ag-490 and Inflammation

Potassium (K(+)) channels are membrane proteins expressed in most living cells that selectively control the flow of K(+) ions. More than 80 genes encode the K(+) channel subunits in the human genome. The TWIK-related K(+) channel (TREK-1) belongs to the two-pore domain K(+) channels (K2P) and displays various properties including sensitivity to physical (membrane stretch, acidosis, temperature) and chemical stimuli (signaling lipids, volatile anesthetics). The distribution of TREK-1 in the central nervous system, coupled with the physiological consequences of its opening and closing, leads to the emergence of this channel as an attractive therapeutic target. We review the TREK-1 channel, its structural and functional properties, and the pharmacological agents (agonists and antagonists) able to modulate its gating.

Topics: Arrhythmias, Cardiac; Depression; Epilepsy; Humans; Inflammation; Models, Molecular; Molecular Structure; Neuroprotective Agents; Pain; Potassium Channels, Tandem Pore Domain; Structure-Activity Relationship

Infertility is a common disease among women of childbearing age and seriously endangers the reproductive health of human beings.. We aimed to study the active effect and mechanism of betulonic acid (BTA) on tubal inflammatory infertility.. An inflammatory model was established in isolated rat oviduct epithelial cells. Immunofluorescence of cytokeratin 18 was performed in cells. The therapeutic effect of BTA on cells was observed. Subsequently, we added JAK/STAT inhibitor AG490 and MAPK inhibitor U0126 and measured the levels of inflammatory factors via enzyme-linked immunosorbent assay and qRT-PCR. CCK-8 assay was applied to test cell proliferation, whereas flow cytometry was used to measure apoptosis. The levels of TLR4, IκBα, JAK1, JAK2, JAK3, Tyk2, STAT3, p38, ERK and the phosphorylation of p65 were determined by Western blotting.. Betulonic acid inhibited the activation of TLR4 and NF-κB signalling pathways, and significantly downregulated IL-1β, IL-6, and TNF-α, with high doses being the most effective. Furthermore, high-dose BTA promoted the proliferation of oviduct epithelial cells and inhibited apoptosis. In addition, BTA inhibited the activation of JAK/STAT signalling pathway to perform effectively in oviduct epithelial cells inflammation. The addition of AG490 led to the inhibition of the JAK/STAT signalling pathway. BTA also inhibited the activation of MAPK signalling pathway in oviduct epithelial cells inflammation. Under U0126 treatment, the inhibition of proteins in MAPK pathway by BTA was weakened.. Therefore, BTA inhibited the TLR, JAK/STAT and MAPK signalling pathways.. Our study provided a new therapeutic strategy for infertility caused by oviduct inflammation.

Topics: Animals; Epithelial Cells; Female; Humans; Inflammation; NF-kappa B; Oviducts; Rats; Toll-Like Receptor 4

Sepsis-induced myocardial dysfunction (SIMD) represents one of the serious complications secondary to sepsis, which is a leading cause of the high mortality rate among septic cases. Subsequent cardiomyocyte apoptosis, together with the uncontrolled inflammatory response, has been suggested to be closely related to SIMD. Piceatannol (PIC) is verified with potent anti-apoptotic and anti-inflammatory effects, but its function and molecular mechanism in SIMD remain unknown so far. This study aimed to explore the potential role and mechanism of action of PIC in resisting SIMD. The interaction of PIC with JAK2 proteins was evaluated by molecular docking, molecular dynamics (MD) simulation and surface plasmon resonance imaging (SPRi). The cecal ligation and puncture-induced septicemia mice and the LPS-stimulated H9C2 cardiomyocytes were prepared as the models in vivo and in vitro, separately. Molecular docking showed that JAK2-PIC complex had the -8.279 kcal/mol binding energy. MD simulations showed that JAK2-PIC binding was stable. SPRi analysis also showed that PIC has a strong binding affinity to JAK2. PIC treatment significantly ameliorated the cardiac function, attenuated the sepsis-induced myocardial loss, and suppressed the myocardial inflammatory responses both in vivo and in vitro. Further detection revealed that PIC inhibited the activation of the JAK2/STAT3 signaling, which was tightly associated with apoptosis and inflammation. Importantly, pre-incubation with a JAK2 inhibitor (AG490) partially blocked the cardioprotective effects of PIC. Collectively, the findings demonstrated that PIC restored the impaired cardiac function by attenuating the sepsis-induced apoptosis and inflammation via suppressing the JAK2/STAT3 pathway both in septic mice and H9C2 cardiomyocytes.

Topics: Animals; Apoptosis; Cardiomyopathies; Cardiotonic Agents; Cell Line; Disease Models, Animal; Inflammation; Janus Kinase 2; Male; Mice, Inbred C57BL; Molecular Docking Simulation; Molecular Dynamics Simulation; Myocytes, Cardiac; Rats; Sepsis; Signal Transduction; STAT3 Transcription Factor; Stilbenes; Tyrphostins

As an alternative to bisphenol A (BPA), bisphenol F (BPF) has been increasingly used in manufacturing various consumer products. Exposured to BPF may lead to imbalanced immune homeostasis, yet the underlying mechanisms have not been fully elucidated. The present study was aimed to investigate the effects of BPF on macrophages and the underlying mechanism in regard to its association with estrogen receptor (ER), janus kinase 2/signal transducer and activator of transcription 3/suppressor of cytokine signaling 3 (JAK2/STAT3/SOCS3) pathway. In this study, after treatment of RAW264.7 macrophages with BPF (0, 5, 10, 20 μM), the macrophage M1 polarization was promoted, and the gene expression of M1 functional markers and pro-inflammatory cytokines was upregulated, which suggested the involvement of a vicious circle associated with chronic inflammation. Moreover, BPF facilitated SOCS3 expression in the cells in a dose-dependent manner, via activation of the JAK2/STAT3 signaling pathway, which may promote the transcription of many pro-inflammatory factors. Additionally, the above effects of BPF were blocked by either JAK2/STAT3 inhibitor AG490 (10 μM) or ER antagonist ICI 182,780 (10 μM). Taken together, the results of this study indicate that BPF promotes macrophage polarization toward pro-inflammatory M1 subtype, through activation of the ER-JAK2/STAT3/SOCS3 signaling pathway. Our finding may provide a new insight into the link between bisphenol exposure and immune dysfunction.

Topics: Animals; Benzhydryl Compounds; Cytokines; Dose-Response Relationship, Drug; Fulvestrant; Inflammation; Janus Kinase 2; Macrophages; Mice; Phenols; RAW 264.7 Cells; Receptors, Estrogen; Signal Transduction; STAT3 Transcription Factor; Suppressor of Cytokine Signaling 3 Protein; Tyrphostins

To investigate the preclinical pharmacodynamics and mechanism of JLX001 against myocardial ischemia reperfusion (MI/R) for clinical application.. In vivo, SD rats were given intragastric administration for 5 days, and the MI/R model was established by ligating/releasing the left anterior descending coronary artery. In vitro, the oxygen-glucose deprivation/reperfusion (OGD/R) model was established after the drug was pre-incubated for 24 h in H9C2 cells. The infract size was determined by TTC staining. Left ventricular function of MI/R rats was detected by echocardiography. The level of histopathological score was determined by hematoxylin-eosin (HE) staining. The level of superoxide dismutase (SOD), malondialdehyde (MDA), creatine kinase (CK), lactic dehydrogenase (LDH), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) were determined by relevant kits. The level of apoptosis was measured by Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and Hoechst staining. The expression of p-Jak2, p-Stat3, Bax, Bcl-2, TNF-α, IL-1β protein were determined by western blot.. JLX001 can significantly improve left ventricular function, reduce myocardial infract size, histopathological score, the level of MDA, CK, LDH, TNF-α, IL-1β and the expression of Bax protein, significantly increase the activity of SOD, Bcl-2 protein expression, p-Jak2 protein expression, p-Stat3 protein expression in rat heart tissues and H9C2 cells. These effects can be reversed by AG490 which is a specific inhibitor of Jak2-Stat3 pathway.. JLX001 can alleviate MI/R injury by inhibiting myocardial apoptosis, inflammation, and oxidative stress via Jak2-Stat3 pathway in vivo and in vitro.

Topics: Animals; Apoptosis; Cell Line; Inflammation; Janus Kinase 2; Male; Myocardial Reperfusion Injury; Oxidative Stress; Rats; Rats, Sprague-Dawley; STAT3 Transcription Factor; Triterpenes; Tyrphostins

Congenital obstructive nephropathy is the main cause of end-stage renal disease in infants and children. Renal insufficiency is due to impaired growth and maturation in the developing kidney with obstruction. Congenital obstructive nephropathy leads to cytokine mediated inflammation and the development of interstitial fibrosis. The Janus kinase-2 (JAK-2) and Signal Transducer and Activator of Transcription'-3 (STAT3) are involved in cytokine production, inflammation, and interstitial fibrosis.. We studied the role of JAK2/STAT3 in a model of congenital obstructive nephropathy using unilateral ureteral obstruction (UUO) in neonatal mice at the second day of life. Cytokine production, inflammation, and interstitial fibrosis were analyzed in obstructed and sham operated kidneys of neonatal mice treated with or without JAK2/STAT3 inhibitor Tyrphostin AG490. To mimic obstruction and distension, proximal tubular cells were stretched in vitro.. We show that STAT3 is highly activated in the developing kidney with obstruction and in proximal tubular cells following stretch. JAK2/STAT3 activation mediates cytokine release and leukocyte recruitment into neonatal kidneys after UUO. Pharmacological blockade of JAK2/STAT3 by Tyrphostin AG490 reduced inflammation, tubular apoptosis, and interstitial fibrosis. JAK2/STAT3 blockade decreased pro-inflammatory and profibrotic mediators in tubular cells.. Our findings provide evidence that JAK2/STAT3 mediates inflammation and fibrosis in the developing kidney with obstruction. Blocking JAK2/STAT3 may prove beneficial in congenital obstructive nephropathy in children.

Topics: Animals; Animals, Newborn; Enzyme Inhibitors; Fibrosis; Inflammation; Janus Kinase 2; Mice; STAT3 Transcription Factor; Tyrphostins; Ureteral Obstruction

Interstitial cystitis (IC) is a heterogeneous syndrome with unknown etiology, and microRNAs (miRs) were found to be involved in IC. In our study, we aim to explore the role of miR-132 in the inflammatory response and detrusor fibrosis in IC through the Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway in rat models. A rat model of IC was established and treated with the miR-132 mimic, miR-132 inhibitor, and/or JAK-STAT signaling pathway inhibitor AG490. Enzyme-linked immunosorbent assay was applied to measure the expression of interleukin (IL)-6, IL-10, interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α), and intercellular adhesion molecule-1 (ICAM-1). The urodynamic test was performed to assess urodynamic parameters, and reverse transcription quantitative polymerase chain reaction and Western blot analysis for the expression of miR-132, STAT4, suppressors of cytokine signaling 3 (SOCS3), JAK2, vascular endothelial growth factor (VEGF), IFN-γ, and TNF-α. IC rats treated with miR-132 inhibitor and AG490 had decreased collagen fiber, inflammatory cell infiltration, and mast cells, lower expression of IL-6, IL-10, IFN-γ, TNF-α, ICAM-1, collagens I and III, and alleviated urodynamic parameters and decreased expression of STAT4, VEGF, JAK2, IFN-γ, TNF-α, and increased expression of SOCS3. Taken together, our data indicate that downregulation of miR-132 alleviates inflammatory response and detrusor fibrosis in IC via the inhibition of the JAK-STAT signaling pathway.

Topics: Animals; Cystitis, Interstitial; Female; Humans; Immunohistochemistry; In Vitro Techniques; Inflammation; Janus Kinase 2; Janus Kinases; MicroRNAs; Rats; Rats, Sprague-Dawley; Signal Transduction; STAT4 Transcription Factor; Tumor Necrosis Factor-alpha; Tyrphostins; Vascular Endothelial Growth Factor A

To explore AG490 (the inhibitor of Janus kinase (JAK) 2/signal transducer and activator of transcription (STAT) 3 pathway) in cisplatin (DDP)-induced acute kidney injury (AKI) in mice with lung cancer.. Mice were randomly divided into normal, model, AG490, DDP and DDP + AG490 groups. The lung cancer models were established except for Normal group. The levels of blood urea nitrogen (BUN) and creatinine and the status of oxidative stress were detected. Then, histological changes were assessed by HE and PAS staining and apoptosis by TUNEL experiment. The molecule expressions were detected by qRT-PCR and western blot, and immunohistochemistry, respectively.. DDP inhibited the tumor growth in mice with lung cancer, which was further promoted by the combination with AG490. Mice in the DDP group had elevated levels of BUN and creatinine than those in the Normal group with the increased inflammatory cytokines (TNF-α, IL-6, MCP-1 and CXCL-1) and malondialdehyde (MDA) level and the decreased glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT). In addition, DDP could activate the JAK2/STAT3 pathway to promote the apoptosis by upregulating Bax, cleaved caspase-9 and cleaved caspase-3 while downregulating the Bcl-2 in the kidney tissues. DDP + AG490 group showed the alleviated AKI and the improvements in oxidative stress, inflammatory responses and apoptosis in the kidney tissues, as compared to DDP group.. AG490 alleviated DDP-induced AKI in lung cancer mice with improved oxidative stress and inflammation, and the suppression of JAK2/STAT3 pathway.

Topics: Acute Kidney Injury; Animals; Apoptosis; Blood Urea Nitrogen; Carcinoma, Lewis Lung; Cisplatin; Creatinine; Female; Inflammation; Janus Kinase 2; Lung Neoplasms; Male; Mice; Mice, Inbred C57BL; Oxidative Stress; Signal Transduction; STAT3 Transcription Factor; Tyrphostins

Accounting for high mortality and morbidity rates, intracerebral hemorrhage (ICH) remains one of the most detrimental stroke subtypes lacking a specific therapy. Neuroinflammation contributes to ICH-induced brain injury and is associated with unfavorable outcomes. This study aimed to evaluate whether

Topics: alpha7 Nicotinic Acetylcholine Receptor; Animals; Blood Transfusion, Autologous; Brain Injuries; Bridged Bicyclo Compounds, Heterocyclic; Cerebral Hemorrhage; Collagenases; Disease Models, Animal; Humans; Inflammation; Janus Kinase 2; Mice; Neurons; Peroxidase; Quinuclidines; Rats; STAT3 Transcription Factor; Tumor Necrosis Factor-alpha; Tyrphostins

Interleukin (IL)‑23, as a novel pro‑inflammatory cytokine, is important in several inflammatory diseases, including myocardial ischemia and reperfusion (I/R) injury, however, the underlying mechanism remains to be elucidated. The present study was designed to investigate the specific role of IL‑23 in myocardial I/R injury, and whether the Janus kinase 2/signal transducer and activator of transcription 3 (JAK2‑STAT3) signaling pathway, one of the important downstream signaling pathways of IL‑23, and the IL‑17A downstream pro‑inflammatory cytokine, were involved. Anesthetized rats underwent different treatments with adenovirus (Ad) vectors (Ad‑GFP, Ad‑IL‑23, Anti‑IL‑23 or Ad‑IL‑23+AG490) and were then subjected to ischemia for 30 min prior to 4 h reperfusion. The effects of the upregulation and downregulation of IL‑23 on myocardial injury, inflammatory responses in myocardial tissue, and myocardial apoptosis were measured accordingly. In addition, the levels of phosphorylated (P‑)JAK2 and P‑STAT3 were measured to assess the activity of the JAK2‑STAT3 signaling pathway. The results demonstrated that there was an increased expression of IL‑23 in the myocardial tissue exposed to myocardial I/R injury (P<0.05). The upregulation of IL‑23 significantly increased the infarct size and the expression levels of lactate dehydrogenase and creatine kinase (P<0.05). The upregulation of IL‑23 significantly increased inflammatory responses, as reflected by the high expression levels of IL‑17A, IL‑6, tumor necrosis factor‑α in the myocardial tissues (P<0.05). Furthermore, the upregulation of IL‑23 significantly facilitated the decrease in the B‑cell lymphoma 2 (Bcl‑2)/Bcl‑2‑associated X protein ratio, and the increases in the myocardial apoptotic index and expression of caspase‑3 induced by myocardial I/R (P<0.05). IL‑23 also activated the JAK2‑STAT3 signaling pathway, upregulating the expression levels of P‑JAK2 and P‑STAT3 in the myocardial tissues (P<0.05). Treatment with AG490, an inhibitor of JAK2‑STAT3, partially attenuated the pro‑inflammatory and pro‑apoptotic effects of IL‑23 (P<0.05). The results of the present study suggested that IL‑23 aggravated myocardial I/R injury by promoting inflammatory responses and myocardial apoptosis, which may be associated with high expression levels of IL‑17A and upregulation of the JAK2‑STAT3 signaling pathway.

Topics: Adenoviridae; Animals; Apoptosis; HEK293 Cells; Humans; Inflammation; Interleukin-17; Interleukin-23; Janus Kinase 2; Male; Myocardial Reperfusion Injury; Myocardium; Phosphorylation; Rats, Sprague-Dawley; Signal Transduction; STAT3 Transcription Factor; Tyrphostins; Up-Regulation

Zfp637 is a recently identified zinc finger protein, and its functions remain largely unknown. Here, we innovatively demonstrate the effects of Zfp637 on the differentiation of mouse spermatogonia and on its downstream target gene SOX2 in vitro. Obesity has been recognized as a chronic inflammatory disease that leads to decreased sexual function and sexual development disorders. We observed higher levels of IL-6 in serum and testis homogenates from obese mice compared with control mice. We also demonstrated that high levels of IL-6 inhibited Zfp637 expression, and we elucidated the underlying mechanisms. SOCS3 overexpression and STAT3 phosphorylation inhibitor (AG490) were used to investigate the function of the SOCS3/STAT3 pathway during this process. Our results showed that exposure of mouse spermatogonial cells to high levels of IL-6 inhibited Zfp637 expression by increasing SOCS3 expression and inhibiting the phosphorylation of STAT3, further reducing cellular differentiation. Consistent with the in vitro results, we observed increasing expression levels of SOCS3 and SOX2, but a reduction of Zfp637 expression, in obese mouse testes. In conclusion, Zfp637 plays a crucial role in spermatogenesis by downregulating SOX2 expression, and IL-6 can decrease the expression of Zfp637 through the SOCS3/STAT3 signaling pathway.

Topics: Animals; Cell Line; DNA-Binding Proteins; Down-Regulation; Inflammation; Interleukin-6; Leptin; Male; Mice; Mice, Obese; Obesity; Phosphorylation; RNA Interference; Signal Transduction; SOXB1 Transcription Factors; Spermatogenesis; Spermatogonia; STAT3 Transcription Factor; Suppressor of Cytokine Signaling 3 Protein; Testis; Tyrphostins

Objective To observe the effect of lipid derivative of benzylidene malononitrile AG490 on the airway inflammation in a mouse model of neutrophilic asthma (NA). Methods Fifty-four specific pathogen-free (SPF) female C57BL/6 mice were randomly divided into 3 groups: NA group, AG490-treated NA (NAAG) group, and normal control (NC) group, 18 mice in each group. The NA group and the NAAG group were sensitized by airway instillation of ovalbumin (OVA) and lipopolysaccharide (LPS) on day 0, 6 and 13. The NAAG group was injected with AG490 (500 μg/mouse, i.p.) three times a week, from day 0 after the first sensitization, for 3 weeks. Mice were challenged on day 21, 22 for 1 hour/time with an aerosol of 10 g/L OVA. At 24 hours after the final challenge, bronchoalveolar lavage fluid (BALF) was collected. The total number and differential counts of nucleated cells and the percentage of each type were determined. HE staining and PAS staining was employed for observing the lung pathological changes. The percentages of Th17 cells and regulatory T cells (Treg) in the lung issue were determined by flow cytometry. The level of interleukin-17 (IL-17) in BALF was measured using ELISA. Results Compared with the NA group, the total number of nucleated cells, the percentage of neutrophils and the percentage of eosinophils in BALF in the NAAG group were obviously reduced; lung tissue pathologic changes were improved in the NAAG group; goblet cell hyperplasia and the level of IL-17 in BALF in the NAAG group were significantly down-regulated; the proportion of Treg in the lung increased and the proportion of Th17 cells in the lung decreased in the NAAG group. Conclusion After NA mice are treated with AG490 during the sensitization phase, the proportion of Treg in the lung would increase and the proportion of Th17 cells in the lung would decrease. AG490 could attenuate the airway inflammation in the mouse model of NA.

Topics: Animals; Asthma; Bronchoalveolar Lavage Fluid; Enzyme Inhibitors; Female; Flow Cytometry; Inflammation; Interleukin-17; Lipids; Lipopolysaccharides; Lung; Mice, Inbred C57BL; Neutrophils; Ovalbumin; Random Allocation; Respiratory System; Th17 Cells; Tyrphostins

Recent research indicates that the Janus kinase/signal transducer and activator of transcription 3 (JAK/STAT3) pathway may play an important role in chronic inflammation which promotes cancer progression, yet the mechanism is not clear. The present study aimed to investigate the role of the JAK/STAT3 pathway in the growth and cancer-related inflammation (CRI) of esophageal squamous cell carcinoma (ESCC) by studying the crosstalk between the JAK/STAT3 pathway and nuclear factor-κB (NF-κB) and cyclooxygenase-2 (COX-2) which are important inflammatory factors associated with tumorigenesis. Cell growth and the cell cycle were assessed by CCK-8 assays and flow cytometry, respectively. The protein levels of STAT3, phosphorylated STAT3, VEGF, NF-κB p65, phosphorylated NF-κB p65 and COX-2 in ESCC cells following treatment with JAK2 inhibitor for 48 h or interleukin-6 (IL-6) for 24 h were detected. RT-PCR was performed to study the interaction among STAT3, NF-κB and COX-2 by transfection of siRNAs targeted at STAT3 and NF-κB. STAT3 was activated in 3 ESCC cell lines at different levels. Blocking the JAK/STAT3 pathway inhibited cancer growth through regulation of cell growth, cell cycle and angiogenesis. Likewise, abrogation of the JAK/STAT3 pathway decreased CRI by downregulating levels of NF-κB p65 phosphorylation, COX-2 and IL-6 concentration. In addition, CRI and cancer growth were accelerated by IL-6 through stimulation of the JAK/STAT3 and NF-κB p65 pathway. Moreover, STAT3 and NF-κB both regulated COX-2 as a downstream gene. The JAK/STAT3 pathway is an important pathway which links CRI and cancer growth through IL-6 and crosstalk with the NF-κB p65 subunit and COX-2. The STAT3 pathway could be a novel target both for cancer treatment and prevention in ESCC.

Topics: Carcinoma, Squamous Cell; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclooxygenase 2; Enzyme Inhibitors; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; Humans; Inflammation; Interleukin-6; Janus Kinase 2; NF-kappa B; Phosphorylation; Signal Transduction; STAT3 Transcription Factor; Tyrphostins

Human β-defensin-2(HBD-2) is one of the two major vertebrate antimicrobial peptide families (α and β), which is highly expressed by proinflammatory induction in the lung and exhibit broad-spectrum antimicrobial activity. We observed that IL-22 receptors high expressed on the membrane of A549 cells; HBD-2 mRNA was expressed in a time- and concentration-dependent manners in A549 cells when treated with IL-22; further studies demonstrated that HBD-2 expression was attenuated by AG490, but to JSH-23, inhibitors of p-STAT3 DNA binding and NF-κB/p65 subunit nuclear translocation, respectively. These results support that IL-22-mediated signalling pathway of HBD-2 gene expression involved STAT3 but not NF-κB in human alveolar epithelium. These findings provide a new insight into how IL-22 may play an important link between innate and adaptive immunity, thereby anti-infection locally in the alveolar epithelium.

Topics: beta-Defensins; Cell Line; DNA-Binding Proteins; Epithelial Cells; Humans; Inflammation; Interleukin-22; Interleukins; Phenylenediamines; Pulmonary Alveoli; Receptors, Interleukin; Respiratory Mucosa; RNA, Messenger; Signal Transduction; STAT3 Transcription Factor; Transcription Factor RelA; Tyrphostins

Heatstroke not only directly induces cell injury, but also causes large amounts of inflammatory mediators release and cells with extensive biological activities to induce a systemic inflammatory response and immune dysfunction. This study aimed to observe the effects of JAK2 inhibitor AG490 on the brain injury and inflammatory responses of rats with systemic heatstroke. Under the light microscope, the hippocampus tissues of rat with heatstroke were edema and apoptotic rate was increased. Up-regulation of malondialdehyde (MDA), nitric oxide synthase (iNOS), reactive oxygen species (ROS) and down-regulation of superoxide dismutase (SOD) were also found after heatstroke in rats, which compared with that of the control group. Heatstroke induced inflammation factors secretions and up-regulated levels of matrix metallopeptidase 2 and 9 (MMP2 and MMP-9) and systemic inflammatory response molecules including intercellular adhesion molecule-1 (ICAM-1), tumor necrosis factor-beta 1 (TNF-β1) and cyclooxygenase-2 (COX-2). However, the JAK2 inhibitor AG490 was significantly attenuated the brain injury and inflammatory responses induced by heatstroke in rats. The survival time of heatstroke rats showed that AG490 notably lived longer than heatstroke rats without AG490 treatment. These findings suggest that AG490 may prevent the occurrence of heatstroke via inhibiting the JAK2/STAT3 pathway and the systemic inflammatory responses.

Topics: Animals; Anti-Inflammatory Agents; Apoptosis; Brain Edema; Cyclooxygenase 2; Heat Stroke; Hippocampus; Inflammation; Intercellular Adhesion Molecule-1; Janus Kinase 2; Male; Malondialdehyde; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Neuroprotective Agents; Nitric Oxide Synthase Type II; Oxidative Stress; Protein Kinase Inhibitors; Rats, Sprague-Dawley; Reactive Oxygen Species; Signal Transduction; STAT3 Transcription Factor; Superoxide Dismutase; Time Factors; Transforming Growth Factor beta1; Tyrphostins

Tyrphostin AG490 is a Janus kinase (JAK) 2 inhibitor that is clinically used as an anticancer agent and is also effective in various models of inflammatory and autoimmune diseases. In this study, we examined the effects of tyrphostin AG490 on the development of collagenase-induced osteoarthritis (CIOA). Our results showed that tyrphostin-ameliorated cartilage and bone destructions. This effect was associated with decreased expression of signal transducers and activators of transcription 3 (STAT3), phosphorylated JAK2, Dickkopf homolog 1, and receptor activator of nuclear factor κB ligand (RANKL) in the joints of arthritic mice. Tyrphostin AG490 suppressed STAT3 phosphorylation and the expression of tumor necrosis factor-related apoptosis-inducing ligand and RANKL by synovial fluid cells. The drug inhibited RANKL-induced osteoclast differentiation in vitro. Molecules, such as tyrphostin AG490 that limit bone erosion and influence osteoclast generation, might have therapeutic utility in joint degenerative disorders.

Topics: Animals; Animals, Newborn; Bone and Bones; Bone Remodeling; Cell Differentiation; Cytokines; Enzyme Inhibitors; Inflammation; Intercellular Signaling Peptides and Proteins; Interleukin-17; Interleukin-6; Janus Kinase 2; Joints; Mice; Mice, Inbred ICR; Osteoclasts; Phosphorylation; Protein-Tyrosine Kinases; RANK Ligand; STAT3 Transcription Factor; Synovial Fluid; Tyrphostins

The aryl hydrocarbon receptor (AhR) plays a suppressive role in cecal carcinogenesis by CUL4B/AhR-mediated ubiquitylation and degradation of β-catenin, which is activated by xenobiotics and natural ligands. AhR-deficient (AhR(-)(/-)) mice develop cecal tumors with severe inflammation. To elucidate whether the tumors develop autonomously in AhR(-/-) mice due to impaired β-catenin degradation or in association with accelerated inflammation, we performed two kinds of experiments using germ-free (GF) AhR(-/-) mice and compound mutant mice lacking genes for AhR and apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), which plays an essential role in caspase-1 activation in inflammasomes. Both GF AhR(-/-) and AhR(-/-)•ASC(-/-) mice showed considerably reduced tumor development compared with that in AhR(-/-) mice albeit in a 'cancer-prone' state with aberrant β-catenin accumulation. Blocking of the interleukin (IL)-1β signaling pathway by treatment with a caspase-1 inhibitor, YVAD, reduced cecal tumorigenesis in AhR(-/-) mice. Signal transducers and activators of transcription 3 (STAT3) activation was detected in the cecal epithelium of the AhR(-/-) mice due to enhanced IL-6 production. An inhibitor of the STAT3 signaling pathway, AG490 suppressed the tumor formation. ASC-mediated inflammation was also found to play a critical role in tumor development in Apc(Min/+) mice, a mouse model of familial adenomatous polyposis. Collectively, these results revealed an important role of the bacteria-triggered or ASC-mediated inflammation signaling pathway in the intestinal tumorigenesis of mice and suggest a possible chemical therapeutic intervention, including AhR ligands and inhibitors of the inflammation pathway.

Topics: Adenomatous Polyposis Coli; Amino Acid Chloromethyl Ketones; Animals; Basic Helix-Loop-Helix Transcription Factors; beta Catenin; CARD Signaling Adaptor Proteins; Caspase 1; Caspase Inhibitors; Cecal Neoplasms; Cell Line; Enzyme Activation; Female; Germ-Free Life; Inflammasomes; Inflammation; Interleukin-1beta; Interleukin-6; Intestines; Macrophages; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Receptor Cross-Talk; Receptors, Aryl Hydrocarbon; Signal Transduction; STAT3 Transcription Factor; Tyrphostins

IL-27, a member of IL-6/IL-12 cytokine family, plays pro- and anti-inflammatory functions in immune responses. It can promote inflammation by inducing Th1 differentiation and exert the inhibitory effects on Th2 and Th17 mediated immune responses. Moreover, IL-27 suppresses CD28-mediated IL-2 production from mouse naive CD4(+) T cells. In the present study, we demonstrate that IL-27 inhibits the production of IL-22 and induces the expression of IFN-γ in CD4(+) T cells from human umbilical cord blood mononuclear cells (CBMCs) stimulated with anti-CD3 and anti-CD28 in dose-dependent manner. In addition, the suppression of IL-22 is not dependent on the production of IFN-γ and IL-10. Importantly, IL-27 promotes the expression of SOCS1, which could be inhibited by a Jak2/STAT inhibitor, AG490. Importantly, the expression of IL-22 could not be inhibited under the circumstances with the lower expression of SOCS1. Moreover, IL-27 inhibits the production of IL-22 in CD4(+)CD45RA(+) and CD4(+)CD45RO(+) T cells from PBMCs. These data identify that IL-27 may suppress the production of IL-22 by inducing the expression of SOCS1 in human CD4(+) T cells. Furthermore, it demonstrates that IL-27 may be a therapeutic approach in the treatment of IL-22-mediated diseases.

Topics: CD4-Positive T-Lymphocytes; Cells, Cultured; Fetal Blood; Humans; Infant, Newborn; Inflammation; Interferon-gamma; Interleukin-10; Interleukin-22; Interleukins; Leukocyte Common Antigens; Leukocytes, Mononuclear; Lymphocyte Activation; Signal Transduction; Suppressor of Cytokine Signaling 1 Protein; Suppressor of Cytokine Signaling Proteins; Tyrphostins

This protocol describes microsphere-based protease assays for use in flow cytometry and high-throughput screening. This platform measures a loss of fluorescence from the surface of a microsphere due to the cleavage of an attached fluorescent protease substrate by a suitable protease enzyme. The assay format can be adapted to any site or protein-specific protease of interest and results can be measured in both real time and as endpoint fluorescence assays on a flow cytometer. Endpoint assays are easily adapted to microplate format for flow cytometry high-throughput analysis and inhibitor screening.

Topics: Animals; Biotinylation; Flow Cytometry; Fluorescence Resonance Energy Transfer; Green Fluorescent Proteins; High-Throughput Screening Assays; Humans; Inflammation; Kinetics; Microspheres; Peptide Hydrolases; Peptides; Reproducibility of Results; Temperature

Cytokines are known to play an important role in the pathogenesis of lupus nephritis (LN) and the Jak/STAT (Janus kinase-signal transducer and activator of transcription factor) pathway is important in mediating signal transduction of cytokines. This study examined the pathogenic role of Jak/STAT signaling in LN. MRL/lpr mice were either treated with a selective Jak2 inhibitor tyrphostin AG490 or with vehicle alone from 12 weeks of age until being sacrificed at week 20. AG490 significantly inhibited the phosphorylation of Jak2 and STAT1 (p < 0.05). Compared with the vehicle-treated mice, AG490 treatment significantly reduced proteinuria, improved renal function and suppressed histological lesions of the kidneys and salivary glands (p < 0.05). AG490 treatment significantly inhibited the renal expression of monocyte chemotactic protein (MCP)-1, interferon (IFN)-gamma and class II MHC, which was accompanied by reduced renal infiltration of T cells and macrophages (p < 0.05). In addition, AG490 treatment resulted in a decrease in serum anti-double-stranded DNA (anti-dsDNA) antibody and attenuated the deposition of IgG and C3 in the kidneys (p < 0.05). This study demonstrated that Jak/STAT pathway is implicated in the progression of renal inflammation in MRL/lpr mice and targeting this pathway may provide a potential therapeutic approach for LN.

Topics: Animals; Antibodies, Antinuclear; Disease Models, Animal; Disease Progression; DNA; Enzyme Inhibitors; Female; Inflammation; Janus Kinase 2; Janus Kinases; Kidney; Lupus Nephritis; Mice; Mice, Inbred MRL lpr; Phosphorylation; Salivary Glands; Signal Transduction; STAT Transcription Factors; STAT1 Transcription Factor; Tyrphostins

Our laboratories have previously identified the alpha7 nAChR-JAK2 pathway as playing a central role in nicotine-induced neuroprotection. We have also reported that the angiotensin II (Ang II) AT(2) receptor induced activation of SHP-1 induces the tyrosine dephosphorylation of JAK2 that results in a complete neutralization of the alpha7 nAChR-JAK2 pro-survival cascade. In this study, we investigated the effects of inhibiting the alpha7 nAChR-JAK2 pro-survival cascade on the nicotine-induced production of the survival factor Bcl-2 and the transcriptional activation of NF-kappaB, AP-1, STAT1, STAT3, and STAT5. We report that nicotine induced the production of Bcl-2 and increased the transcriptional activation of NF-kappaB, AP-1, STAT1, and STAT3, and with the exception of AP-1, the other transcription factors (NF-kappaB, STAT1, and STAT3) were significantly reduced by JAK2 inhibition. We also demonstrate that, via transfection of either Bcl-2 antisense or NF-kappaB, STAT1 and STAT3 transcription factor decoys oligodeoxyribonucleotides into PC12 cells, nicotine induces its neuroprotection in PC12 cells via activation of the alpha7 nAChR-JAK2-(NF-kappaB; STAT3)-Bcl-2 pro-survival pathway. Finally, the neuroprotective nicotine-induced production of Bcl-2 appears to fully counteract the Abeta (1-42)-induced apoptosis of PC12 cells by blocking Abeta (1-42)-induced mitochondrial release of cytosolic cytochrome C.

Topics: alpha7 Nicotinic Acetylcholine Receptor; Amyloid beta-Peptides; Animals; Apoptosis; Cytochromes c; Enzyme Activation; Gene Expression; Inflammation; Janus Kinase 2; Mitochondria; Neuroprotective Agents; NF-kappa B; Nicotine; PC12 Cells; Peptide Fragments; Proto-Oncogene Proteins c-bcl-2; Rats; Receptors, Nicotinic; Signal Transduction; STAT1 Transcription Factor; STAT3 Transcription Factor; Transcription Factor AP-1; Tyrphostins

Both interleukin 1 beta (IL-1beta) and interleukin-6 (IL-6) are pro-inflammatory cytokines that play a major role in inflammatory diseases as well as cancer. In this work we investigated the signaling pathway involving lipopolysaccharide (LPS)-mediated IL-1beta and IL-6 production in murine macrophage cell lines and primary macrophages. We show that in response to LPS, the JAK/STAT pathway is activated, leading to tyrosine phosphorylation at residue 705 on STAT3 and at residue 701 on STAT1, respectively. A newly developed STAT3 specific inhibitor (stattic) blocked LPS-mediated STAT3 tyrosine phosphorylation and led to inhibition of LPS-mediated IL-1beta and IL-6 production but not TNF-alpha production. Knockdown of STAT3 expression via small interfering RNA (siRNA) decreased the level of STAT3 expression in Raw 264.7 cells and decreased STAT3 tyrosine phosphorylation in response to LPS treatment. Quantitative real time PCR and Western analysis of cells treated with inhibitor or STAT3 siRNA after LPS treatment showed a significant reduction of IL-1beta and IL-6 mRNA and protein compared to cells treated with LPS alone. Moreover stattic abrogated IL-1beta formation in response to extracellular bacteria Staphylococcus aureus and Escherichia coli in murine peritoneal macrophages. This inhibition did not affect caspase-1 activation. These results highlight the complex role of STAT3 in cytokine production and the key role of STAT3 tyrosine phosphorylation in IL-1beta and IL-6 production in response to inflammation.

Topics: Animals; Bacteria; Bacterial Physiological Phenomena; Cells, Cultured; Cyclic S-Oxides; Enzyme Inhibitors; Female; Inflammation; Interleukin-1beta; Interleukin-6; Lipopolysaccharides; Male; Mice; Mice, Inbred BALB C; Microbial Viability; Phosphorylation; Protein-Tyrosine Kinases; STAT3 Transcription Factor; Tumor Necrosis Factor-alpha; Tyrosine; Tyrphostins

Tyrphostins, derivatives of benzylidene malononitrile are recognized as tyrosine kinase inhibitors that have been applied in some models of acute inflammatory conditions, like LPS and zymosan-induced shock. In the present study, we have investigated the effects of tyrphostin AG-490, on the development of multiple organ failure induced by i.p. injection of zymosan (1 mg/g body weight) in mice. Organ dysfunction and systemic inflammation was estimated 24 h after zymosan administration. Treatment of mice with AG-490 (dose, 5 mg/kg i.p. simultaneously with zymosan) decreased the number of cells and the level of NO in the peritoneal lavage. The substance attenuated the elevation of creatinine (indicator of renal failure), alanine aminotransferase (ALT), aspartate aminotransferase (AST) and bilirubin (indicators for liver dysfunction) and prevented the accelerated coagulation time. The injection of zymosan resulted in a substantial increase in the serum level of TNF-alpha and IL-6, which was strongly inhibited by AG-490. Tyrphostin abolished the expression of iNOS and TNF-alphaR in the liver. Moreover, immunohistochemistry of liver showed decreased phosphorylation of Stat1 and Stat3. In conclusion, the administration of tyrphostin AG-490 in zymosan-induced nonseptic shock significantly improved the rate of survival and lead to less exerted signs of multiple organ failure.

Topics: Alanine Transaminase; Animals; Aspartate Aminotransferases; Bilirubin; Creatinine; Inflammation; Interleukin-6; Macrophages; Male; Mice; Multiple Organ Failure; Nitric Oxide; Nitric Oxide Synthase Type II; Protein Kinase Inhibitors; STAT Transcription Factors; Tumor Necrosis Factor-alpha; Tyrphostins; Whole Blood Coagulation Time; Zymosan

Adipose tissue is a prominent source of plasminogen activator inhibitor-1 (PAI-1), the primary physiological inhibitor of plasminogen activation. Increased PAI-1 expression acts as a cardiovascular risk factor, and plasma levels of PAI-1 strongly correlate with body mass index (BMI). Elevated serum levels of interleukin-6 (IL-6), an inflammatory cytokine and a member of the glycoprotein 130 (gp130) ligand family, are found in obese patients and might indicate low-grade systemic inflammation. Another gp130 ligand, oncostatin M (OSM), upregulates PAI-1 in cardiac myocytes, astrocytes, and endothelial cells. We used tissue explants and primary cultures of preadipocytes and adipocytes from human subcutaneous and visceral adipose tissue to investigate whether IL-6 and OSM affect PAI-1 expression in fat.. Human subcutaneous and visceral adipose tissue responded to treatment with IL-6 and OSM with a significant increase in PAI-1 production. Human preadipocytes were isolated from subcutaneous and visceral adipose tissue. Adipocyte differentiation was induced by hormone supplementation. All cell types expressed receptors for IL-6 and OSM and produced up to 12-fold increased levels of PAI-1 protein and up to 9-fold increased levels of PAI-1 mRNA on stimulation with IL-6 and OSM. AG-490, a janus kinase/signal transducer and activator of transcription inhibitor, abolished the OSM-dependent PAI-1 induction almost completely.. We have for the first time established a link between the gp130 ligands, the proinflammatory mediators IL-6 and OSM, and the expression of PAI-1 in human adipose tissue. Thus, we speculate that IL-6 and OSM, by upregulating PAI-1 in adipose tissue, can contribute to the increased cardiovascular risk of obese patients.

Topics: Adipose Tissue; Adult; Aged; Antigens, CD; Cells, Cultured; Cytokine Receptor gp130; Enzyme Inhibitors; Humans; Inflammation; Interleukin-6; Ligands; Membrane Glycoproteins; Middle Aged; Oncostatin M; Peptides; Plasminogen Activator Inhibitor 1; Receptors, Cytokine; Receptors, Interleukin-6; Receptors, Oncostatin M; RNA, Messenger; Tyrphostins; Up-Regulation

Hepatitis C virus (HCV) infection is a leading cause a of chronic liver disease worldwide. The main therapeutic regimen is the combination of interferon alpha (IFN) and the nucleoside analog, Ribavirin. IFN initiates an intracellular antiviral state by the JAK-STAT signaling pathway, including a presumed role for STAT1 and STAT2. We have previously shown that the STAT3 activation occurs during IFN treatment of human hepatoma cells, suggesting that the STAT3-mediated pathway is relevant to IFN-induced antiviral activity. In this study, we investigate the role of activated STAT3 in the induction of anti-HCV activity in human hepatoma cells. We demonstrate that the STAT3 activation is involved in efficient IFN-induced anti-HCV activity. Using an inducible, cytokine-independent, STAT3 activation system, in which the entire coding region of STAT3 is fused with the ligand-binding domain of the estrogen receptor, we demonstrate that: activated STAT3 is tightly regulated in a stably transfected cell line by an estrogen analog, 4-HT; activated STAT3 initiates efficient anti-HCV activity in a HCV subgenomic replicon cell line; and activation of STAT3 is associated with the induction of a potential antiviral gene, 1-8U. In addition, we show that the cytokine IL-6, a potent STAT3 activator, inhibits HCV subgenomic RNA replication through STAT3 activation and ERK pathway. These results strongly suggest that STAT3 activation is capable of initiating intracellular antiviral pathways.

Topics: Antiviral Agents; Blotting, Northern; Blotting, Western; Carcinoma, Hepatocellular; Cell Line; Cell Line, Tumor; Cytokines; DNA-Binding Proteins; Dose-Response Relationship, Drug; Enzyme Inhibitors; Genes, Dominant; Hepacivirus; Humans; Inflammation; Interferons; Interleukin-6; Ligands; Liver; Liver Neoplasms; Luciferases; Plasmids; Protein Structure, Tertiary; Reverse Transcriptase Polymerase Chain Reaction; Ribavirin; RNA; RNA, Messenger; STAT3 Transcription Factor; Time Factors; Trans-Activators; Transfection; Tyrphostins