined that TSA increased basal ERK phosphorylation, and attenuated TZD-mediated suppression of TNFα-induced ERK phosphorylation, consistent with TSA’s effects on lipolysis. These studies suggest that TSA, through down-regulating PPARγ, attenuates TZD-mediated suppression of TNFα-induced ERK phosphorylation and lipolysis in adipocytes.

Anti-obesity effect of Lactobacillus gasseri SBT2055 accompanied by inhibition of pro-inflammatory gene expression in the visceral adipose tissue in diet-induced obese mice

Probiotic Lactobacillus gasseri SBT2055 (LG2055) has an anti-obesity effect although it is unknown whether the effect influences inflammatory responses in adipose tissue and lipid metabolism in the liver, which are considered substantially relevant to adiposity. C57BL/6 mice were fed a 10%-fat diet containing LG2055 cells for 24 weeks. We then studied body weight, fat tissue mass, liver fat content and inflammatory genes in the adipose tissue, and lipogenic and lipolytic genes in the liver. Consumption of LG2055 resulted in a significant reduction in body weight and fat tissue mass (epididymal and perirenal/retroperitoneal), with a lowered level of triglyceride content in the liver. DNA microarray analysis showed that LG2055 generally inhibited the up-regulation of pro-inflammatory genes, including CCL2 and CCR2, in the epididymal adipose tissue. In the liver, LG2055 tended to inhibit lipogenic gene up-regulation, including ACC1, FAS and SREBP1, but LG2055 did not markedly alter lipolytic genes. Real-time PCR analysis confirmed the DNA microarray results in part, showing a significant reduction in the mRNA expression of CCL2 in the epididymal adipose tissue, and a downward tendency in FAS mRNA expression in the liver, in the LG2055-fed group. LG2055 significantly prevented body weight gain, fat accumulation and pro-inflammatory gene expression in the adipose tissue. Relatively lower triglyceride levels and reduced expression of lipogenic genes were also observed in the liver. It is suggested that improvement in the inflammatory state of the adipose tissue might be a possible mechanism underlying the anti-obesity effect of LG2055.

Targeting Activation of Specific NF-κB Subunits Prevents Stress-Dependent Atherothrombotic Gene Expression

Psychosocial stress has been shown to be a contributing factor in the development of atherosclerosis. Although the underlying mechanisms have not been elucidated entirely, it has been shown previously that the transcription factor nuclear factor-κB (NF-κB) is an important component of stress-activated signaling pathway. In this study, we aimed to decipher the mechanisms of stress-induced NF-κB-mediated gene expression, using an in vitro and in vivo model of psychosocial stress. Induction of stress led to NF-κB-dependent expression of proinflammatory (tissue factor, intracellular adhesive molecule 1 [ICAM-1]) and protective genes (manganese superoxide dismutase [MnSOD]) via p50, p65 or cRel. Selective inhibition of the different subunits and the respective kinases showed that inhibition of cRel leads to the reduction of atherosclerotic lesions in apolipoprotein(-/-) (ApoE(-/-)) mice via suppression of proinflammatory gene expression. This observation may therefore provide a possible explanation for ineffectiveness of antioxidant therapies and suggests that selective targeting of cRel activation may provide a novel approach for the treatment of stress-related inflammatory vascular disease.

Preliminary safety and biological efficacy studies of ethyl pyruvate in normal mature horses

Endotoxaemia causes substantial morbidity and mortality in horses with colic and sepsis. Ethyl pyruvate is a novel anti-inflammatory medication that improved survival in preclinical models of severe sepsis endotoxaemia and intestinal ischaemia and reperfusion in rodents, swine, sheep and dogs and may be a useful medication in horses. Ethyl pyruvate has no adverse effects in normal horses and is biologically active based on suppression of proinflammatory gene expression in endotoxin stimulated whole blood, in vitro. Physical and neurological examinations, behaviour scores, electrocardiograms and clinicopathological tests were performed on 5 normal healthy horses receiving 4 different doses of ethyl pyruvate. Doses included 0, 50, 100 and 150 mg/kg bwt administered in a randomised crossover design with a 2 week washout period between doses. Biological efficacy was assessed by stimulating whole blood with endotoxin from the horses that received ethyl pyruvate prior to and 1 and 6 h after drug infusion. Gene expression for TNFα, IL-1β and IL-6 was assessed. There were no effects of drug or dose (0, 50, 100 or 150 mg/kg bwt) on any of the physical or neurological examination, behaviour factors, electrocardiogram or clinical pathological results collected from any of the horses. All parameters measured remained within the normal reference range. There was a significant reduction in TNFα, IL-1β and IL-6 gene expression in endotoxin stimulated whole blood from horses 6 h after receiving 150 mg/kg bwt ethyl pyruvate. There were no detectable effects on gene expression of any of the other doses of ethyl pyruvate tested. We were unable to detect any detrimental effects of ethyl pyruvate administration in normal horses. Ethyl pyruvate significantly decreased proinflammatory gene expression in endotoxin stimulated blood 6 h after drug administration. Ethyl pyruvate may be a safe, effective medication in endotoxaemic horses.

B‐vitamin deficiency is protective in experimental colitis

Methionine (Met) cycle activity is critical for normal cell functions and requires B‐vitamin (B6/B12) as cofactors. S‐adenosylhomocysteine (SAH) is a Met cycle intermediates that is known to inhibit methyltransferases. Met metabolism is altered in patients with inflammatory bowel disease (IBD), but Met's role in IBD is poorly understood. We hypothesize that accumulation of Met metabolites due to B‐vitamin deficiency is protective in experimental colitis. Mice received either a control diet (C) or a B6/B12‐deficient diet (D) to disrupt Met metabolism for 2 weeks and were challenged for 5 days with 3% dextran sulfate sodium (DSS) to induce colitis. Clinical and histological disease indices were reduced in D compared to C, along with the colonic expression of the proinflammatory genes (TNFα and iNOS) and myeloperoxidase activity. Whole body Met flux was assessed by infusing 13C‐2H3‐Met and measuring transmethylation (TM), transsulfuration (TS) and remethylation (RM). Plasma TS was reduced but RM was increased in D mice. TM was not different between groups. Colonic SAH and plasma Met abundance also were elevated in D mice compared to C. The protective effect of B‐vitamin deficiency is associated with reduced inflammation and tissue injury. We postulate that colonic SAH accumulation reduces methyltransferase activity and leads to epigenetic suppression of proinflammatory gene expression. Funded by USDA.Grant Funding Source: USDA