Tumor Necrosis Factor-α Synthesis Research Articles

The role of regulatory micro-RNAs in inflammatory processes and production of tumor necrosis factor-alpha in patients with rheumatoid arthritis

Background. Micro-RNAs are fundamental agents of post-transcriptional control of gene expression. In recent years many works have appeared on the possible role of micro-RNAs in rheumatoid arthritis (RA). Studies of the role of micro-RNA and the relationship with the synthesis of tumor necrosis factor-α (TNF-α) are very promising for understanding the pathogenesis and treatment of RA and other autoimmune diseases. The purpose of the research was to study the role of regulatory micro-RNAs in inflammatory processes and the possible connection with the production of TNF-α in patients with RA. Materials and methods. 29 patients with active RA and 20 healthy individuals (control) were examined. All subjects were examined for 16 micro-RNAs. The choice of micro-RNA was based on previous studies and theoretical conclusions (according to the miRWalk database). Rheumatoid factor, the level of antibodies to cyclic peptides containing citrulline, C-reactive protein (СRP), levels of TNF-α (serum, spontaneous, and stimulated) were determined in the blood of patients. Results. Statistical analysis demonstrated significant overexpression of miR-221, miR-203, miR-146b, miR-132, miR-21 and miR-17-3p and inhibition of miR-223 synthesis in RA patients. The activation of TNF-α synthesis at rest and the increased production of TNF-α by mononuclear cells after stimulation in RA were shown. Differences in the levels of relative expression of some micro-RNAs between seropositive and seronegative groups of RA patients were found, but only hyperexpression of miR-155 was highly reliable. For the first time, a possible relationship between TNF-α production and miR-29 and miR-155 micro-RNAs, as well as a correlation between miR-16, miR-99b and miR-203 and CRP levels, was revealed. Conclusions. The obtained data on the profile of micro-RNAs in RA makes it possible to distinguish the most “interesting” micro-RNAs for further study of pathogenesis, their role in inflammation, to study the choice of TNF-α inhibitors, and predicting the effectiveness of that treatment.

Jujuboside B Inhibited High Mobility Group Box Protein 1-Mediated Severe Inflammatory Responses in Human Endothelial Cells and Mice.

High mobility group box protein 1 (HMGB1) is a biomolecule that acts as an alerting signal of late sepsis by accelerating the production of proinflammatory cytokines, and eventually leads to various inflammation-related symptoms. When released into plasma at high concentration, it disrupts precise diagnosis and prognosis and worsens the survival of patients with systemic inflammatory conditions. Jujuboside B (JB) is a natural compound pressed from the seed of Zizyphi Spinosi Semen, which is known for its medical efficacies in treating various conditions such as hyperlipidemia, hypoxia, and platelet aggregation. Nevertheless, the medicinal activity of JB on HMGB1-involved inflammatory response in vascular cells in the human body is still ambiguous. Therefore, we hypothesized that JB could regulate the lipopolysaccharide (LPS)-induced dynamics of HMGB1 and its mediated cascade in inflammatory responses in human umbilical vein endothelial cells (HUVECs). In this experiment, JB and HMGB1 were administered in that order. In vitro and in vivo permeability, and cell viability, adhesion, and excavation of leukocytes, development of cell adhesion molecules, and lastly production of proinflammatory substances were investigated on human endothelial cells and mouse disease models to investigate the efficacy of JB in inflammatory condition. JB substantially blocked the translocation of HMGB1 from HUVECs and controlled HMGB1-induced adhesion and extravasation of the neutrophils through LPS-treated HUVECs. Moreover, JB decreased the formation of HMGB1 receptors and continually prevented HMGB1-induced proinflammatory mechanisms by blocking transcription of nuclear factor-κB and synthesis of tumor necrosis factor-α. In conclusion, JB demonstrated preventive effects against inflammatory pathologies and showed the potential to be a candidate substance for various inflammatory diseases by regulating HMGB1-mediated cellular signaling.

Hericium erinaceus Mycelium Ameliorates In Vivo Progression of Osteoarthritis

Osteoarthritis (OA) is an age-related disorder that affects the joints and causes functional disability. Hericium erinaceus is a large edible mushroom with several known medicinal functions. However, the therapeutic effects of H. erinaceus in OA are unknown. In this study, data from Sprague-Dawley rats with knee OA induced by anterior cruciate ligament transection (ACLT) indicated that H. erinaceus mycelium improves ACLT-induced weight-bearing asymmetry and minimizes pain. ACLT-induced increases in articular cartilage degradation and bone erosion were significantly reduced by treatment with H. erinaceus mycelium. In addition, H. erinaceus mycelium reduced the synthesis of proinflammatory cytokines interleukin-1β and tumor necrosis factor-α in OA cartilage and synovium. H. erinaceus mycelium shows promise as a functional food in the treatment of OA.

Captopril reduces lung inflammation and accelerated senescence in response to thoracic radiation in mice.

ABSTRACTThe lung is sensitive to radiation and exhibits several phases of injury, with an initial phase of radiation-induced pneumonitis followed by delayed and irreversible fibrosis. The angiotensin-converting enzyme inhibitor captopril has been demonstrated to mitigate radiation lung injury and to improve survival in animal models of thoracic irradiation, but the mechanism remains poorly understood. Here we investigated the effect of captopril on early inflammatory events in the lung in female CBA/J mice exposed to thoracic X-ray irradiation of 17–17.9 Gy (0.5–0.745 Gy min–1). For whole-body + thoracic irradiation, mice were exposed to 7.5 Gy (0.6 Gy min–1) total-body 60Co irradiation and 9.5 Gy thoracic irradiation. Captopril was administered orally (110 mg kg–1 day–1) in the drinking water, initiated 4 h through to150 days post-irradiation. Captopril treatment increased survival from thoracic irradiation to 75% at 150 days compared with 0% survival in vehicle-treated animals. Survival was characterized by a significant decrease in radiation-induced pneumonitis and fibrosis. Investigation of early inflammatory events showed that captopril significantly attenuated macrophage accumulation and decreased the synthesis of radiation-induced interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) pro-inflammatory cytokines in the lungs of irradiated mice. Suppression of IL-1β and TNF-α correlated with an increase of the anti-inflammatory cytokine IL-10 in the spleen with captopril treatment. We also found that captopril decreased markers for radiation-induced accelerated senescence in the lung tissue. Our data suggest that suppression of inflammation and senescence markers, combined with an increase of anti-inflammatory factors, are a part of the mechanism for captopril-induced survival in thoracic irradiated mice.

Anti-inflammatory effects of rhaponticin on LPS-induced human endothelial cells through inhibition of MAPK/NF-κβ signaling pathways.

The untreated systemic chronic inflammation leads to autoimmune diseases, hyperglycemia, cardiovascular diseases, type 2 diabetes, hypertension, osteoporosis, and so on. Phytochemicals effectively inhibitthe inflammation, and numerous studies have proved that the phytocomponents possessanti-inflammatory property via inhibiting the cyclooxygenase and lipoxygenase signaling pathways. Rhaponticin is one such phytochemical obtained from the perennial plant Rheum rhaponticum L. belonging to Polygonaceae family. We assessed the anti-inflammatory potency of rhaponticin in endothelial cells induced with lipopolysaccharides (LPS). Four different endothelial cells induced with LPS were treated with rhaponticin and assessed for the nitric oxide generation. The cytotoxic potency of rhaponticin was evaluated in endothelial cells using the3-(4,5-dimethylthizaol-2yl)-2,5-diphenyl tetrazolium bromide assay. The tumor necrosis factor-α (TNF-α) synthesis was quantified using the commercially available assay kit. The inflammatory signaling protein gene expression of TNF-α, inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX2), and interleukin-1β (IL-1β) was analyzed with quantitative polymerase chain reaction (PCR) analysis. The gene expression of NADPH oxidase (NOX) cytoplasmic catalytic subunits gp91phox , p47phox , and p22phox was assessed with real-time PCR analysis. Finally, to confirm the anti-inflammatory potency of rhaponticin, the nuclear factor kappa B (NFκB) and mitogen-activated protein kinase (MAPK) signaling protein expressionwas analyzed with immunoblotting analysis. Rhaponticin treatment significantly decreased the levels of nitric oxide and TNF-α synthesis in LPS-induced endothelial cells. It significantly decreased the gene expression of inflammatory proteins and NOX signaling protein. The protein expression of NFκB and MAPK signaling proteins was drastically decreased in rhaponticin-treated endothelial cells induced with LPS. Overall, our results confirm that rhaponticin effectively inhibited the inflammation triggered by LPS in endothelial cells via downregulating iNOS, COX2, and NFκB and MAPK signaling pathways.

Slit2 Regulates Hyaluronan & Cytokine Synthesis in Fibrocytes: Potential Relevance to Thyroid-Associated Ophthalmopathy.

CD34+ fibrocytes have been implicated in development of thyroid-associated ophthalmopathy (TAO), a consequential autoimmune manifestation of Graves disease (GD). In TAO, CD34+ fibrocytes appear to masquerade as CD34+ orbital fibroblasts mixed with CD34- OF (collectively, GD-OF). Slit2, an axon guidance glycoprotein, is expressed by CD34- OF and attenuates GD-OF gene expression. Cardinal features of TAO include hyaluronan (HA) accumulation and cytokine-driven inflammation. Compare expression of HA synthase isoenzymes (HAS1-3), UDP-glucose dehydrogenase (UGDH), synthesis of HA, interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) in fibrocytes and GD-OF. Determine whether Slit2 alters gene expression patterns. Patients with TAO and healthy donors were recruited from an academic practice. Real-time polymerase chain reaction, HA, IL-6, and TNF-α immunoassays. HA synthesis and release from fibrocytes is substantially lower than in GD-OF. HAS1 expression dominates in fibrocytes while HAS2 in GD-OF. In contrast, HAS2 and UGDH expression dominate GD-OF and localize to CD34- OF. Recombinant human Slit2 (rhSlit2) substantially upregulates HA synthesis and HAS2 expression in fibrocytes but attenuates IL-6 and TNF-α production in these cells. In contrast, knocking down Slit2 in GD-OF reduces HA synthesis and HAS2 and UGDH expression while upregulating IL-6 and TNF-α. The dramatic differences in HA, IL-6, and TNF-α production, and HAS and UGDH expression found in fibrocytes and GD-OF appear, at least in part, to be attributable to Slit2. These findings provide novel insight into the differences in gene expression exhibited by CD34+ fibrocytes and CD34+ OF and therefore reveal important aspects of disease pathogenesis.

N-acetyl cysteine inhibits lipopolysaccharide-mediated synthesis of interleukin-1β and tumor necrosis factor-α in human periodontal ligament fibroblast cells through nuclear factor-kappa B signaling.

The aim of this study was to investigate the role of n-acetyl cysteine (NAC) in the lipopolysaccharide (LPS)-mediated induction of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) synthesis by human periodontal ligament fibroblast cells (hPDLFs). In addition, we aimed to determine the involvement of the nuclear factor-kappa B (NF-κB) pathway in any changes in IL-1β and TNF-α expression observed in response to LPS and NAC. HPDLFs were obtained by primary culture. The culture medium used in this experiment was Dulbecco's Modified Eagle Medium (DMEM low-glucose). Cells were stimulated with various concentrations of NAC or LPS. Cell proliferation was measured at various time-points with the cell Counting Kit 8 (CCK-8) assay. mRNA levels of IL-1β and TNF-α were determined by real-time quantitative polymerase chain reaction (RT-qPCR) analysis. Protein levels of IL-1β and TNF-α were measured by enzyme-linked immunosorbent assay (ELISA). Protein and mRNA expression levels of NF-κB were measured by western blot and RT-qPCR. The results showed that LPS treatment in hPDLFs induced mRNA and protein expression of IL-1β, TNF-α, and NF-κB. However, these effects were eliminated by pretreatment with NAC. Pretreatment with both NAC (1 mmol/L) and BAY11-7082 (10 μmol/L) significantly inhibited the NF-κB activity induced by LPS. NAC inhibits the LPS-mediated synthesis of tumor TNF-α and IL-1β in hPDLFs, through the NF-κB pathway.

CDDO-Me Attenuates Vasogenic Edema and Astroglial Death by Regulating NF-κB p65 Phosphorylations and Nrf2 Expression Following Status Epilepticus.

2-Cyano-3,12-dioxo-oleana-1,9(11)-dien-28-oic acid methyl ester (CDDO-Me) is a triterpenoid analogue of oleanolic acid that has anti-inflammatory, antioxidant, and neuroprotective activities. In the present study, we evaluate the effects of CDDO-Me on serum extravasation and astroglial death in the rat piriform cortex (PC) induced by status epilepticus (a prolonged seizure activity, SE) in order to propose an underlying pharmacological mechanism of CDDO-Me and its availability for treatment of vasogenic edema. CDDO-Me effectively mitigated serum extravasation and a massive astroglial loss in the PC following SE. CDDO-Me abrogated tumor necrosis factor-α (TNF-α) synthesis in activated microglia by inhibiting nuclear factor-κB (NF-κB) p65 serine 276 phosphorylation. CDDO-Me also abolished NF-κB threonine 435 phosphorylation in endothelial cells and TNF-α-mediated-phosphatidylinositol-3-kinase (PI3K)/AKT/endothelial nitric oxide synthase (eNOS) signaling cascades, which trigger vasogenic edema following SE. Furthermore, CDDO-Me increased astroglial viability via the up-regulation of nuclear factor-erythroid 2-related factor 2 (Nrf2) expression. Therefore, our findings suggest that CDDO-Me may ameliorate SE-induced vasogenic edema formation by regulating NF-κB p65 phosphorylations in microglia as well as endothelial cells and enhancing Nrf2 expression in astrocytes, respectively.

Alpha-naphthoflavone attenuates non-alcoholic fatty liver disease in oleic acid-treated HepG2 hepatocytes and in high fat diet-fed mice

Non-alcoholic fatty liver disease (NAFLD) is a chronic liver disease. The literature suggests that the aryl hydrocarbon receptor (AHR) may be a key player in the pathogenesis of NAFLD, and it can modulate the synthesis of cytochrome P450 1A1 (CYP1A1) and tumor necrosis factor-α (TNF-α). Previous studies have shown that CYP1A1 is a key enzyme of oxidative stress, TNF-α is involved in the formation of insulin resistance (IR), oxidative stress and insulin resistance are the key factors for the formation of NAFLD. Therefore, it can be said that AHR may participate in contributing to NAFLD by regulating CYP1A1 and TNF-α. Alpha-naphthoflavone (ANF) is an effective AHR inhibitor. The present study was designed to explore the hepatoprotective effect of ANF in high fat diet (HFD)-induced NAFLD mice and oleic acid (OA)-treated HepG2 hepatocytes. Mice were fed HFD to induce NAFLD, HepG2 cells were exposed to OA to induce hepatocyte injury, and ANF significantly reduced mouse and cellular liver damage compared to the HFD-induced NAFLD and OA-treated HepG2 hepatocytes. ANF treatment reduces liver damage by reducing ROS and IR, the data show that ANF inhibits the expression of AHR, CYP1A1 and TNF-α in NAFLD. Taken together, these findings show that ANF alleviate NAFLD via regulation of AHR/CYP1A1 and AHR/TNF-α pathways, which may have potential for further development as novel therapeutic agents for NAFLD.

Demethylation of MicroRNA-124a Genes Attenuated Proliferation of Rheumatoid Arthritis Derived Fibroblast-Like Synoviocytes and Synthesis of Tumor Necrosis Factor-α.

ObjectiveTo examine the impact of 5-Aza-2ʹ-deoxycytidine (5-AzadC) on methylation status of miR-124a genes in rheumatoid arthritis (RA) associated fibroblast-like synoviocytes (FLS) and its effect on RA-FLS proliferation and TNF-α expression.Materials and MethodsFLS were isolated from seven RA-derived synovial tissues and cultured in vitro. The expression of miR-124a was measured by real time quantitative polymerase chain reaction (PCR) in FLS with or without 5-AzadC treatment. MiR-124a gene methylation was detected by methylation-specific PCR. FLS were divided into three groups as control, IL-1β and IL-1β/5-AzadC, respectively. The cells in the IL-1β group were treated with 5 μg/L of IL-1β for 24 hours, whereas the cells in the IL-1β/5-AzadC group were first treated with IL-1β exactly as those in the IL-1β group for 24 h but further treated with 1μM 5-AzadC for additional 3 days. The cell growth was estimated based on absorbance at UV450nm. Secreted TNF-α from the cells was evaluated by enzyme-linked immunosorbent assay. After that, RA-FLS treated with IL-1β plus 5-AzadC were further transfected with miR-124a inhibitor or scrambled control. After culturing for 3 days, cell growth and TNF-α concentrations were measured.ResultsAfter 5-AzadC treatment, the expression of miR-124a was significantly increased compared with the control group (1.545 ± 0.189 vs 0.836 ± 0.166, p = 0.001). On the other hand, 5-AzadC significantly reduced IL-1β-mediated cell proliferation by nearly 2.5 fold (p = 0.006). Also, the level of TNF-α secreted from the cells treated with IL-1β plus 5-AzadC was considerably less than that from the cells treated with IL-1β alone (324.99 ± 22.73 ng/L vs 387.91 ± 58.51 ng/L, p = 0.022). After transfection with miR-124a inhibitor in RA-FLS treated with IL-1β plus 5-AzadC, the cell proliferation was increased by 18.2% and the TNF-α expression was increased by 19.0% (p = 0.001 and 0.011, respectively).ConclusionMethylation of miR-124a genes contributed to IL-1β-mediated RA-FLS proliferation and TNF-α expression.

Role of interleukin-1 receptor 1/MyD88 signalling in the development and progression of pulmonary hypertension.

Pulmonary artery smooth muscle cell (PA-SMC) proliferation and inflammation are key components of pulmonary arterial hypertension (PAH). Interleukin (IL)-1β binds to IL-1 receptor (R)1, thereby recruiting the molecular adaptor myeloid differentiation primary response protein 88 (MyD88) (involved in IL-1R1 and Toll-like receptor signal transduction) and inducing IL-1, IL-6 and tumour necrosis factor-α synthesis through nuclear factor-κB activation.We investigated the IL-1R1/MyD88 pathway in the pathogenesis of pulmonary hypertension.Marked IL-1R1 and MyD88 expression with predominant PA-SMC immunostaining was found in lungs from patients with idiopathic PAH, mice with hypoxia-induced pulmonary hypertension and SM22-5-HTT(+) mice. Elevations in lung IL-1β, IL-1R1, MyD88 and IL-6 preceded pulmonary hypertension in hypoxic mice. IL-1R1(-/-), MyD88(-/-) and control mice given the IL-1R1 antagonist anakinra were protected similarly against hypoxic pulmonary hypertension and perivascular macrophage recruitment. Anakinra reversed pulmonary hypertension partially in SM22-5-HTT(+) mice and markedly in monocrotaline-treated rats. IL-1β-mediated stimulation of mouse PA-SMC growth was abolished by anakinra and absent in IL-1R1(-/-) and MyD88(-/-) mice. Gene deletion confined to the myeloid lineage (M.lys-Cre MyD88(fl/fl) mice) decreased pulmonary hypertension severity versus controls, suggesting IL-1β-mediated effects on PA-SMCs and macrophages. The growth-promoting effect of media conditioned by M1 or M2 macrophages from M.lys-Cre MyD88(fl/fl) mice was attenuated.Pulmonary vessel remodelling and inflammation during pulmonary hypertension require IL-1R1/MyD88 signalling. Targeting the IL-1β/IL-1R1 pathway may hold promise for treating human PAH.

Synthesis, Anti-inflammatory Activities and Mechanisms of 3,5- dihydroxycinnamic Acid Derivatives.

3,4-dihydroxycinnamic acid and its derivatives exhibit numerous biologic activities. Such activities have not previously been reported for 3,5-dihydroxycinnamic acid derivatives. In this study, ten derivatives of 3,5- dihydroxycinnamic acid were synthesized and their anti-inflammatory activities were tested in 12-O-tetradecanoylphorbol 13-acetate-induced mouse ear edema. Molecular biological studies have shed lights on their anti-inflammatory mechanism. Anti-inflammatory activities of ten new synthesized derivatives of 3,5-dihydroxycinnamic acid were tested in 12-O-tetradecanoylphorbol 13-acetate-induced mouse ear edema, and their anti-inflammatory mechanism was studied by ELISA, real-time RT-PCR, MPO assay and AA-induced mouse ear edema. Compound 7 showed a pronounced anti-inflammatory effect and the inhibition rate was 65.6% at a dose of 1.6mg/ear. This compound acted by reducing mRNA and protein synthesis of tumor necrosis factor-α, interleukins 1β and 6, and also by decreasing the levels of activated neutrophil infiltrates. Furthermore, compound 7 significantly suppressed arachidonic acid-induced edema as well. Cell-based assays showed that compound 7 inhibited the production of cyclooxygenase- 2-catalyzed prostaglandin E2 from lipopolysaccharide-treated RAW 264.7 cells, and also inhibited 5-lipoxygenase production from A23187-treated RBL-1 cells, and consequently reduced leukotriene B4 production. This investigation revealed that some of the derivatives of 3,5-dihydroxycinnamic acid exhibit a more pronounced anti-inflammatory effect than 3,4-dihydroxycinnamic acid. Therefore, 3,5-dihydroxycinnamic acid derivatives, especially compound 7, represent potential value for antiinflammatory drug development.

Intracellular NAD+ levels are associated with LPS-induced TNF-α release in pro-inflammatory macrophages.

Metabolism and immune responses have been shown to be closely linked and as our understanding increases, so do the intricacies of the level of linkage. NAD(+) has previously been shown to regulate tumour necrosis factor-α (TNF-α) synthesis and TNF-α has been shown to regulate NAD(+) homoeostasis providing a link between a pro-inflammatory response and redox status. In the present study, we have used THP-1 differentiation into pro- (M1-like) and anti- (M2-like) inflammatory macrophage subset models to investigate this link further. Pro- and anti-inflammatory macrophages showed different resting NAD(+) levels and expression levels of NAD(+) homoeostasis enzymes. Challenge with bacterial lipopolysaccharide, a pro-inflammatory stimulus for macrophages, caused a large, biphasic and transient increase in NAD(+) levels in pro- but not anti-inflammatory macrophages that were correlated with TNF-α release and inhibition of certain NAD(+) synthesis pathways blocked TNF-α release. Lipopolysaccharide stimulation also caused changes in mRNA levels of some NAD(+) homoeostasis enzymes in M1-like cells. Surprisingly, despite M2-like cells not releasing TNF-α or changing NAD(+) levels in response to lipopolysaccharide, they showed similar mRNA changes compared with M1-like cells. These data further strengthen the link between pro-inflammatory responses in macrophages and NAD(+). The agonist-induced rise in NAD(+) shows striking parallels to well-known second messengers and raises the possibility that NAD(+) is acting in a similar manner in this model.

Kallistatin treatment attenuates lethality and organ injury in mouse models of established sepsis.

IntroductionKallistatin levels in the circulation are reduced in patients with sepsis and liver disease. Transgenic mice expressing kallistatin are resistant to lipopolysaccharide (LPS)-induced mortality. Here, we investigated the effect of kallistatin on survival and organ damage in mouse models of established sepsis.MethodsMice were rendered septic by cecal ligation and puncture (CLP), or endotoxemic by LPS injection. Recombinant human kallistatin was administered intravenously six hours after CLP, or intraperitoneally four hours after LPS challenge. The effect of kallistatin treatment on organ damage was examined one day after sepsis initiation, and mouse survival was monitored for four to six days.ResultsHuman kallistatin was detected in mouse serum of kallistatin-treated mice. Kallistatin significantly reduced CLP-induced renal injury as well as blood urea nitrogen, serum creatinine, interleukin-6 (IL-6), and high mobility group box-1 (HMGB1) levels. In the lung, kallistatin decreased malondialdehyde levels and HMGB1 and toll-like receptor-4 (TLR4) synthesis, but increased suppressor of cytokine signaling-3 (SOCS3) expression. Moreover, kallistatin attenuated liver injury, serum alanine transaminase (ALT) levels and hepatic tumor necrosis factor-α (TNF-α) synthesis. Furthermore, delayed kallistatin administration improved survival in CLP mice by 38%, and LPS-treated mice by 42%. In LPS-induced endotoxemic mice, kallistatin attenuated kidney damage in association with reduced serum creatinine, IL-6 and HMGB1 levels, and increased renal SOCS3 expression. Kallistatin also decreased liver injury in conjunction with diminished serum ALT levels and hepatic TNF-α and TLR4 expression. In cultured macrophages, kallistatin through its active site increased SOCS3 expression, but this effect was blocked by inhibitors of tyrosine kinase, protein kinase C and extracellular signal-regulated kinase (ERK), indicating that kallistatin stimulates a tyrosine-kinase-protein kinase C-ERK signaling pathway.ConclusionsThis is the first study to demonstrate that delayed human kallistatin administration is effective in attenuating multi-organ injury, inflammation and mortality in mouse models of polymicrobial infection and endotoxemia. Thus, kallistatin therapy may provide a promising approach for the treatment of sepsis in humans.

Central Renin-Angiotensin System Activation and Inflammation Induced by High-Fat Diet Sensitize Angiotensin II-Elicited Hypertension.

Obesity has been shown to promote renin-angiotensin system activity and inflammation in the brain and to be accompanied by increased sympathetic activity and blood pressure. Our previous studies demonstrated that administration of a subpressor dose of angiotensin (Ang) II sensitizes subsequent Ang II-elicited hypertension. The present study tested whether high-fat diet (HFD) feeding also sensitizes the Ang II-elicited hypertensive response and whether HFD-induced sensitization is mediated by an increase in renin-angiotensin system activity and inflammatory mechanisms in the brain. HFD did not increase baseline blood pressure, but enhanced the hypertensive response to Ang II compared with a normal-fat diet. The sensitization produced by the HFD was abolished by concomitant central infusions of either a tumor necrosis factor-α synthesis inhibitor, pentoxifylline, an Ang II type 1 receptor blocker, irbesartan, or an inhibitor of microglial activation, minocycline. Furthermore, central pretreatment with tumor necrosis factor-α mimicked the sensitizing action of a central subpressor dose of Ang II, whereas central pentoxifylline or minocycline abolished this Ang II-induced sensitization. Real-time quantitative reverse transcription-polymerase chain reaction analysis of lamina terminalis tissue indicated that HFD feeding, central tumor necrosis factor-α, or a central subpressor dose of Ang II upregulated mRNA expression of several components of the renin-angiotensin system and proinflammatory cytokines, whereas inhibition of Ang II type 1 receptor and of inflammation reversed these changes. The results suggest that HFD-induced sensitization of Ang II-elicited hypertension is mediated by upregulation of the brain renin-angiotensin system and of central proinflammatory cytokines.

Overexpression of COX-2 but not indoleamine 2,3-dioxygenase-1 enhances the immunosuppressive ability of human umbilical cord-derived mesenchymal stem cells

Owing to their immunosuppressive properties mesenchymal stem cells (MSCs) are widely applicable in the treatment of autoimmune disease. The aim of this study was to investigate whether the indoleamine 2,3-dioxygenase-1 (IDO-1) and cyclooxygenase-2 (COX-2) genes enhanced the immunosuppressive functional ability of MSCs following stable transfection. To strengthen the immunomodulatory ability of MSCs, IDO-1 and COX-2 were overexpressed in umbilical cord progenitor cell-derived MSCs using recombinant plasmids and electroporation. RT-qPCR analysis and western blotting confirmed the expression of IDO-1 and COX-2 in transfected MSCs. Further functional assays in co-culture experiments, including lymphocyte proliferation and cyto-toxicity assays showed that COX-2-transfected MSCs possessed more potent immunomodulatory cells than the untreated MSCs, or MSCs transfected with IDO-1. Additionally, synthesis of interferon-γ and tumor necrosis factor-α (TNF-α) was significantly inhibited in lymphocytes co-cultured with COX-2-transfected MSCs, which was consistent with changes in immune-related genes in MSCs. An enhanced expression of IDO-1, COX-2, heme-oxygenase-1, inducible nitric-oxide synthase, TNF-α-stimulated gene/protein-6, transforming growth factor-β (TGF-β), human leukocyte antigen molecule 5 (HLA-G5) and interleukin-10 (IL-10) was identified following COX-2 transfection. We showed that the overexpression of COX-2 enhanced the immunosuppressive function of MSCs. COX-2-modified MSCs more potently inhibited the activation and proliferation of peripheral blood mononuclear cells.

Transiently lowering tumor necrosis factor-α synthesis ameliorates neuronal cell loss and cognitive impairments induced by minimal traumatic brain injury in mice.

BackgroundThe treatment of traumatic brain injury (TBI) represents an unmet medical need, as no effective pharmacological treatment currently exists. The development of such a treatment requires a fundamental understanding of the pathophysiological mechanisms that underpin the sequelae resulting from TBI, particularly the ensuing neuronal cell death and cognitive impairments. Tumor necrosis factor-alpha (TNF-α) is a cytokine that is a master regulator of systemic and neuroinflammatory processes. TNF-α levels are reported to become rapidly elevated post TBI and, potentially, can lead to secondary neuronal damage.MethodsTo elucidate the role of TNF-α in TBI, particularly as a drug target, the present study evaluated (i) time-dependent TNF-α levels and (ii) markers of apoptosis and gliosis within the brain and related these to behavioral measures of ‘well being’ and cognition in a mouse closed head 50 g weight drop mild TBI (mTBI) model in the presence and absence of post-treatment with an experimental TNF-α synthesis inhibitor, 3,6′-dithiothalidomide.ResultsmTBI elevated brain TNF-α levels, which peaked at 12 h post injury and returned to baseline by 18 h. This was accompanied by a neuronal loss and an increase in astrocyte number (evaluated by neuronal nuclei (NeuN) and glial fibrillary acidic protein (GFAP) immunostaining), as well as an elevation in the apoptotic death marker BH3-interacting domain death agonist (BID) at 72 h. Selective impairments in measures of cognition, evaluated by novel object recognition and passive avoidance paradigms - without changes in well being, were evident at 7 days after injury. A single systemic treatment with the TNF-α synthesis inhibitor 3,6′-dithiothalidomide 1 h post injury prevented the mTBI-induced TNF-α elevation and fully ameliorated the neuronal loss (NeuN), elevations in astrocyte number (GFAP) and BID, and cognitive impairments. Cognitive impairments evident at 7 days after injury were prevented by treatment as late as 12 h post mTBI but were not reversed when treatment was delayed until 18 h.ConclusionsThese results implicate that TNF-α in mTBI induced secondary brain damage and indicate that pharmacologically limiting the generation of TNF-α post mTBI may mitigate such damage, defining a time-dependent window of up to 12 h to achieve this reversal.

The role of TNF-alpha/NF-kappa B pathway on the up-regulation of voltage-gated sodium channel Nav1.7 in DRG neurons of rats with diabetic neuropathy

Diabetic neuropathy (DN) is a common form of peripheral neuropathy, yet the mechanisms responsible for chronic pain in this disease are poorly understood. The up-regulation of the expression and function of voltage-gated sodium channel Nav1.7 has been implicated in DN, however, the exact mechanism is unclear. In the present study, we found that a proportion of streptozotocin (STZ)-treated rats suffered from mechanical allodynia and thermal hyperalgesia for a long-lasting time. Nav1.7 was up-regulated in spinal dorsal root ganglia (DRG) of rats with DN, double immunofluorescence staining showed that the increased Nav1.7 was co-localized with large and small sized neurons but not satellite glial cells. Inhibiting the synthesis of tumor necrosis factor-α (TNF-α) by thalidomide prevented DN, accompanied by strongly blocking the up-regulation of Nav1.7, TNF-α and p-nucleus factor-kappa B (p-NF-κB) in DRG. Intrathecal injection of NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC) significantly attenuated the pain behaviors and over-expression of Nav1.7 in DRG neurons. These data suggest that increased TNF-α may be responsible for up-regulation of Nav1.7 in DRG neurons of rats with DN, and NF-κB signal pathway is involved in this process. The findings might provide potential target for preventing diabetic neuropathy.

Physical exercise protects myenteric neurons and reduces parasitemia in Trypanosoma cruzi infection

To evaluate the parasitemia, nitrergic neurons, and cytokines in Trypanosoma cruzi-infected mice subjected to moderate physical exercise, forty male Swiss mice, 30days of age, were divided: Trained Control (TC), Trained Infected (TI), Sedentary Control (SC), and Sedentary Infected (SI). The moderate physical exercise program on a treadmill lasted 8weeks. Three days after completing the moderate physical exercise program, the TI and SI groups were inoculated with 1300 blood trypomastigotes of the Y strain of T. cruzi, and parasitemia was evaluated from day 4 to day 22 after inoculation. After 75days of infection, cytokines were measured and colonic neurons were quantified using immunofluorescence to identify neuronal nitric oxide synthase (nNOS). The results were analyzed using analysis of variance – Tukey and Kruskal–Wallis tests, to 5% significance. Moderate physical exercise reduced the parasite peak on day 8 of infection and total parasitemia (p<0.05), contributed to survival of number of nNOS-immunoreactive neurons (p<0.01) and promoted neuronal hypertrophy of the neurons (p<0.05), increased the synthesis of tumor necrosis factor-α (p<0.01) and transforming growth factor-β (p>0.05), providing beneficial effects to the host by acting on the immune system to preserve nitrergic neurons.

An imbalance in the production of proinflammatory and anti-inflammatory cytokines is observed in whole blood cultures of preeclamptic women in comparison with healthy pregnant women

Objective: To compare the production of pro- and anti-inflammatory cytokines by whole blood samples stimulated with lipopolysaccharide from normotensive and preeclamptic women. Methods: The synthesis of tumor necrosis factor-α, interleukyn-1β, interleukin-6 and interleukin-10 were assayed by immunosorbent assays in 56 and 50 preeclamptic and normotensive women, respectively. Results: Preeclamptic women showed a higher synthesis (p < 0.05) of interleukin-1β and interleukin-6 versus normotensive group. Interleukin-10 synthesis from samples of normotensive women showed a higher production versus preeclamptic group (p < 0.05). Conclusion: Preeclamptic women showed an imbalance in the production of pro and anti-inflammatory cytokines in comparison with normotensive women.