P65 Subunit Research Articles

Mechanisms of Nitroalkene Inhibition of TLR4 Mediated Macrophage Activation

Fatty acid nitroalkenes, such as nitro‐oleic acid (OA‐NO2), are endogenously formed signaling mediators. They have demonstrated anti‐inflammatory properties but have not been extensively studied in the lung. We have previously shown that intratracheal administration of OA‐NO2 reduces bleomycin‐mediated acute lung injury histopathologically and impacts pulmonary macrophage populations and activation. TLR4 is the principal damage receptor in acute lung injury and its activation results in pro‐inflammatory signaling. We hypothesize that changes to macrophage phenotype with OA‐NO2 administration are through inhibition of TLR4 responses specifically through NF‐κB inhibition. To investigate this hypothesis, OA‐NO2 effects on macrophage activation were examined in LPS‐activated RAW 267.4 cells. Following LPS treatment cells were collected either 6h, for gene expression analysis, or 24h for protein and metabolic analysis. qPCR showed OA‐NO2 inhibited TLR4 target gene expression IL‐6, PDGS2, and NOS2 in response to LPS (51±9.3* vs 19±12.3†; 45±15.0* vs 6±1.0†; 30±7.3* vs 1.4±0.8†‐fold change, respectively). OA‐NO2 also inhibited iNOS protein expression when compared to LPS (443±52.9* vs 88±5.5†% density). To examine OA‐NO2 effects on transcription factor function, we tested its effects on LPS‐mediated increases in NF‐κB activity in RAW Blue cells, where there was a reduction in SEAP activity (10±0.7* vs 4±0.1† rate). To determine if reduced activity could be attributed to OA‐NO2 stabilization of NF‐κB inhibitory proteins Iκ‐B protein levels were assessed. Iκ‐B was not stabilized, as protein concentrations were decreased with OA‐NO2 regardless of LPS stimulation. This indicated OA‐NO2 may directly bind to NF‐κB, preventing its activation and marking Iκ‐B for degradation. To test this hypothesis, an alkyne‐containing OA‐NO2 derivative was used to identify proteins that are adducted by OA‐NO2. After reaction with biotinylated azide, streptavidin was used to capture alkynyl‐OA‐NO2 adducted proteins. This revealed that OA‐NO2 adducts both the p50 and p65 subunits of NF‐κB, a reaction that does not prevent nuclear translocation, as determined by NF‐κB subunit detection following nuclear and cytoplasmic fractioning but does prevents its ability to transcribe downstream targets. This demonstrates that OA‐NO2 inhibits TLR4 mediated pro‐inflammatory gene expression responses through direct post‐translational modification and inhibition of NF‐κB. OA‐NO2 has the potential to alter other thiol containing proteins, meaning it may also act as a metabolic regulator. Our early data suggests that OA‐NO2 reduces LPS stimulation of glycolysis mediated acidification (6±1.1* vs 2±0.3†mpH/min). OA‐NO2 also reduces mitochondrial respiration regardless of LPS activation (9±1.5* 3±0.6*; 1±0.2#†pmol/min). Therefore, OA‐NO2 may inhibit macrophage activation through metabolic inhibition. In summary, OA‐NO2 is a potent regulator of pro‐inflammatory macrophage signaling and metabolism, indicating it potential therapeutic use in acute lung injury.

Regulation of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) by the Nuclear Bile Acid Receptor, Farnesoid X Receptor

Introduction and AimsCFTR, a Cl‐ channel important in regulating intestinal fluid and electrolyte secretion, is implicated in the pathogenesis of a number of intestinal disorders. We have previously shown that bile acids, acting via the nuclear receptor, farnesoid X receptor (FXR), inhibit colonic epithelial CFTR expression. Dietary phytochemicals have been reported to have the capacity to modulate FXR signalling. Here, we set out to investigate mechanisms underlying FXR regulation of CFTR expression, and the potential for therapeutically targeting the receptor with dietary phytochemicals.MethodsMonolayers of T84 colonic epithelial cells were treated with the FXR agonist, GW4064 (5 µM) in the absence or presence of a plant phytochemical, designated here as KFS1 (5 µM). mRNA and protein expression were measured by qPCR and Western blotting. FXR binding to the CFTR promoter was investigated by chromatin immunoprecipitation (ChIP). Electrophysiological studies of T84cells were conducted in Ussing chambers. Studies on human and murine enteroids were carried out with ethical approval from Johns Hopkins University School of Medicine Institutional Review Board and the Institutional Review Board of the University of California San Diego, respectively.ResultsTreatment of T84 monolayers with GW4064 downregulated CFTR mRNA to 0.51 ± 0.06 fold after 12 hrs (n = 12; p < 0.001) and protein levels to 0.28 ± 0.06 fold after 48 hrs, compared to controls (n = 8; p < 0.001). Studies in Ussing chambers showed that GW4064 treatment for 48 hrs inhibited Cl‐ secretory responses to the Ca2+‐ and cAMP‐dependent agonists, carbachol (CCh; 100mM) and forskolin (FSK; 10mM), by 79.9 ± 7.5 % and 74.2 ± 8.9 %, respectively. Transcriptomic analysis of human colonic enteroids revealed FXR to be robustly expressed in secretory (crypt‐like) cells and that its activation also induced CFTR downregulation. FXR activation did not alter expression or phosphorylation of the p65 subunit of NF‐κB, or inhibit its translocation to the nucleus. ChIP‐qPCR analysis appears to show enhanced binding of FXR to the CFTR promoter upon GW4064 treatment. The phytochemical, KFS1 (5 mM; 24hrs), upregulated FXR mRNA and protein expression in T84 cells and enhanced GW4064‐induced downregulation of CFTR mRNA by 0.28 ± 0.05 fold (n = 8; p <0.01) and protein by 0.25 ± 0.11 fold (n = 4) after 24 hours. Similarly, KFS1 significantly upregulated FXR mRNA expression in murine colonic epithelial enteroids (2.3 ± 0.2; n = 4; p < 0.01) and enhanced GW4064‐induced downregulation of CFTR mRNA by 0.5 ± 0.1 fold (n = 4; p < 0.05) compared to GW4064 alone. Finally, KFS1 enhanced FXR inhibition of agonist‐induced Cl‐ secretory responses across T84 cells mounted in Ussing chambers.ConclusionFXR regulates colonic epithelial CFTR expression and function by a mechanism which appears to involve direct binding of FXR to the CFTR promoter. By virtue of their ability to upregulate FXR expression, and thereby enhance its antisecretory actions, plant extracts containing KFS1 have excellent potential to be developed as FXR‐targeted nutraceuticals for the treatment and prevention of intestinal disease.

Bombesin receptor-activated protein exacerbates cisplatin-induced AKI by regulating the degradation of SIRT2.

Acute kidney injury (AKI) is a public health problem with no specific therapies in the clinic and the underlying pathogenesis of AKI remains obscure. Bombesin receptor-activated protein (BRAP, C6ORF89 protein) was initially discovered as a ligand for a previously orphan G-protein-coupled receptor bombesin-like receptor-3. At present, accepted biological effects of BRAP include cell cycle progression, wound repair and the activation of histone deacetylases. However, its role in kidney disease is unknown. In this study we have investigated the role of BRAP and underlying mechanisms involved in cisplatin (CP)-induced AKI. Here we used Bc004004 (homologous of C6ORF89 in mice) knockout mice and HK2 cells to investigate the effect of BRAP on AKI in vitro and in vivo. We analyzed ChIP-Seq and RNA-Seq data to search for the upstream regulators of BRAP and downstream mediators of BRAP action in AKI. Immunostaining, real-time polymerase chain reaction (PCR), co-immunoprecipitation, a dual-luciferase reporter assay and ChIP-PCR assay were applied to reveal the upstream and downstream regulation mechanism of BRAP during cisplatin-induced AKI. BRAP was downregulated in mice and human kidneys with AKI. Global Bc004004 deletion alleviated tubular cell apoptosis and necroptosis in CP-induced AKI mice, whereas local overexpression of BRAP in kidneys aggravated them. Pan-caspase inhibitor Z-VAD pretreatment attenuated CP-induced blood creatinine increase and kidney injury in wild-type mice but not in BRAP -/- mice. The activation of mixed lineage kinase like-domain was magnified by Z-VAD in CP-treated mice, especially in BRAP -/- mice. The cytoprotective effect of Z-VAD was more substantial than necrostatin-1 (Nec-1, an inhibitor of necroptosis) in CP-treated human kidney proximal tubular epithelial (HK2) cells. Furthermore, Nec-1 pretreatment reduced the CP-induced cell death in BRAP overexpression HK2 cells but did not work in cells with normal BRAP levels. We determined that CP treatment activated the nuclear factor-κB subunit P65 and inhibition of P65 increased the messenger RNA (mRNA) levels of BRAP in HK2 cells. The chromatin immunoprecipitation assay and dual-luciferase reporter gene assay verified P65 binding to the C6ORF89 promoter and reduced its mRNA expression upon CP treatment. Next we found that sirtuin 2 (SIRT2) was downregulated in CP-induced AKI and BRAP levels directly impacted the protein levels of SIRT2. Our findings further confirmed that BRAP regulates the SIRT2 protein levels by affecting SIRT2's interactions with E3 ubiquitin ligase HRD1 and subsequent proteasomal degradation. Our results demonstrated that BRAP played an important role in tubular cell apoptosis and necroptosis during CP-induced AKI. Safe and efficient BRAP inhibitors might be effective therapeutic options for AKI.

Tanreqing Injection Attenuates Macrophage Activation and the Inflammatory Response via the lncRNA-SNHG1/HMGB1 Axis in Lipopolysaccharide-Induced Acute Lung Injury.

The etiology of acute lung injury (ALI) is not clear, and the treatment of ALI presents a great challenge. This study aimed to investigate the pathogenesis and potential therapeutic targets of ALI and to define the target gene of Tanreqing (TRQ), which is a traditional Chinese medicine formula composed of five medicines, scutellaria baicalensis, bear bile powder, goat horn powder, honeysuckle and forsythia. Macrophage activation plays a critical role in many pathophysiological processes, such as inflammation. Although the regulation of macrophage activation has been extensively investigated, there is little knowledge of the role of long noncoding RNAs (lncRNAs) in this process. In this study, we found that lncRNA-SNHG1 expression is distinctly regulated in differently activated macrophages in that it is upregulated in LPS. LncRNA-SNHG1 knockdown attenuates LPS-induced M1 macrophage activation. The SNHG1 promoter was bound by NF-κB subunit p65, indicative of SNHG1 being a direct transcriptional target of LPS-induced NF-κB activation. SNHG1 acts as a proinflammatory driver that leads to the production of inflammatory cytokines and the activation of macrophages and cytokine storms by physically interacting with high-mobility group box 1 (HMGB1) in ALI. TRQ inhibited NF-κB signaling activation and binding of NF-κB to the SNHG1 promoter. In conclusion, this study defined TRQ target genes, which can be further elucidated as mechanism(s) of TRQ action, and provides insight into the molecular pathogenesis of ALI. The lncRNA-SNHG1/HMGB1 axis is an ideal therapeutic for ALI treatment.

Bone marrow granulocytes downregulate IL-1β and TNF production and the microbicidal activity of inflammatory macrophages.

Macrophages play critical roles in inflammation and defense against pathogens, as well as in the return to tissue homeostasis. Macrophage subpopulations displaying antagonistic phenotypes are generally classified as proinflammatory M1, implicated in antipathogen and antitumoral activities, or as anti-inflammatory M2, associated with tissue repair. Granulocytic and monocytic myeloid-derived suppressor cells recruited from the bone marrow to tissues and phagocytosis of apoptotic neutrophils can attenuate macrophage microbicidal activity. Here, we showed that bone marrow neutrophils, but not thioglycollate-recruited neutrophils, directly suppress the responses of macrophages that were previously committed to an inflammatory phenotype. Cocultures of inflammatory macrophages with bone marrow CD11b+Ly6Ghi granulocytes led to reduced release of IL-1β, TNF-α, and IL-6 by macrophages after lipopolysaccharide stimulation. The suppressive activity was unrelated to granulocyte apoptosis or to secreted factors and required cell-to-cell contact. The suppressive effect was paralleled by reduction in the nuclear levels of the NF-κB p65 subunit, but not of the p50 subunit. Furthermore, bone marrow granulocytes decreased the phagocytic activity of macrophages and their capacity to kill intracellular Escherichia coli. Taken together, these results show that bone marrow granulocytes can function as suppressors of the proinflammatory activity and microbial-killing responses of macrophages.

Proprotein convertase furin is a driver and potential therapeutic target in proliferative diabetic retinopathy.

Furin converts inactive proproteins into bioactive forms. By activating proinflammatory and proangiogenic factors, furin might play a role in pathophysiology of proliferative diabetic retinopathy (PDR). We studied vitreous samples from PDR and nondiabetic patients, epiretinal membranes from PDR patients, retinal microvascular endothelial cells (HRMECs), retinal Müller cells and rat retinas by ELISA, Western blot analysis, immunohistochemistry and immunofluorescence microscopy. We performed in vitro angiogenesis assays and assessed adherence of monocytes to HRMECs. Furin levels were significantly increased in PDR vitreous samples. In epiretinal membranes, immunohistochemistry analysis revealed furin expression in monocytes/macrophages, vascular endothelial cells and myofibroblasts. Furin was significantly upregulated in diabetic rat retinas. Hypoxia and TNF-α induced significant upregulation of furin in Müller cells and HRMECs. Furin induced upregulation of phospho-ERK1/2, p65 subunit of NF-κB, ADAM17 and MCP-1 in cultured Müller cells and phospho-ERK1/2 in cultured HRMECs and induced HRMECs migration. Treatment of monocytes with furin significantly increased their adhesion to HRMECs. Intravitreal administration of furin in normal rats induced significant upregulation of p65 subunit of NF-κB, phospho-ERK1/2 and ICAM-1 in the retina. Inhibition of furin with dec-CMK significantly decreased levels of MCP-1 in culture medium of Müller cells and HRMECs and significantly attenuated TNF-α-induced upregulation of p65 subunit of NF-κB, ICAM-1 and VCAM-1 in HRMECs. Dec-CMK significantly decreased adherence of monocytes to HRMECs and TNF-α-induced upregulation of adherence of monocytes to HRMECs. Treatment of HRMECs with dec-CMK significantly attenuated migration of HRMECs. Furin is a potential driver molecule of PDR-associated inflammation and angiogenesis.

P52 signaling promotes cellular senescence

BackgroundNuclear factor-κB is a multi-subunit transcription factor that plays a central role in cellular senescence. We previously reported that an increase in the p52 subunit is seen in senescent cells and aged tissue. In the current work, we examined the mechanism by which p52 is activated and whether the increase in p52 promotes senescence.ResultsUsing both primary mouse embryonic fibroblasts (MEFs) and WI-38 human lung fibroblasts, we examined cells after serial passage and following prolonged culture. An increase in p52 was found in the nucleus relative to pre-senescent cells. The increase in p52 protein was not reflected by an increase in NFKB2 mRNA or by an increase in the abundance of upstream activating kinases, IKKα and NIK. To examine whether p52 promotes senescence, we over-expressed mature p52 in primary MEFs. Significantly more senescence was seen compared to control, a finding not seen with p52 mutated at critical DNA binding residues. In addition, blocking p52 nuclear translocation with the peptide inhibitor, SN52, decreased β-galactosidase (β-gal) formation. Subsequent filtration studies demonstrated that proteins in conditioned media (CM) were necessary for the increase in p52 and mass spectrometry identified S100A4 and cyclophilin A (CYPA) as potential factors in CM necessary for induction of p52. The requirement of these proteins in CM for induction of p52 was confirmed using depletion and supplementation studies. In addition, we found that activation of STAT3 signaling was required for the increase in p52. Finally, genome wide ChIP-sequencing analysis confirmed that there is an increase in p52 chromatin enrichment with senescence and identified several downstream factors whose expression is regulated by increased p52 binding.ConclusionsThese results demonstrate that p52 nuclear translocation is increased in senescent cells by factors in conditioned media and that mature p52 induces cellular senescence. The data are consistent with the prior observation that p52 is elevated in aged tissue and support the hypothesis that p52 contributes to organismal aging.

Bioactive Components of Houttuynia cordata Thunb and Their Potential Mechanisms Against COVID-19 Using Network Pharmacology and Molecular Docking Approaches.

We investigated the molecular mechanism by which Houttuynia cordata Thunb (HCT) may intervene in coronavirus disease 2019 (COVID-19) and COVID-19-induced cytokine storms using network pharmacology and molecular docking approaches. Using the Traditional Chinese medicine Systems Pharmacology Database and Analysis Platform (TCMSP), a "component-target-pathway" topology map of HCT for COVID-19 treatment was constructed using Cytoscape. Core target genes were analyzed using the STRING database, and the signal pathway map and biological mechanism of COVID-19 therapy were obtained using cluster profilers. Active components of HCT were docked with severe respiratory syndrome coronavirus 2 (SARS-CoV-2) 3C-like protease (3CLpro) and RNA-dependent RNA polymerase (RdRp) using AutoDockTools. Data visualization and statistical analysis were conducted using the R program. A molecular dynamic simulation was carried out with the Groningen Machine for Chemical Simulation program. HCT had six active anti-COVID-19 ingredients and 45 molecular targets. Their crucial target proteins for COVID-19 treatment were the RELA (nuclear factor kappa B [NF-κB] p65 subunit), interleukin 6, and mitogen-activated protein kinase 1. In functional enrichment analysis, the potential molecular targets of active components of HCT for COVID-19 treatment belonged to 18 signaling pathways (adjusted P = 2.12E-11). Gene ontology obtained by Kyoto Encyclopedia of Genes and Genome enrichment screening showed that the primary mechanism of COVID-19 treatment was upregulation of protein kinase C followed by downregulations of T cell differentiation and proliferation and NF-κB signaling. Molecular docking showed that the active components of HCT (quercetin and kaempferol) had similar binding affinities for SARS-CoV-2 3CLpro and SARS-CoV-2 RdRp, primary COVID-19 target proteins as did clinically used drugs. These results were confirmed with molecular dynamics simulation. In conclusion, multiple components of HCT, especially quercetin and kaempferol, have the potential to treat COVID-19 infection and COVID-19-induced cytokine storm by targeting multiple proteins.

CARD9 Mediates Pancreatic Islet Beta-Cell Dysfunction Under the Duress of Hyperglycemic Stress.

Background/Aims:Published evidence implicates Caspase recruitment domain containing protein 9 (CARD9) in innate immunity. Given its recently suggested roles in obesity and insulin resistance, we investigated its regulatory role(s) in the onset of islet beta cell dysfunction under chronic hyperglycemic (metabolic stress) conditions.Methods:Islets from mouse pancreas were isolated by the collagenase digestion method. Expression of CARD9 was suppressed in INS-1 832/13 cells by siRNA transfection using the DharmaFect1 reagent. The degree of activation of Rac1 was assessed by a pull-down assay kit. Interactions between CARD9, RhoGDIβ and Rac1 under metabolic stress conditions were determined by co-immunoprecipitation assay. The degree of phosphorylation of stress kinases was assessed using antibodies directed against phosphorylated forms of the respective kinases.Results:CARD9 expression is significantly increased following exposure to high glucose, not to mannitol (both at 20 mM; 24 hrs.) in INS-1 832/13 cells. siRNA-mediated knockdown of CARD9 significantly attenuated high glucose-induced activation of Rac1 and phosphorylation of p38MAPK and p65 subunit of NF-κB (RelA), without significantly impacting high glucose-induced effects on JNK1/2 and ERK1/2 activities. CARD9 depletion also suppressed high glucose-induced CHOP expression (a marker for endoplasmic reticulum stress) in these cells. Co-immunoprecipitation studies revealed increased association between CARD9-RhoGDIβ and decreased association between RhoGDIβ-Rac1 in cells cultured under high glucose conditions.Conclusion:Based on these data, we conclude that CARD9 regulates activation of Rac1-p38MAPK-NFκB signaling pathway leading to functional abnormalities in beta cells under metabolic stress conditions.

Inhibition of the CEBP\u03b2-NF\u03baB interaction by nanocarrier-packaged Carnosic acid ameliorates glia-mediated neuroinflammation and improves cognitive function in an Alzheimer\u2019s disease model

Neuroinflammation occurs early in Alzheimer’s disease (AD). The initial stage of AD is related to glial dysfunction, which contributes to impairment of Aβ clearance and disruption of synaptic connection. CEBPβ, a member of the CCAAT-enhancer-binding protein (CEBP) family, modulates the expression of inflammation-associated genes, and its expression is elevated in brains undergoing degeneration and injured brains. However, the mechanism underlying CEBPβ-mediated chronic inflammation in AD is unclear. In this study, we observed that increases in the levels of nuclear CEBPβ facilitated the interaction of CEBPβ with the NFκB p65 subunit, increasing the transcription of proinflammatory cytokines in the APP/PS1 mouse brain. Oral administration of nanocarrier-packaged carnosic acid (CA) reduced the aberrant activation of microglia and astrocytes and diminished mature IL-1β, TNFα and IL-6 production in the APP/PS1 mouse brain. CA administration reduced β-amyloid (Aβ) deposition and ameliorated cognitive impairment in APP/PS1 mice. We observed that CA blocked the interaction of CEBPβ with NFκB p65, and chromatin immunoprecipitation revealed that CA reduced the transcription of the NFκB target genes TNFα and IL-6. We confirmed that CA alleviated inflammatory mediator-induced neuronal degeneration and reduced Aβ secretion by inhibiting the CEBPβ-NFκB signalling pathway in vitro. Sulfobutyl ether-beta-cyclodextrin (SBEβCD) was used as the encapsulation agent for the CA-loaded nanocarrier to overcome the poor water solubility and enhance the brain bioavailability of CA. The CA nanoparticles (NPs) had no obvious toxicity. We demonstrated a feasible SBEβCD-based nanodelivery system targeting the brain. Our data provide experimental evidence that CA-loaded NPs are potential therapeutic agents for AD treatment.

Euphorbia factor L3 ameliorates rheumatoid arthritis by suppressing the inflammatory response by targeting Rac family small GTPase 1

ABSTRACT Euphorbia factor L3 (EFL3) is extracted from Euphorbia lathyris and is known for its anti-inflammatory properties. This study focused on the potential anti-inflammatory and therapeutic effects of EFL3 on rheumatoid arthritis (RA) using fibroblast-like synoviocytes (FLSs) and arthritis animal models. Functional analysis showed that EFL3 could ameliorate the inflammatory phenotype of FLSs derived from RA patients, as evidenced by the decreases in cell viability, migration, invasion and cytokine production. Luciferase activity, Western blotting and immunofluorescence assays demonstrated that EFL3 inhibited the nuclear translocation of the p65 subunit and the subsequent activation of the nuclear factor kappa-Β (NF-κB) pathway. Furthermore, the therapeutic effects of EFL3 against arthritic progression were evidenced by decreases in joint swelling, arthritis scores, inflammatory factor production, synovial hyperplasia, and bone destruction in collagen-induced arthritis (CIA) and tumor necrosis factor-α (TNF-α) transgenic (TNF-tg) mouse models. Molecular analysis identified Rac family small GTPase 1 (Rac1) as the potential target that was required for EFL3-mediated suppression of the inflammatory RA FLS phenotype. In summary, this study uncovered the therapeutic potential of EFL3 in RA, which suggests its future clinical use.

N,N-Dimethylformamide Delays LPS-Induced Preterm Birth in a Murine Model by Suppressing the Inflammatory Response.

Preterm birth accounts for the majority of perinatal mortality worldwide, and there remains no FDA-approved drug to prevent it. Recently, we discovered that the common drug excipient, N,N-dimethylacetamide (DMA), delays inflammation-induced preterm birth in mice by inhibiting NF-κB. Since we reported this finding, it has come to light that a group of widely used, structurally related aprotic solvents, including DMA, N-methyl-2-pyrrolidone (NMP) and dimethylformamide (DMF), have anti-inflammatory efficacy. We show here that DMF suppresses LPS-induced TNFα secretion from RAW 264.7 cells and IL-6 and IL-8 secretion from HTR-8 cells at concentrations that do not significantly affect cell viability. Like DMA, DMF protects IκBα from degradation and prevents the p65 subunit of NF-κB from translocating to the nucleus. In vivo, DMF decreases LPS-induced inflammatory cell infiltration and expression of TNFα and IL-6 in the placental labyrinth, all to near baseline levels. Finally, DMF decreases the rate of preterm birth in LPS-induced pregnant mice (P<.0001) and the rate at which pups are spontaneously aborted (P<.0001). In summary, DMF, a widely used solvent structurally related to DMA and NMP, delays LPS-induced preterm birth in a murine model without overt toxic effects. Re-purposing the DMA/DMF/NMP family of small molecules as anti-inflammatory drugs is a promising new approach to delaying or reducing the incidence of inflammation-induced preterm birth and potentially attenuating other inflammatory disorders as well.

Blueberry Polyphenols Increase Nitric Oxide and Attenuate Angiotensin II-Induced Oxidative Stress and Inflammatory Signaling in Human Aortic Endothelial Cells.

Accumulating evidence indicate that blueberries have anti-hypertensive properties, which may be mainly due to its rich polyphenol content and their high antioxidant capacity. Thus, we aimed to investigate the mechanisms by which blueberry polyphenols exert these effects. Human aortic endothelial cells (HAECs) were incubated with 200 µg/mL blueberry polyphenol extract (BPE) for 1 h prior to a 12 h treatment with angiotensin (Ang) II, a potent vasoconstrictor. Our results indicate that Ang II increased levels of superoxide anions and decreased NO levels in HAECs. These effects were attenuated by pre-treatment with BPE. Ang II increased the expression of the pro-oxidant enzyme NOX1, which was not attenuated by BPE. Pre-treatment with BPE attenuated the Ang II-induced increase in the phosphorylation of the redox-sensitive MAPK kinases, SAPK/JNK and p38. BPE increased the expression of the redox-transcription factor NRF2 as well as detoxifying and antioxidant enzymes it transcribes including HO-1, NQO1, and SOD1. We also show that BPE attenuates the Ang II-induced phosphorylation of the NF-κB p65 subunit. Further, we show that inhibition of NRF2 leads to a decrease in the expression of HO-1 and increased phosphorylation of the NF-κB p65 subunit in HAECs treated with BPE and Ang II. These findings indicate that BPE acts through a NRF2-dependent mechanism to reduce oxidative stress and increase NO levels in Ang II-treated HAECs.

Inhibitory effects of luteolin on TLR3-mediated inflammation caused by TAK/NF-κB signaling in human corneal fibroblasts.

To study the role of luteolin (LUT) in the expression of toll-like receptors 3 (TLR3) ligand polyI:C stimulated inflammatory factors in human corneal fibroblasts (HCFs). HCFs cells were cultivated with or without LUT or polyI:C. The expression levels of interleukin (IL)-6, IL-8, monocyte chemotactic protein-1 (MCP-1), vascular cell adhesion molecule (VCAM)-1, as well as intercellular adhesion molecule (ICAM)-1 were measured using enzyme-linked immunosorbent assay (ELISA), immunoblotting or reverse transcription-quantitative polymerase chain reaction (PCR) analyses. Immunoblotting was used to assess toll-interleukin-1 receptor-domain-containing adapter-inducing interferon-β (TRIF), TLR3, transforming growth factor-b-activated kinase 1 (TAK1), tumor necrosis factor receptor-associated factor 6 (TRAF6), the transcription factor AP-1, as well as transcription factor nuclear factor (NF-κB)-inhibitory protein IκB-α degradation and phosphorylation. Immunofluorescence assays were used to localize the cellular location of the p65 subunit of NF-κB. Corneal fibroblasts exposed to polyI:C demonstrated decreased VCAM-1, ICAM-1, MCP-1, IL-6, and IL-8 expression levels upon exposure to LUT in a time-dependent and concentration-dependent manner. LUT was observed to suppress polyI:C-triggered expression of TLR3, the translocation of NF-κB p65 into cell nuclei, as well as the phosphorylation of TAK, c-Jun, and IκB-α, while no impact on the expression levels of TRIF and TRAF6 were observed. LUT suppress the expression of proinflammatory adhesion molecules, chemokines, and cytokines in polyI:C exposed HCFs. These effects are likely mediated through TAK/NF-κB signal attenuation. Therefore, LUT is a candidate molecule that can prevent the TLR3-mediated inflammation response associated with corneal viral infection.

Protease-activated receptor 2 enhances innate and inflammatory mechanisms induced by lipopolysaccharide in macrophages from C57BL/6 mice.

This study was conducted to investigate the effects of the synthetic PAR2 agonist peptide (PAR2-AP) SLIGRL-NH2 on LPS-induced inflammatory mechanisms in peritoneal macrophages. Peritoneal macrophages obtained from C57BL/6 mice were incubated with PAR2-AP and/or LPS, and the phagocytosis of zymosan fluorescein isothiocyanate (FITC) particles; nitric oxide (NO), reactive oxygen species (ROS), and cytokine production; and inducible NO synthase (iNOS) expression in macrophages co-cultured with PAR-2-AP/LPS were evaluated. Co-incubation of macrophages with PAR2AP (30µM)/LPS (100ng/mL) enhanced LPS-induced phagocytosis; production of NO, ROS, and the pro-inflammatory cytokines interleukin (IL)-1β, tumour necrosis factor (TNF)-α, IL-6, and C-C motif chemokine ligand (CCL)2; and iNOS expression and impaired the release of the anti-inflammatory cytokine IL-10 after 4h of co-stimulation. In addition, PAR2AP increased the LPS-induced translocation of the p65 subunit of the pro-inflammatory transcription factor nuclear factor kappa B (NF-κB) and reduced the expression of inhibitor of NF-κB. This study provides evidence of a role for PAR2 in macrophage response triggered by LPS enhancing the phagocytic activity and NO, ROS, and cytokine production, resulting in the initial and adequate macrophage response required for their innate response mechanisms.

Nuclear factor kappa B signaling within the rat nucleus accumbens core sex-dependently regulates cue-induced cocaine seeking and matrix metalloproteinase-9 expression

Chronic drug self-administration and withdrawal are associated with distinct neuroimmune adaptations that may increase drug craving and relapse vulnerability in humans. The nuclear factor kappa-B (NF-κB) pathway is a critical regulator of many immune- and addiction-related genes such as the extracellular matrix enzyme matrix metalloproteinase-9 (MMP-9), which is a known modulator of learning, memory, and synaptic plasticity. While some studies suggest striatal NF-κB signaling may regulate drug-conditioned behavior, no studies to date have examined whether NF-κB signaling within the nucleus accumbens core (NAc core) alters downstream neuroimmune function and cue-motivated cocaine seeking following a period of forced abstinence, whether any effects are specific to cocaine over other reinforcers, or whether sex differences exist. Here, we examined whether viral-mediated knockdown of the p65 subunit of NF-κB within the NAc core would alter MMP-9 expression and cue-induced cocaine- and sucrose-seeking behavior following a period of forced abstinence in male and female rats. We demonstrate that NAc core p65 knockdown results in a significant decrease in cue-induced cocaine seeking in males but not females. This effect was specific to cocaine, as p65 knockdown did not significantly affect cue-induced sucrose seeking in either males or females. Moreover, we demonstrate that males express higher levels of MMP-9 within the NAc core and nucleus accumbens shell (NAcSh) compared to females, and that p65 knockdown significantly decreases MMP-9 in the NAc core of males but not females among cocaine cue-exposed animals. Altogether, these results suggest that NAc core NF-κB signaling exerts modulatory control over cue-motivated drug-seeking behavior and downstream neuroimmune function in a sex-specific manner. These findings highlight the need to consider sex as an important biological variable when examining immunomodulatory mechanisms of cocaine seeking.

Chondroprotective Effects of 4,5-Dicaffeoylquinic Acid in Osteoarthritis through NF-κB Signaling Inhibition.

Osteoarthritis (OA) is characterized by cartilage degradation, inflammation, and pain. The dicaffeoylquinic acid (diCQA) isomer, 4,5-diCQA, exhibits antioxidant activity and various other health-promoting benefits, but its chondroprotective effects have yet to be elucidated. In this study, we aimed to investigate the chondroprotective effects of 4,5-diCQA on OA both in vitro and in vivo. Primary rat chondrocytes were pre-treated with 4,5-diCQA for 1 h before stimulation with interleukin (IL)-1β (5 ng/mL). The accumulation of nitrite, PGE2, and aggrecan was observed using the Griess reagent and ELISA. The protein levels of iNOS, COX-2, MMP-3, MMP-13, ADMATS-4, MAPKs, and the NF-κB p65 subunit were measured by Western blotting. In vivo, the effects of 4,5-diCQA were evaluated for 2 weeks in a destabilization of the medial meniscus (DMM)-surgery-induced OA rat model. 4,5-diCQA significantly inhibited IL-1β-induced expression of nitrite, iNOS, PGE2, COX-2, MMP-3, MMP-13, and ADAMTS-4. 4,5-diCQA also decreased the IL-1β-induced degradation of aggrecan. It also suppressed the IL-1β-induced phosphorylation of MAPKs and translocation of the NF-κB p65 subunit to the nucleus. These findings indicate that 4,5-diCQA inhibits DMM-surgery-induced cartilage destruction and proteoglycan loss in vivo. 4,5-diCQA may be a potential therapeutic agent for the alleviation of OA progression. In this study, diclofenac was set to be administered once every two days, but it showed an effect on OA. These results may be used as basic data to suggest a new dosing method for diclofenac.

Treatment with Commelina communis Extract Exerts Anti-inflammatory Effects in Murine Macrophages via Modulation of the Nuclear Factor-κB Pathway.

The incidence of severe inflammatory diseases caused by chronic inflammation has increased owing to unprecedented changes brought about by industrialization. In this study, we aimed to assess the effect of treatment of lipopolysaccharide- (LPS-) induced murine macrophages with Commelina communis Linne extract (CCE) on synthesis of nitric oxide (NO), hypersecretion of proinflammatory cytokines, intranuclear transition of the p65 subunit of nuclear factor- (NF-) κB, and degradation of the NF-κB inhibitor IκBα. Notably, CCE treatment did not affect cell viability even at a final concentration of 1.5 mg/mL. At a high concentration of CCE, the LPS-induced high levels of NO, tumor necrosis factor-α, interleukin- (IL-) 1β, and IL-6 were decreased via downregulation of inducible NO synthase and proinflammatory cytokine mRNA expression. Furthermore, phosphorylation of IκBα was significantly decreased upon CCE treatment, and the intranuclear transition of NF-κB p65 triggered by LPS was inhibited at a high concentration of CCE. Polyphenols and flavonoids, secondary metabolites in CCE that regulate the NF-κB pathway, may be responsible for its anti-inflammatory activity. We suggest that CCE has anti-inflammatory effects related to suppression of the NF-κB pathway and can be used to treat chronic inflammation.

Biochemical and subcellular characterization of a squid hnRNPA/B-like protein 2 in osmotic stress activated cells reflects molecular properties conserved in this protein family.

We have identified endogenous p65 to be an SDS-stable dimer protein composed of ~ 37kDa hnRNPA/B-like subunits. We have investigated molecular properties involved in the stability of dimeric form, and their regulation in the transition between monomeric and dimeric forms of hnRNPA/B-like protein 2. We also investigated a cellular property conserved between squid hnRNPA/B-like protein 2 and human hnRNPA1 protein in a neuronal context. Here we show biochemical properties of a recombinant hnRNPA/B-like protein 2 (rP2) in vitro experiments, as one of p65 subunit. We found that interaction between rP2 and RNA molecules interfered with the dynamics of rP2 dimers formation, involved in disulfide bonds and/or postranslational alterations in distinct stage of SDS-stable dimers formation. In addition, we have performed immunofluorescence in SH-SY5Y cells and observed that the pEGFP-P2 fusion protein was expressed in the nucleus, similar to what is observed for human hnRNPA1 protein. Our results reinforce the idea that p65 is an SDS-stable dimer. Thus, a deeper understanding between monomeric and dimeric transition dynamic is critical into evolution of several neurodegenerative disease.

Beneficial effect of trimetazidine on folic acid-induced acute kidney injury in mice: Role of HIF-1α/HO-1.

Acute kidney injury (AKI) is a complex syndrome associated with a decrease in renal function and a significant impact on patient outcomes. Injection of folic acid (FA) in mice is used for studying the pathogenesis of AKI. This study investigated the impact of trimetazidine (a metabolic modulator-antianginal drug; TMZ), against FA-induced AKI.AKI was induced by FA (250 mg/kg, ip) in mice. Two doses of TMZ were administered orally for 10 days.Administration of TMZ at a high dose (20 mg/kg) exhibited significant decreases in the renal somatic index (RSI), serum levels of lactate dehydrogenase (LDH), creatinine (Cr), blood urea nitrogen (1), and proteins level in urine. Moreover, TMZ significantly increased creatinine clearance (CCr), serum albumin, urine creatinine, and urine urea levels. This improvement in markers of kidney damage was associated with marked renal antioxidant effects (↓NO and ↓lipid peroxidation, normalized reduced glutathione (GSH) level and superoxide dismutase (SOD) activity, and increased HIF-1α/HO-1 level). Furthermore, TMZ significantly decreased FA-induced expression of MPO and inflammatory cytokine IL-18, TNF-α, and NF-κB p65 subunit. Renal apoptosis, along with apoptotic markers, were enhanced by FA injection and suppressed by TMZ administration (↓Caspase-3, ↓Bax, and ↑Bcl2 expression). Finally, TMZ amended FA-induced histopathological changes in kidneys.By mitigating functional alteration, oxidative stress, and preventing the development of inflammatory and apoptosis signals, TMZ provides dose-dependent defense against FA-induced AKI mainly via stimulation of hypoxia-inducible factor-1 alpha (HIF-1α)/heme oxygenase-1 (HO-1) pathway.