Tumor Necrosis Factor Protein Research Articles

Transcriptomic responses of Caco-2 cells to Lactobacillus rhamnosus GG and Lactobacillus plantarum J26 against oxidative stress

Oxidative stress is the basic reason for aging and age-related diseases. In this study, we investigated the protective effect of 2 strains of lactic acid bacteria (LAB), Lactobacillus rhamnosus GG and L. plantarum J26, against oxidative stress in Caco-2 cells, and gave an overview of the mechanisms of lactic acid bacteria antioxidant activity using digital gene expression profiling. The 2 LAB strains provided significant protection against hydrogen peroxide (H2O2)-induced reduction in superoxide dismutase activity and increase in glutathione peroxidase activity in Caco-2 cells. However, inactive bacteria had little effect on alleviating oxidation stress in Caco-2 cells. Eight genes related to oxidative stress-FOSB, TNF, PPP1R15A, NUAK2, ATF3, TNFAIP3, EGR2, and FBN2-were significantly upregulated in H2O2-induced Caco-2 cells compared with untreated Caco-2 cells. After incubation of the H2O2-induced Caco-2 cells with L. rhamnosus GG and L. plantarum J26, 5 genes (TNF, EGR2, NUAK2, FBN2, and TNFAIP3) and 2 genes (NUAK2 and FBN2) were downregulated, respectively. In addition, the Kyoto Encyclopedia of Genes and Genomes indicated that some signaling pathways associated with inflammation, immune response, and apoptosis, such as Janus kinase/signal transducers and activators of transcription (Jak-STAT), mitogen-activated protein kinase (MAPK), nuclear factor-κB, and tumor necrosis factor, were all negatively modulated by the 2 strains, especially L. rhamnosus GG. In this paper, we reveal the mechanism of LAB in relieving oxidative stress and provide a theoretical basis for the rapid screening and evaluation of new LAB resources.

Inflammaging: Age and Systemic, Cellular, and Nuclear Inflammatory Biology in Older Adults.

Systemic inflammation is associated with increasing age. Yet, there are limited data about the association between age and systemic inflammation within older adults, and whether older age is also associated with cellular and nuclear signaling markers of inflammation. In community-dwelling older adults (N = 262, 60-88 years), systemic levels of C-reactive protein, interleukin-6, and soluble tumor necrosis factor receptor II; levels of toll-like receptor-4-stimulated monocytic production of interleukin-6 and tumor necrosis factor α; and resting nuclear levels of activated nuclear factor kappa B and signal transducer and activator of transcription (STAT1, STAT3, STAT5) were evaluated. Adjusting for demographic and clinical factors, multivariate linear regression tested the association between age and each inflammatory marker. Age was positively associated with increased levels of interleukin-6 and soluble tumor necrosis factor receptor II (p's < .05) and with increases in STAT1, STAT3, and STAT5 activation (p's < .05). However, no relationship was found between age and C-reactive protein, toll-like receptor-4-stimulated interleukin-6/tumor necrosis factor alpha α production, or nuclear factor kappa B. Within a community-dwelling sample of older adults, older age is associated with increases in STAT activation, along with increases of systemic inflammatory cytokines. In older adults, heterogeneity in age-related increases in inflammatory disease risk may be related to individual variability in inflammation.

The transcriptomic responses of the ark shell, Anadara broughtonii, to sulfide and hypoxia exposure.

Sulfide and hypoxia threaten marine organisms in various ways. Anadara broughtonii, a commercial marine bivalve in China which has great potential exposure to sulfide and hypoxia, was selected to test the responses to these stresses. Digital gene expression profile (DGE) analysis was performed on the juveniles' gills after exposed to normal condition (CG group), hypoxia (LO group), and low/high concentration of sulfide (LS/HS group, administered in hypoxia), respectively, using RNA-seq technology. A total of over 30 million clean reads were filtered from each DGE library and over 90% of them were annotated successfully. In total, 774 significant differentially expressed genes (DEGs) were detected and assigned to Gene ontology (GO) classification and KEGG Pathway enrichment analysis. The results show that many of the upregulated DEGs are related to hemoglobin, immunology, and stress responding. In the stressed A. broughtonii, cytochrome P450 and phosphoenolpyruvate carboxykinase may stimulate the glycolysis process to reduce oxygen consumption; Aminoacyl-tRNA synthetases, heat shock protein and protein disulfide isomerase probably help to maintain the genome integrity; Baculoviral IAP repeat-containing protein 2/3, mitogen-activated protein kinase and tumor necrosis factor pathways were probably responsible for protein repair, proteolysis, apoptosis and immune responses to high concentration of sulfide. Combined challenges also induced alternative oxidase and sushi repeat-containing protein, which have indistinct but probably indispensable function in invertebrates. For the first time, comprehensive transcriptome information on A. broughtonii in response to sulfide and hypoxia were provided. Our research offers new insights into the molecular mechanism behind the resistance of shellfish to sulfide and hypoxia.

Axonal Protection by Tacrolimus with Inhibition of NFATc1 in TNF-Induced Optic Nerve Degeneration

Tacrolimus, a calcineurin (CaN) inhibitor, has been used for treatment of refractory allergic ocular disease, although its role in optic nerve degeneration remains to be elucidated. In this study, we investigated whether tacrolimus modulates tumor necrosis factor (TNF)-mediated axonal degeneration and whether it alters nuclear factor of activated T cells (NFATc), a downstream effector of CaN signaling. Immunoblot analysis showed no significant difference in CaNAα protein levels in optic nerve on day 3, 7, or 14 after TNF injection compared with PBS injection. However, a significant increase in NFATc1 protein level was observed in optic nerve 7 days after TNF injection. This increase was negated by simultaneous administration of tacrolimus. Administration of tacrolimus alone did not change the NFATc1 protein level in comparison to that observed after PBS injection. A significant increase in TNF protein level was observed in optic nerve 14 days after TNF injection and this increase was prevented by tacrolimus. Immunohistochemical analysis showed the immunoreactivity of NFATc1 to be increased in optic nerve after TNF injection. This increased immunoreactivity was colocalized with glial fibrillary acidic protein and was suppressed by tacrolimus. Treatment of tacrolimus significantly ameliorated the TNF-mediated axonal loss. These results suggest that tacrolimus is neuroprotective against axon loss in TNF-induced optic neuropathy and that the effect arises from suppression of the CaN/NFATc1 pathway.

MicroRNA-146a-5p Downregulates the Expression of P-Glycoprotein in Rats with Lithium-Pilocarpine-Induced Status Epilepticus.

Increasing evidence supports that the efflux transporters, especially P-glycoprotein (P-gp), have vital roles on drug resistance in epilepsy. Overexpression of P-gp in the brain could reduce the anti-epileptic drugs (AEDs) concentration in the epileptogenic zone, resulting in drug resistance. Studies have demonstrated that recurrent seizures induce the expression of P-gp and status epilepticus (SE) could upregulate the expression of P-gp, resulting in drug resistance. MicroRNAs (miRNAs), as endogenous regulators, represent small regulatory RNA molecules that have been shown to act as negative regulators of gene expression in different biological processes. We investigated the impact of miR-146a-5p on the expression of P-gp in status epilepticus rat model. The expression of miR-146a-5p in rat cortex and hippocampus was measured by quantitative RT-PCR at 2 weeks after induction of SE. Meanwhile, we detected the expression of P-gp in the brain of SE rats using Western blotting and immunohistochemistry. Upregulation of miR-146a-5p and overexpression of P-gp were evident at 2 weeks after SE. Moreover, the expression of P-gp was downregulated by injection of miR-146a mimic into the hippocampus. We also detected the expression of interleukin-1 receptor-associated protein kinases-1 (IRAK1) and tumor necrosis factor receptor-associated factor 6 (TRAF6) and nuclear factor-kappaB (NF-κB) p65 using Western blotting and immunohistochemistry, which indicated the expression of IRAK1, TRAF6 and NF-κB p-p65/p65 increased in the brain of SE rats, and overexpression of miR-146a-5p could downregulate the expression of IRAK1, TRAF6, NF-κB p-p65/p65 and P-gp. Our study indicated that miR-146a-5p may decrease the expression of P-gp in status epilepticus rats via NF-κB signaling pathway.

Coffee consumption and plasma biomarkers of metabolic and inflammatory pathways in US health professionals

Coffee consumption has been linked to lower risk of various health outcomes. However, the biological pathways mediating the associations remain poorly understood. The aim of this study was to assess the association between coffee consumption and concentrations of plasma biomarkers in key metabolic and inflammatory pathways underlying common chronic diseases. We investigated the associations of total, caffeinated, and decaffeinated coffee consumption with 14 plasma biomarkers, including C-peptide, insulin-like growth factor 1 (IGF-1), IGF binding protein (IGFBP) 1, IGFBP-3, estrone, total and free estradiol, total and free testosterone, sex hormone-binding globulin (SHBG), total adiponectin, high-molecular-weight (HMW) adiponectin, leptin, C-reactive protein (CRP), interleukin 6 (IL-6), and soluble tumor necrosis factor receptor 2 (sTNFR-2). Data were derived from 2 cohorts of 15,551 women (Nurses' Health Study) and 7397 men (Health Professionals Follow-Up Study), who provided detailed dietary data before blood draw and were free of diabetes, cardiovascular disease, or cancer at the time of blood draw. Multivariable linear regression was used to calculate the percentage difference of biomarker concentrations comparing coffee drinkers with nondrinkers, after adjusting for a variety of demographic, clinical, and lifestyle factors. Compared with nondrinkers, participants who drank ≥4 cups of total coffee/d had lower concentrations of C-peptide (-8.7%), IGFBP-3 (-2.2%), estrone (-6.4%), total estradiol (-5.7%), free estradiol (-8.1%), leptin (-6.4%), CRP (-16.6%), IL-6 (-8.1%), and sTNFR-2 (-5.8%) and higher concentrations of SHBG (5.0%), total testosterone (7.3% in women and 5.3% in men), total adiponectin (9.3%), and HMW adiponectin (17.2%). The results were largely similar for caffeinated and decaffeinated coffee. Our data indicate that coffee consumption is associated with favorable profiles of numerous biomarkers in key metabolic and inflammatory pathways. This trial was registered at clinicaltrials.gov as NCT03419455.

Transcriptional Analysis of Immunohistochemically Defined Subgroups of Non-Muscle-Invasive Papillary High-Grade Upper Tract Urothelial Carcinoma.

Immunohistochemical (IHC) staining for CK5/6 and CK20 was reported to be correlated with the prognosis of early urothelial carcinoma in a way contrary to that of advanced tumors for unknown reasons. We aimed to characterize the gene expression profiles of subgroups of non-muscle-invasive papillary high-grade upper tract urothelial carcinoma (UTUC) classified by CK5/6 and CK20 expression levels: group 1 (CK5/6-high/CK20-low), group 2 (CK5/6-high/CK20-high), and group 3 (CK5/6-low/CK20-high). Expression of group 3 was predictive of worse prognosis of non-muscle-invasive papillary high-grade UTUC. Transcriptional analysis revealed 308 differentially expressed genes across the subgroups. Functional analyses of the genes identified cell adhesion as a common process differentially enriched in group 3 compared to the other groups, which could explain its high-risk phenotype. Late cell cycle/proliferation signatures were also enriched in group 3 and in some of the other groups, which may be used as a prognostic biomarker complementary to CK5/6 and CK20. Group 2, characterized by low levels of genes associated with mitogen-activated protein kinase and tumor necrosis factor signaling pathways, was hypothesized to represent the least cancerous subtype considering its normal urothelium-like IHC pattern. This study would facilitate the application of easily accessible prognostic biomarkers in practice.

Effects of early life stress on cocaine conditioning and AMPA receptor composition are sex-specific and driven by TNF

Exposure to early life adversity can predispose adolescents to the formation of substance abuse disorders. In rodents, early stressors such as repeated maternal separation (MS) impact AMPAR activity in the prefrontal cortex (PFC) and nucleus accumbens (NAc), regions involved in drug-cue association after cocaine-induced conditioned place preference (CPP). Notably, previous reports suggest that the pro-inflammatory cytokine tumor necrosis factor (TNF) regulates AMPAR subunit composition; increased TNF levels are reported to reduce GluA2-positive AMPARs. Since MS can elevate adolescent TNF levels, the stressor may therefore alter AMPAR subunit composition via neuroimmune signaling, thereby affecting cocaine-induced CPP. We tested the specific role of soluble TNF in MS-induced GluA2 loss and cocaine-induced CPP with biologic disruption of TNF signaling. TNF gene and protein expression were elevated in both PFC and NAc of MS males, but not females. GluA2 expression was reduced in both regions in only male MS rats, and systemic treatment with either ibudilast – a phosphodiesterase inhibitor, or XPro1595 – a blood-brain barrier-permeable blocker of soluble TNF – reversed such loss. MS males also formed greater preference for a cocaine-paired environment, the expression of which returned to control levels after XPro1595 administration. These data suggest a sex-specific mechanistic link between TNF signaling and changes in GluA2 expression and drug-cue conditioning, thereby providing further evidence for a role of MS and neuro-immune activity in cortical and striatal AMPAR changes. Moreover, manipulation of the TNF signaling pathway represents a novel approach for influencing response to reinforcing effects of drug use.

Aspergeillus Fumigatus: The Effects of Adiponectin on Inflammatory Cytokine Production

Background and Hypothesis: Although inflammatory cytokines are important for antifungal defenses, excessive production significantly increases host immune pathology. It is therefore important to identify host pathways that limit detrimental inflammation in invasive fungal infection. Our prior results showed that mice with invasive aspergillosis (IA) that were deficient in the metabolic cytokine production produced more of the cytokine Tumor Necrosis Factor (TNF) than alveolar in wild-type control mice. Therefore, we hypothesize that adiponectin inhibits antifungal cytokine secretion in alveolar macrophages. &#x0D; Experimental Design or Project Methods: To test this hypothesis, the commonly used A. fumigatus strain Af293 was purchased from the Fungal Genetics Stock Center and grown on and harvested from agar. The alveolar macrophage cell line MH-S and cytokine ELISA kits was obtained from MilliporeSigma and ThermoFisher, respectively. MH-S cells were stimulated with swollen, fixed Af293 conidia for 24 hours in the presence or absence of recombinant mouse adiponectin. After 24 hours, supernatants and cells was were collected and assayed for ILs 1a, 6, and TNF protein and mRNA , by ELISA and quantitative RT-PCR, respectively. &#x0D; Results: Although our preliminary results suggest possible inhibition of cytokine secretion by adiponectin in response to A. fumigatus, significant differences have thus far not been observed. &#x0D; Conclusion and Potential Impact: We are therefore currently optimizing our experimental conditions to improve antifungal cytokine secretion. These studies may ultimately assist in the discovery of novel therapeutic targets and improve the prognosis of A. fumigatus infections

Phytomodulatory proteins promote inhibition of hepatic glucose production and favor glycemic control via the AMPK pathway

Phytomodulatory proteins from the latex of the medicinal plant Calotropis procera has been shown to be implicated in many pharmacological properties. However there is no current information about their activity on glucose metabolism, although the latex is used in folk medicine for treating diabetes. In this study the phytomodulatory proteins (LP) from C. procera latex were assessed on glycemic homeostasis.Control animals received a single intravenous dose (5 mg/kg) of LP or saline solution (CTL). Four hours after treatment, the animals were euthanized and their livers were excised for analysis by western blot and RT-PCR AMP-activated protein kinase (AMPK), phosphoenolpyruvate carboxykinase (PEPCK) and tumor necrosis factor alpha (TNF-α). In vivo tests of intraperitoneal tolerance to insulin, glucose and pyruvate were also performed, and the effect of LP administration on fed glycemia was studied followed by blood analysis to determine serum insulin levels.Treatment with LP reduced glycemia two hours after glucose administration (LP: 87.2 ± 3.70 mg/dL versus CTL: 115.6 ± 8.73 mg/dL). However, there was no change in insulin secretion (CTL: 14.16 ± 0.68 mUI/mL and LP: 14.96 ± 0.55 mUI/mL). LP improved the insulin sensitivity, represented by a superior glucose decay constant during an insulin tolerance test (kITT) (4.17 ± 0.94%/min) compared to the CTL group (0.82 ± 0.72%/min), and also improved glucose tolerance at 30 min (105.2 ± 12.4 mg/dL versus 154.2 ± 18.51 mg/dL), while it decreased hepatic glucose production at 15 and 30 min (LP: 75.5 ± 9.31 and 52.5 ± 12.05 mg/dL compared to the CTL: 79.0 ± 3.02 and 84.5 ± 7.49 mg/dL). Furthermore, there was a significant inhibition of gene expression of PEPCK (LP: 0.66 ± 0.06 UA and CTL: 1.14 ± 0.22 UA) and an increase of phosphorylated AMPK (LP: 1.342 ± 0.21 UA versus CTL: 0.402 ± 0.09 UA). These findings confirm the effect of LP on glycemic control and suggest LP may be useful in diabetes treatment. However, the pharmacological mechanism of LP in PEPCK modulation still needs more clarification.

Ameliorative effects of a fusion protein dual targeting interleukin 17A and tumor necrosis factor α on imiquimod-induced psoriasis in mice.

In recent decades, biological agents such as tumor necrosis factor-α (TNF-α) inhibitors, have revolutionized the treatment of psoriasis. However, inhibition of a single cytokine may not achieve satisfactory therapeutic results. It is against this background that this research was undertaken to investigate the anti-psoriatic effect of a novel fusion protein (DTF) dual targeting TNF-α and interleukin-17 A (IL-17 A). Imiquimod (IMQ) was topically applied to the skin of mice to develop psoriasis-like skin and treated with etanercept or different doses of DTF. Results showed that DTF treatment (1 mg/kg, 3 mg/kg, 5 mg/kg) significantly attenuated IMQ-induced typical psoriasis-like inflammation, severity score, and epidermis thickening in a dose-dependent manner, and was again more efficient than etanercept (3 mg/kg) in alleviating all these parameters at the same dose. Furthermore, DTF was more potent than etanercept in suppressing the expression of inflammatory factors (IL-17 A, IL-6, IL-1β, IL-23, IL-22 and IL-12) in the serum, spleen and psoriasis-like skin compared with etanercept at the same dose. In addition, DTF was more efficient than etanercept in reducing the expression of keratins, decreasing the mRNA expression of Ly-6 G and Ly-6C, and enhancing the expression of filaggrin and caspase 14 in IMQ-induced psoriasis-like skin. We conclude that DTF alleviates IMQ-induced psoriasis by attenuating inflammatory cascades, reducing keratinocytes proliferation and improving epidermal barrier function through suppressing TNF-α and IL-17 A signal pathways. These data suggest that DTF has potential to be a novel therapeutic candidate for psoriasis.

Changes in gut microbiota and plasma inflammatory factors across the stages of colorectal tumorigenesis: a case-control study

BackgroundColorectal cancer (CRC) is a common malignant gastrointestinal tumor. In China, CRC is the 5th most commonly diagnosed cancer. The vast majority of CRC cases are sporadic and evolve with the adenoma-carcinoma sequence. There is mounting evidence indicating that gut microbiota and inflammation play important roles in the development of CRC although study results are not entirely consistent. In the current study, we investigated the changes in the CRC-associated bacteria and plasma inflammatory factors and their relationships based on data from a case-control study of Han Chinese. We included 130 initially diagnosed CRC patients, 88 advanced colorectal adenoma patients (A-CRA), 62 patients with benign intestinal polyps and 130 controls.ResultsFecal microbiota composition was obtained using 16S ribosomal DNA (16S rDNA) sequencing. PCOA analysis showed structural differences in microbiota among the four study groups (P = 0.001, Unweighted Unifrac). Twenty-four CRC-associated bacteria were selected by a two-step statistical method and significant correlations were observed within these microbes. CRC-associated bacteria were found to change with the degree of malignancy. Plasma C-reactive protein (CRP) and soluble tumor necrosis factor II (sTNFR-II) displayed significant differences among the four study groups and increased with adenoma-carcinoma sequence. The correlations of CRP and sTNFR-II with several CRC-associated microbes were also explored.ConclusionsCRC-associated species and plasma inflammatory factors tended to change along the adenoma-carcinoma sequence. Several CRC-associated bacteria were correlated with CRP and sTNFR-II. It is likely that gut microbiome and inflammation gradually form a microenvironment that is associated with CRC development.

Proposing novel TNFα direct inhibitor Scaffolds using fragment-docking based e-pharmacophore modeling and binary QSAR-based virtual screening protocols pipeline

Tumor necrosis factor alpha (TNFα) is a homotrimer protein that plays a pivotal role for critical immune functions, including infection, inflammation and antitumor responses. It also plays a primary role in autoimmune diseases like rheumatoid arthritis (RA). So far, only biological therapeutics like infliximab, etanercept, and adalimumab are available as treatment of inflammatory diseases. They directly bind to TNFα and interrupt its binding to its receptor protein tumor necrosis factor receptor (TNFR). However, they may also cause serious side effects such as activating an autoimmune anti-antibody response or the weakening of the body's immune defenses. Thus, small molecule-based therapies can be considered as alternative methods. In this study, a novel method is applied to develop energetically optimized, structure-based pharmacophore models for rapid in silico drug screening. Fragment-based docking results were used in the construction of an universal e-pharmacophore model development. The developed model is then used for screening of small-molecule library Specs-screening compounds (Specs-SC) which includes more than 200.000 drug-like molecules. In another approach, binary QSAR-based models were used to screen Specs-SC, as well as Specs-natural products (NP) which has around 750 compounds, and a library of drugs registered or approved for use in humans NIH's NCGC pharmaceutical collection (NPC) which has around 7500 molecules. The MetaCore/MetaDrug platform was used for binary QSAR models for therapeutic activity prediction as well as pharmacokinetic and toxicity profile predictions of screening molecules. This platform is constructed based on a manually curated database of molecular interactions, molecular pathways, gene-disease associations, chemical metabolism, and toxicity information. Molecular docking and molecular dynamics (MD) simulations were performed for the selected hit molecules. As target protein, both homodimer and homotrimer forms of TNFα were considered. The screening results showed that indinavir and medroxalol from NPC chemical library and a set of compounds (AT-057/43115940, AP-970/42897107, AK-968/41925665, AI-204/31679053, AN-648/41666950, AN-698/42006940) from Specs-SC database were identified as safe and active direct inhibitors of TNFα.

Characteristics of pulmonary function and pathological changes following acute lung injury in rats

Objective To investigate the characteristics of pulmonary function and pathological changes in rats with acute lung injury (ALI) and provide experimental basis for further study on the mechanism of ALI. Methods Twenty five male SD rats were randomly divided into the control group (n=5) and acute lung injury (ALI) group (n=20). Lipopolysaccharide (LPS) (4.5 mg/kg) were injected into the ALI group rats to establish the ALI rat model. The rats in control group were given 150 μl isotonic saline. At 12, 24, 48, and 72 hours after injury, lung function of the rats were tested by Buxco small animal lung function test system, including the dynamic lung compliance (Cdyn), forced vital capacity (FVC), functional residual gas (FRC), quasi static compliance (Cchord), 100th millisecond expiratory volume (FEV100), and airway resistance (RI). In addition, the bronchoalveolar lavage fluid (BALF) was collected for detection of protein level and tumor necrosis factor (TNF-alpha) concentration. At the same time, the changes of lung tissues were recorded, and the pathological changes were observed by HE staining. Results Compared with the control group, Cdyn, FVC, FRC, and FEV100 in ALI group were significantly decreased at each time point after injury (P 0.05). The protein level and TNF-alpha concentration of BALF in ALI group were increased significantly (P<0.05 or <0.01) 12-72 hours after injury (P<0.01). Compared with the control group, the whole lung was dark red in ALI group 12 hours after injury, and the most serious bleeding occurred in the pulmonary hilum area with single or multiple hemorrhagic foci of different sizes. Multiple punctate and focal bleeding of different sizes were seen on the lung surface, which were radially distributed around the pulmonary hilum. The color of lung tissue was gradually restored to normal at 72 hours after injury. Under the light microscope, pulmonary interstitial edema, inflammatory cell infiltration, pulmonary vascular congestion and focal pulmonary hemorrhage were observed 12 h after injury, showing typical ALI pathological changes. The pathological changes were the most significant at 24 hours and reduced obviously at 72 hours. Conclusions A single intratracheal injection of LPS can induce typical ALI pathological changes. There is a similar trend between the pulmonary function indexes, lung pathology characteristics, and the protein level of BALF and proinflammatory cytokine level, suggesting that the pulmonary function test parameters can provide reference for evaluation of ALI. Key words: Lipopolysaccharide; Acute lung injuries; Respiratory function tests; Tumor necrosis factor α

Plant-derived verbascoside and isoverbascoside regulate Toll-like receptor 2 and 4-driven neutrophils priming and activation

BackgroundNeutrophils have a short live in circulation and accelerate greatly local immune responses via increased granulopoiesis and migration at high numbers to infected or inflamed tissue. HypothesisSince neutrophils produce a variety of factors with destructive and pro-inflammatory potential the regulation of their homeostasis and functions might be eventually beneficial in inflammation-related pathological conditions. Herein we investigated the effect of natural-derived verbascoside (Verb) and its positional isomer isoverbascoside (IsoVerb) on neutrophil functions. MethodsWe used purified murine bone marrow (BM) neutrophils to study cell responsiveness to priming or activation via Toll-like receptors (TLRs) 2 and 4. The expression of CD11b, chemokine (CXC motif) receptor 2 (CXCR2), the intracellular level of phosphorylated p38 mitogen-activated protein kinase (MAPK) and tumor necrosis factor (TNF)-α in neutrophils were determined by flow cytometry while the release of macrophage inflammatory protein (MIP)-2 in culture supernatant was determined by enzyme-linked immunosorbent assay (ELISA). ResultsWe found that Verb appeared less powerful inhibitor of TLR2 and TLR4-mediated apoptosis than IsoVerb. However at concentrations below 16 µM and in LPS priming conditions Verb was more selective inhibitor of CD11b and CXCR2 expression than IsoVerb. Both compounds showed similar activity on integrin/chemokine receptor expression when neutrophils were stimulated with ZY or were activated with LPS. Verb sustained CXCR2 expression and turnover via regulation of the cell responsiveness to its ligand KC (CXCL1) and via the release of MIP-2 (CXCL2). Both Verb and IsoVerb increased TNF-α production and inhibited p38 phosphorylation in TNF-α+ cells. We fail to discriminate sharply between Verb's and IsoVerb's efficacy when studying p38 phosphorylation in LPS stimulated neutrophils. The multi-parametric analysis provides critical insight on the range of on-target effects of Verb and IsoVerb. ConclusionThe strength and selectivity of Verb and IsoVerb depended on the degree of activation and functional state of neutrophils, and both compounds are with potential to affect neutrophil-related pathologies/conditions in heterogenic populations.

Tumor Necrosis Factor Inhibition in the Acute Management of Traumatic Optic Neuropathy.

PurposeTo determine the effectiveness of etanercept, a tumor necrosis factor (TNF) inhibitor, in conferring neuroprotection to retinal ganglion cells (RGCs) and improving visual outcomes after optic nerve trauma with either optic nerve crush (ONC) or sonication-induced traumatic optic neuropathy (SI-TON) in mice.MethodsMouse optic nerves were unilaterally subjected to ONC (n = 20) or SI-TON (n = 20). TNF expression was evaluated by using immunohistochemistry and quantitative RT-PCR (qRT-PCR) in optic nerves harvested 6 and 24 hours post ONC (n = 10) and SI-TON (n = 10). Mice in each injury group received daily subcutaneous injections of either etanercept (10 mg/kg of body weight; five mice) or vehicle (five mice) for 7 days. Pattern electroretinograms were performed on all mice at 1 and 2 weeks after injury. ONC mice were killed at 2 weeks after injury, while SI-TON mice were euthanized at 4 weeks after injury. Whole retina flat-mounts were used for RGC quantification.ResultsImmunohistochemistry and qRT-PCR showed upregulation of TNF protein and gene expression within 24 hours after injury. In both models, etanercept use immediately following optic nerve injury led to higher RGC survival when compared to controls, which was comparable between the two models (24.23% in ONC versus 20.42% in SI-TON). In both models, 1 and 2 weeks post injury, mice treated with etanercept had significantly higher a-wave amplitudes than untreated injured controls.ConclusionsTreatment with etanercept significantly reduced retinal damage and improved visual function in both animal models of TON. These findings suggest that reducing TNF activity in injured optic nerves constitutes an effective therapeutic approach in an acute setting.

Transcriptome sequencing analysis reveals the effect of combinative treatment with low‑intensity pulsed ultrasound and magnesium ions on hFOB1.19 human osteoblast cells.

Biodegradable magnesium (Mg) materials are considered ideal as osteosynthesis implants. However, clinical application has proven complex. This is primarily associated with the issue of reducing the extent of implant degradation to a range acceptable for the human body, while simultaneously enhancing osteogenesis or osteoinduction. In the present study, a combination of Mg ions and low-intensity pulsed ultrasound (LIPUS) treatment was applied in hFOB 1.19 human osteoblast cells as a potential strategy to resolve this issue. A total of 7,314 differentially expressed genes (DEGs) and 826 shared DEGs in hFOB1.19 osteoblast cells were identified by microarray analysis following treatment with Mg and/or LIPUS. Gene Ontology analysis demonstrated that among cells treated with a combination of Mg and LIPUS, DEGs were significantly enriched in various functional annotations, including ‘wound healing’, ‘transforming growth factor beta receptor signaling pathway’, ‘transcription, DNA-templated’, ‘receptor complex’, ‘nucleus’, ‘SMAD protein complex’, ‘DNA binding’, ‘metal ion binding’ and ‘GTPase activator activity’. Notably, the transforming growth factor (TGF)-β, mitogen-activated protein kinase (MAPK) and tumor necrosis factor (TNF) signaling pathways were preferentially overrepresented in the Mg and LIPUS combination group, which was subsequently confirmed by reverse transcription-quantitative polymerase chain reaction. Furthermore, genes involved in osteoblast mineralization promotion, including bone morphogenetic protein 6, noggin, bone morphogenetic protein receptor (BMPR)1A, BMPR2 and SMAD 5/8, were significantly upregulated following combination treatment compared with the control group. Genes involved in the promotion of migration, including c-Jun N-terminal kinase, doublecortin, paxillin and Jun proto-oncogene AP-1 transcription factor subunit, were also upregulated in the combination treatment group compared with the control group. The DEG data were supported by morphological observations of the osteoblasts using alizarin red S staining and wound healing assays, which indicated that Mg and LIPUS combinative treatment had a synergistic effect on osteoblast mineralization and migration. Additionally, the combined treatment significantly upregulated metal transporter genes associated with Mg entry, including ATPase Na+/K+-transporting subunit α1, cyclin and CBS domain divalent metal cation transport mediator 2, K+ voltage-gated channel subfamily J member 14, transient receptor potential cation channel (TRP) subfamily M member 7 and TRP subfamily V member 2. In summary, the findings of the present study revealed that combined stimulation with Mg and LIPUS may exhibit a synergistic effect on human osteoblast bone formation through the TGF-β, MAPK and TNF signaling pathways, while also facilitating Mg influx. The present study demonstrated the potential of combinative LIPUS and Mg treatment as a novel therapeutic strategy for enhancing the osteoinduction, biocompatibility and biosafety of biodegradable Mg implants.

Lack of Evidence for a Direct Interaction of Progranulin and Tumor Necrosis Factor Receptor-1 and Tumor Necrosis Factor Receptor-2 From Cellular Binding Studies.

Progranulin (PGRN) is a secreted anti-inflammatory protein which can be processed by neutrophil proteases to various granulins. It has been reported that at least a significant portion of the anti-inflammatory effects of PGRN is due to direct high affinity binding to tumor necrosis factor receptor-1 (TNFR1) and TNFR2 and inhibition of tumor necrosis factor (TNF)-induced TNFR1/2 signaling. Two studies failed to reproduce the interaction of TNFR1 and TNFR2 with PGRN, but follow up reports speculated that this was due to varying experimental circumstances and/or the use of PGRN from different sources. However, even under consideration of these speculations, there is still a striking discrepancy in the literature between the concentrations of PGRN needed to inhibit TNF signaling and the concentrations required to block TNF binding to TNFR1 and TNFR2. While signaling events induced by 0.2–2 nM of TNF have been efficiently inhibited by low, near to equimolar concentrations (0.5–2.5 nM) of PGRN in various studies, the reported inhibitory effects of PGRN on TNF-binding to TNFR1/2 required a huge excess of PGRN (100–1,000-fold). Therefore, we investigated the effect of PGRN on TNF binding to TNFR1 and TNFR2 in highly sensitive cellular binding studies. Unlabeled TNF inhibited >95% of the specific binding of a Gaussia princeps luciferase (GpL) fusion protein of TNF to TNFR1 and TNFR2 and blocked binding of soluble GpL fusion proteins of TNFR1 and TNFR2 to membrane TNF expressing cells to >95%, too. Purified PGRN, however, showed in both assays no effect on TNF–TNFR1/2 interaction even when applied in huge excess. To rule out that tags and purification- or storage-related effects compromise the potential ability of PGRN to bind TNF receptors, we directly co-expressed PGRN, and as control TNF, in TNFR1- and TNFR2-expressing cells and looked for binding of GpL-TNF. While expression of TNF strongly inhibited binding of GpL-TNF to TNFR1/2, co-expression of PGRN had not effect on the ability of the TNFR1/2-expressing cells to bind TNF.

Niacin action in the atherogenic mixed dyslipidemia of metabolic syndrome: Insights from metabolic biomarker profiling and network analysis

Niacin as an adjunct to statin treatment to reduce cardiovascular risk is questioned. To evaluate interrelationships between the effects of niacin on mixed dyslipidemia and a spectrum of metabolic and inflammatory biomarkers. Obese, nondiabetic, hypertriglyceridemic males (n=19) with low high-density lipoprotein-cholesterol levels received extended-release nicotinic acid for 8weeks. Multiple biomarkers were measured using enzyme-linked immunosorbent assay, enzymatic/absorptiometric, or multiplex biochip assays. Treatment effects were determined for each variable and a differential correlation network created on the basis of univariate correlations between baseline and response to niacin treatment for all pairs of variables. Extended-release niacin treatment favoured normalization of plasma lipid and apolipoprotein profile. Plasma markers of inflammation, hepatic function, cellular adhesion and proliferation, and macrophage phenotype were attenuated; however, insulin resistance increased. Differential network analysis revealed that changes in triglycerides and high-density lipoprotein-cholesterol were closely linked; equally, niacin mediated reductions in total cholesterol, apolipoprotein B, low-density lipoprotein-cholesterol and lipoprotein(a) clustered together, as did homeostatic model assessment of insulin resistance, insulin, and interleukin-6 levels. Two clusters of inflammatory markers were identified, involving (1) intercellular adhesion molecule 1 and high-sensitive C-reactive protein and (2) soluble tumor necrosis factor receptors; and novel clusters involving matrix metallopeptidase 9 and apolipoprotein E, and adiponectin and cystatin C, respectively, were equally revealed. At lower stringency, lipid and insulin resistance clusters were linked; a C-reactive protein-centered cluster linked reduction in apolipoprotein CIII to intercellular adhesion molecule 1, gamma-glutamyltransferase, soluble tumor necrosis factor receptors, and E-selectin. A niacin-mediated trend to normalize atherogenic mixed dyslipidemia was intimately linked to attenuation of biomarkers of inflammation, cell adhesion, hepatic dysfunction and cell proliferation, but to enhanced insulin resistance and plasma homocysteine elevation.

Therapeutic efficacy of liraglutide on diabetic nephropathy mice by inhibiting inflammatory factors

This study was to investigate the role and mechanism of liraglutide in the treatment of diabetic nephropathy (DN) mice. A mouse model of streptozotocin-induced DN was established. The mice were intraperitoneally injected with liraglutide at a dose of 200 μg/kg for 6 weeks. The expression of interleukin-6 (IL-6), tumor necrosis factor (TNF), and nuclear factor kappa B (NF-κB) messenger RNA (mRNA) in renal tissue of mice was examined by real-time quantitative polymerase chain reaction (PCR). Meanwhile, the expression of IL-6 and TNF protein in renal tissue of mice was detected by western blot, while the expression of NF-κB protein in renal tissues of each group was detected by immunofluorescence. After 6 weeks of intervention, the blood glucose (GLU), total cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL), and weight of the liraglutide group were significantly lower than those of the DN group ( P &lt; 0.01 or P &lt; 0.05), whereas high-density lipoprotein (HDL) was significantly increased ( P &lt; 0.05). At the same time, the microscale albuminuria (MAU) and N-acetyl-β-d-glucosaminidase (NAG) in the liraglutide group were significantly lower than those in the DN group ( P &lt; 0.05). Moreover, the urea (UR), creatinine (CR), and uric acid (UA) in the liraglutide group were significantly lower than those in the DN group ( P &lt; 0.01 or P &lt; 0.05). In addition, the mRNA and proteins of IL-6, TNF, and NF-κB in the liraglutide group were significantly lower than those in the DN group ( P &lt; 0.05). In conclusion, the mechanism of liraglutide in the treatment of DN may be related to the inhibition of the expression of genes and proteins of inflammatory factors.