Important Inflammatory Factor Research Articles

Prognosis of sepsis induced by cecal ligation and puncture in mice improved by anti-Clonorchis Sinensis cyclopholin a antibodies.

BackgroundCyclophilin A (CyPA), a ubiquitously distributed intracellular protein, is thought to be one of the important inflammatory factors and plays a significant role in the development process of sepsis. In the form of cytokine, CyPA deteriorates sepsis by promoting intercellular communication, apoptosis of endothelial cells and chemotactic effect on inflammatory cells. In our previous study, cyclophilin A of Clonorchis sinensis (CsCyPA), a type of excretory-secretory antigen, could induce the patients infected with Clonorchis sinensis to produce specific anti-CsCyPA antibodies. In this study, we investigated whether anti-CsCyPA antibodies could cross-react with CyPA and then play a protective role against sepsis, just like other anti-cytokine antagonists.MethodsThe mice model with sepsis was established with cecal ligation and puncture (CLP). Fifty mg/kg purified anti-CsCyPA antibodies were injected via the caudal vein 6 h after the CLP operation, and persistent observation was performed for 72 h. Blood samples and tissues were collected at 6 h, 12 h, 24 h, 48 h and 72 h after CLP. Cytokines in serum were measured by ELISA. Lung and mesentery tissues were stained with hematoxylin-eosin. Endothelial cells (ECs) isolated from murine aorta were co-cultured with CyPA of mice (MuCyPA) and anti-CsCyPAs for 24 h, then, viability was measured by Cell Counting Kit-8.ResultsAnti-CsCyPA antibodies could combine with MuCyPA and inhibite its peptidyl prolyl isomerase (PPIase) activity. In the antibodies treatment group, blood coagulation indicators including PT, aPTT, D-dimer and platelet count were obviously more ameliorative, the proinflammary factors like IL-6, TNF-α, IL-1β were significantly lower at 12 h and 24 h after surgery and the viability of ECs was significantly improved compared to those in the control group. Furthermore, the survival rate was elevated, ranging from 10.0 % to 45.0 % compared to the control group.ConclusionsThese antibodies may have a favorable effect on sepsis via inhibition of enzymic activity or protection of endothelial cells.

HMGB1 binding to receptor for advanced glycation end products enhances inflammatory responses of human bronchial epithelial cells by activating p38 MAPK and ERK1/2.

The proinflammatory factor high mobility group box protein 1 (HMGB1) has been implicated as an important mediator of many chronic inflammatory diseases, including asthma. Human bronchial epithelial cells (HBECs) play a central role in the pathogenesis of asthma. However, the effects of HMGB1 on HBECs and the underlying mechanisms remain unknown. Here, we investigated receptor expression and proinflammatory cytokine production by primary cultures of HBECs stimulated by HMGB1. We then examined the effects of specific receptor blockade and inhibition of p38 MAPK, ERK1/2, or PI3-K on HMGB1-induced expression of proinflammatory cytokines. HMGB1 increased the expression and secretion of TNF-α, TSLP, MMP-9, and VEGF in a dose- and time-dependent manner. HMGB1 also induced elevated expression of RAGE protein. Secretion of TNF-α, VEGF, MMP-9, and TSLP was significantly decreased by RAGE blockade and p38 MAPK pathway inhibition, while a less pronounced effect was mediated by ERK1/2 inhibition. These observations suggest that HMGB1 binds RAGE and promotes activities of p38 MAPK and ERK1/2 pathways in HBECs. This then enhances the expression of TNF-α, VEGF, MMP-9, and TSLP, which are the important inflammatory factors in asthma. These results demonstrate that HMGB1 enhances the inflammatory responses of HBECs, which are involved in the modulation of inflammatory processes in asthma.

Lactobacillus casei로 발효한 톳 추출물의 항염증 활성

In this study, we investigated the anti-inflammatory effects of fermented Hizikia fusiformis extracts in lipopolysaccharide (LPS)-stimulated RAW 264.7 mouse macrophages. The fermentation was performed using Lactobacillus casei in mixture of carbon source at 30℃ for 30 days. The sample groups were prepared with/without L. casei group in order to demonstrate the anti-inflammatory activity of fermented H. fusiformis in regard to lactic acid bacteria. As a result, we confirmed the inhibitory effect of H. fusiformis extracts on LPS-stimulated NO production and expression of TNFα, while it had no regulatory effect on the expression of iNOS, COX-2, IL-1β and IL-6 as important inflammatory factors. However, L. casei fermented group significantly suppressed the expression of the above factors. In particular, the difference between the two groups in the matter of mRNA expression of iNOS, which is directly associated with NO production, indicated that the fermentation with lactic acid bacteria effectively suppressed NO production by regulating iNOS expression. Also, effective suppression of pro-inflammatory cytokines showed that the fermentation using L. casei may provide an increment towards extraction of active ingredients that are effective anti-inflammatory agents.

JAK2 inhibitor blocks the inflammation and growth of esophageal squamous cell carcinoma in vitro through the JAK/STAT3 pathway

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.

Interleukin-8 does not influence proliferation of the SGC7901 gastric cancer cell line.

Interleukin-8 (IL-8), an important inflammatory factor, is induced by Helicobacter pylori infection and is clearly associated with gastric cancer. IL-8 levels have been revealed to correlate significantly with the adhesion, migration and invasion of gastric cancer cells. However, whether IL-8 influences cell proliferation in gastric cancer remains unclear. In the present study, the effect of IL-8 on the proliferation of the SGC7901 human gastric cancer cell line was investigated. SGC7901 cells were exposed to various concentrations of IL-8 (0, 0.2, 0.5, 0.8 and 1 ng/ml) for one to seven days. Cell proliferation was detected by Cell Counting Kit-8 assay. In addition, proliferating cell nuclear antigen (PCNA) protein and mRNA levels were measured by immunofluorescence, western blotting and quantitative polymerase chain reaction. Following exposure of SGC7901 cells to the various concentrations of IL-8, no significant changes in terms of cell proliferation were identified. However, IL-8 was shown to regulate PCNA protein and mRNA expression levels; at a concentration of 0.8 ng/ml, IL-8 significantly elevated the PCNA protein and mRNA expression levels, whereas IL-8 significantly inhibited these levels at other concentrations, compared with no treatment. In conclusion, IL-8 does not affect the proliferation of SGC7901 cells. However, IL-8 dosage was associated with PCNA protein and mRNA expression levels.

Detecting multiple cell-secreted cytokines from the same aptamer-functionalized electrode

Inflammatory cytokines are secreted by immune cells in response to infection or injury. Quantification of multiple cytokines in parallel may help with disease diagnosis by illuminating inflammatory pathways related to disease onset and progression. This paper describes development of an electrochemical aptasensor for simultaneous detection of two important inflammatory cytokines, interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α). To enable multiplexing, IFN-γ and TNF-α aptamers were labeled with anthraquinone (AQ) and methylene blue (MB) redox reporters respectively. Random immobilization of two aptamer on gold exhibited redox peaks at −0.37V (AQ) and −0.15V (MB) vs. Ag/AgCl reference. When challenged with either IFN-γ or TNF-α, redox signal of the appropriate reporter changed in concentration dependent manner. To demonstrate one possible application of this sensing approach, electrodes were integrated into microfluidic devices and used to dynamically monitor cytokine release from immune cells. Two cell types, primary human CD4 T-cells and U937 monocytic cells, were used to compare differences in cytokine secretions upon stimulation. These cells were infused into the microfluidic devices and stimulated to commence cytokine production. Release of IFN-γ and TNF-α was monitored concurrently from the same small group of cells over the course of 2h. The strategy of encoding specific aptamer types with unique redox reporters allows sensitive and specific detection of multiple protein biomarkers from the same electrode.

Induction of chemokine (C-C motif) ligand 2 by sphingosine-1-phosphate signaling in neuroblastoma

Background/purposeNeuroblastoma (NB) is the most common extracranial solid tumor of childhood. Preliminary data derived from a human angiogenesis array in NB showed that the bioactive lipid sphingosine-1-phosphate (S1P) induced the secretion of several angiogenesis-related proteins including the important inflammatory factor chemokine (C-C motif) ligand 2 (CCL2). In the present study, we investigated the mechanism of S1P-induced CCL2 expression in NB. MethodsQuantitative real-time PCR and CCL2 ELISA were conducted to detect the mRNA expression and protein secretion of CCL2 in NB cells. Gain and loss of function studies were performed by using specific S1PR antagonists, adenoviral transduction and siRNA transfection. Macrophage F4/80 receptor in NB xenografts was detected by quantitative real-time PCR and immunohistochemistry staining. ResultsS1P induced CCL2 mRNA expression and protein secretion in a time- and concentration-dependent manner in NB cells. Blockade of S1P2 signaling using the selective S1P2 antagonist JTE-013 inhibited S1P-induced CCL2 expression. Overexpression of S1P2 by adenoviral transduction increased CCL2 secretion while knockdown of S1P2 by siRNA transfection decreased S1P-induced CCL2 secretion in NB cells. Macrophage infiltration, as detected by F4/80 staining, was significantly decreased in JTE-013-treated NB xenografts. ConclusionsTaken together, our data for the first time demonstrate that S1P induced the macrophage-recruiting factor CCL2 expression in NB cells via S1P2, providing new insights into the complicated functions of S1P2 in cancer.

Preconditioned hyperbaric oxygenation protects skin flap grafts in rats against ischemia/reperfusion injury

Hyperbaric oxygen (HBO) therapy is an effective therapy for ischemia/reperfusion (I/R) injury of the brain, small intestine, testes and liver. However, the detailed molecular mechanisms underlying the effect of HBO therapy remain undetermined. In the current study, the hypothesis that preconditioning rats with HBO protects grafted skin flaps against subsequent I/R injury was investigated. In addition, the molecular mechanisms underlying HBO therapy were characterized by analyzing the roles of the following important inflammatory factors: High mobility group protein 1 (HMGB1) and nuclear factor-κ B (NF-κB). A total of 40 rats were randomly divided into the following five groups: (i) Sham surgery (SH); (ii) ischemia followed by reperfusion 3 days following surgery (I/R3d); (iii) ischemia followed by reperfusion 5 days following surgery (I/R5d); (iv) HBO preconditioning (HBO-PC) and ischemia followed by reperfusion 3 days following surgery (HBO-PC+3d); and (v) HBO-PC and ischemia followed by reperfusion 5 days following surgery (HBO-PC+5d). For the surgical procedure, all pedicled skin flaps were first measured and elevated (9×6 cm). The feeding vessels of the skin flaps were subsequently clamped for 3 h and released to restore blood flow. The rats in the HBO-PC+3d and HBO-PC+5d groups received 1 h HBO for 3 and 5 consecutive days, respectively, prior to surgery. Following surgery, the rats were euthanized, and grafted tissues were collected for western blotting and immunohistochemistry. HBO-PC increased blood perfusion in epigastric skin flaps and attenuated I/R injury following skin flap graft. Additionally, the elevated expression of HMGB1 and NF-κB proteins during I/R injury was attenuated by HBO-PC treatment. HBO-PC may therefore be applied to reduce I/R injury and improve the survival rate of grafted skin flaps. The molecular mechanisms underlying the effect of HBO therapy are associated with the attenuation of inflammatory responses.

Effect of Quanzhenyiqitang on apoptosis of alveolar macrophages and expression of histone deacetylase 2 in rats with chronic obstructive pulmonary disease.

This study aimed to investigate the effect of Quanzhenyiqitang on alveolar macrophages (AMs) in a rat model of chronic obstructive pulmonary disease (COPD). In addition, the induction of apoptosis and regulation of histone deacetylase 2 (HDAC2) was studied to elucidate the underlying mechanisms of Quanzhenyiqitang treatment of COPD. Quanzhenyiqitang-treated serum was applied to AMs obtained from rats with COPD. A blank (control) group, an untreated serum group and an aminophylline group were also observed to evaluate the differences in AM apoptosis status, as well as the expression levels of caspase-9, caspase-8 and HDAC2. Compared with the control group, Quanzhenyiqitang-treated serum resulted in higher levels of caspase-9 and caspase-8 expression, increased apoptosis of AMs and increased expression of HDAC2 by AMs. In conclusion, Quanzhenyiqitang is capable of inducing apoptosis of AMs, which are the primary inflammatory cells in COPD, and modulating the expression of the important inflammatory factor HDAC2, producing an overall anti-inflammatory effect.

Comparison of high-sensitivity C-reactive protein level between systemic and coronary circulation in patients with acute myocardial infarction

Inflammation plays an important role not only in the initiation and progression of atherosclerosis but also in plaque rupture of coronary artery disease (CAD) [1–3], which leads to acute coronary syndrome (ACS) or even acute myocardial infarction (AMI). It is well known that C-reactive protein (CRP) is one of the most important inflammation markers and has been proved to be an independent risk factor for CAD [4–7]. High level of CRP predicts poor clinical outcomes in patients with both stable CAD and ACS [6–9]. Compared with CRP, high-sensitivity CRP (hs-CRP) is more sensitive that can be examined in serum. However, the source of CRP or hs-CRP in CAD or ACS has not been definitively explored. One hypothesis suggested that elevated hs-CRP, which can be measured in systemic circulation, is released from unstable or ruptured plaque in coronary circulation, and then transported to systemic circulation [10]. Another hypothesis suggested that elevated hs-CRP is released from noncoronary circulation tissue (liver cells and fatty tissues) and then transported to coronary circulation, which transforms stable plaque into an unstable or ruptured one, and finally induces ACS or AMI [11,12]. Whether hs-CRP originates from systemic or coronary circulation is still a controversial issue [10–12]. Besides, monocyte is an important chronic inflammatory factor and secretes cytokines such as interleukin-1, interleukin-6, and tumor necrosis factor-a, which stimulates the production of CRP [1–4]. Large amount of monocytes may induce high level of hs-CRP, which predicates high risk of ACS or AMI for patients with CAD. Here, we compared the level of hs-CRP between systemic and coronary circulation in patients with AMI and stable CAD, as well as the difference of monocyte between patients with AMI and stable CAD. The study included 80 patients (60 patients with AMI and 20 patients with single-vessel stable CAD) hospitalized in the Department of Cardiology, the First Afflicted Hospital of Nanjing Medical University from July 2012 to April 2013. All procedures were in accordance with the ethical standards of the responsible committee on human experimentation and with the Helsinki Declaration of 1975, as revised in 2000 [5]. Informed consent was obtained from all the included patients. In the AMI group, coronary angiography was immediately performed after admission, and the time window from the onset of chest pain to the balloon dilatation ranged from 2 to 12 h. In the stable CAD group, patients received coronary angiography 1–2 days after admission. Blood sample was collected from both systemic and coronary circulation. Blood sample of systemic circulation was taken from peripheral arterial puncture, while intracoronary blood sample was taken by using a ‘Diver’ catheter over the wire to the distal site of plaque in the culprit vessel. Fasting blood sample from each site was collected in the coagulation-promoting tubes, and centrifuged at 2100 g for 10 min. Serum was separated and immediately analyzed. Serum level of hs-CRP (mg/l) was measured by the method of rate nephelometry, using Siemens BN-II Special Protein Analyzer (Bonn, Germany). Meanwhile, the level of monocytes (10/l) from systemic circulation was measured. To explore the source of hs-CRP, the difference of hs-CRP level between systemic and coronary circulation was evaluated in each patient. Paired Student’s t-test revealed that the level of hs-CRP was higher in blood sample from systemic circulation than that from coronary circulation in the AMI group (mean difference (MD): 0.67, n 1⁄4 60, 95% confidence interval (CI): 0.08–1.26, P 1⁄4 0.0266); there was no significant difference between systemic and coronary circulation in the stable CAD group (MD: 0.18, n 1⁄4 20, 95% CI: 20.03 to 0.39, P 1⁄4 0.09) (Fig. 1). There was a trend towards higher MD of hs-CRP between systemic and coronary circulation in the AMI group than that in the stable CAD Acta Biochim Biophys Sin 2014, 46: 161–162 |a The Author 2013. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. DOI: 10.1093/abbs/gmt129. Advance Access Publication 2 December 2013

Beneficial effect of TNF-α inhibition on diabetic peripheral neuropathy

BackgroundTumor necrosis factor-α (TNF-α) is an important inflammatory factor produced by activated macrophages and monocytes and plays an important role in the pathogenesis of diabetic peripheral neuropathy (DPN). To evaluate the effect of TNF-α signaling suppression and the potential of TNF-α in the treatment of DPN, a recombinant human TNF-α receptor-antibody fusion protein (rhTNFR:Fc) was used. We focused on the pathophysiology of the sciatic nerve and examined the expression of myelin basic protein (MBP) under DPN status with or without TNF-α inhibition.MethodsThe DPN rat model was generated by intraperitoneal injection of streptozotocin and by feeding with a high-fat, high-sugar diet. The nerve conduction velocity (NCV) in sciatic nerve of rat was monitored over a period of four weeks. The histopathological changes in nerve tissue were examined through traditional tissue histology and ultrastructure transmission electron microscopy (TEM). The expression of MBP was examined through western blot analysis.ResultsThe DPN induced rats showed significant signs of nerve damage including lower NCV, demyelination of nerve fibers, disorganization of lamellar and axonal structures, and decreased expression of MBP in the nerve tissue. The inhibition of TNF-α in the DPN rats resulted in a significant recovery from those symptoms compared to the DPN rats.ConclusionsOur study demonstrates that TNF-α plays a key role in the pathogenesis of DPN and its inhibition by rhTNFR:Fc can prove to be a useful therapeutic strategy for the treatment of and/or prevention from DPN symptoms.

Inflammatory Response Following Diffuse Axonal Injury

DAI is a leading cause of the patient's death or lasting vegetable state following severe TBI, and up to now the detailed mechanism of axonal injury after head trauma is still unclear. Inflammatory responses have been proved to be an important mechanism of neural injury after TBI. However, most of these studies are concerned with focal cerebral injury following head trauma. In contrast to focal injury, studies on the inflammatory reaction following DAI are only beginning. And in this article, we aimed to review such studies. From the studies reviewed, immune response cells would become reactive around the sites of axonal injury after DAI. Besides, the concentrations of several important inflammatory factors, such as IL-1 family, IL-6 and TNF-ɑ, increased after DAI as well, which implies the participation of inflammatory responses. It can be concluded that inflammatory responses probably participate in the neural injury in DAI, but at present the study of inflammatory responses following DAI is still limited and the clear effects of inflammatory response on axonal injury remain to be more explored.

The significance of visfatin in patients with coronary heart disease

Objective To investigate the change of plasma visfatin in patients with coronary heart disease and its clinical significance.Methods A total of 310 patients with coronary heart disease (ACS group:n =217; SAP group:n =93) and of 90 healthy subjects with a matched age and gender were included in this study.The plasma concentration of visfatin of each subject was measured using ELISA assay,and each patient underwent selective coronary angiography examination.A total of 85 cases of patients in CHD group underwent 64-slice CT coronary imaging to evaluate the main plaque within the coronary.Results The visfatin,LDL,BMI,blood glucose levels was significantly higher in CHD group[ ( 128.18 ± 13.86)ng/ml,(3.63 ± 1.48) mmol/L,( 26.18 ± 1.82) kg/m2,(7.25 ± 2.03 ) mmol/L] than in control group [ (75.96 ± 10.27 )ng/ml,(2.64 ± 0.53 ) mmol/L,( 23.51 ± 0.89 ) kg/m2,(5.11 ± 1.53 ) mmol/L,P ＜ 0.05 ],respectively.The visfatin level in the ACS group [ ( 145.57 ± 19.95 ) ng/ml ] was significantly higher than the SAP group [ (110.79 ±7.78)ng/ml,P ＜0.05].The visfatin concentration gradually increased with the aggravation of the complexity of coronary lesion types and severity of coronary lesions( P ＜ 0.05).The plasma visfatin concentrations in Soft plaque group and the fibrous plaque group were significantly higher than calcified plaque (P ＜ 0.05).HDL-C and Gensini score of coronary lesions were negatively correlated ( r =- 0.055,P ＜0.05) ; LDL-C,Glu,and visfatin was positively correlated with coronary lesions Gensini score ( r =0.464,0.279,0.531,P ＜ 0.05 ),respectively.Conclusions The plasma visfatin level in patients with Coronary heart disease increased and affected its lipid metabolism.It may be an important inflammatory factors promoting arterial atherosclerosis occurs and development,which has a certain value to the judgment of coronary lesions and plaque stability combined with 64-slice spiral CT examination. Key words: Adipokines/blood; Coronary disease/metabolism/etiology

Magnesium sulfate inhibits the secretion of high mobility group box 1 from lipopolysaccharide-activated RAW264.7 macrophages in vitro

BackgroundHigh mobility group box 1 (HMGB1) is an important inflammatory factor that is closely related to mortality in patients with sepsis. High magnesium therapy has been proved to reduce sepsis-related mortality and sepsis-induced pathologic complications. These effects result from reduced expression and release of many inflammatory cytokines, although it is not clear whether high magnesium affects the expression and release of HMGB1. In the present study, we explored the effect of magnesium sulfate on the expression and release of HMGB1 in lipopolysaccharide (LPS)-activated macrophages. MethodsRAW264.7 cells were incubated with LPS in the presence or absence of various concentrations of magnesium sulfate. An enzyme-linked immunosorbent assay was used to detect the levels of HMGB1 in the culture supernatant. Real-time polymerase chain reaction was used to assess the expression of HMGB1 mRNA. The nuclear/cytoplasm extraction kit was used to extract the nuclear and cytoplasmic proteins. Western blotting was used to observe the changes in the translocation of HMGB1 from the nucleus to the cytoplasm. The nuclear factor (NF)-κB p50/p65 Transcription Factor Assay Kit was used to analyze NF-κB activity in the nuclear extract. ResultsWe found that magnesium sulfate inhibited translocation of HMGB1 from the nucleus to the cytoplasm and its extracellular release in LPS-activated macrophages and also suppressed the expression of HMGB1 mRNA. Furthermore, magnesium sulfate inhibited the translocation of NF-κB from the cytoplasm to the nucleus in LPS-activated macrophages in a dose-dependent manner. ConclusionsOur study has demonstrated that magnesium sulfate inhibits the translocation of HMGB1 from the nucleus to the cytoplasm and the expression of HMGB1 mRNA in a dose-dependent manner. The mechanism responsible for these effects involves the NF-κB signaling pathway.

Angiotensin II induces NF-κB, JNK and p38 MAPK activation in monocytic cells and increases matrix metalloproteinase-9 expression in a PKC- andRho kinase-dependent manner

Angiotensin II (ANG II), the main effector of the renin-angiotensin system, is implicated in endothelial permeability, recruitment and activation of the immune cells, and also vascular remodeling through induction of inflammatory genes. Matrix metalloproteinases (MMPs) are considered to be important inflammatory factors. Elucidation of ANG II signaling pathways and of possible cross-talks between their components is essential for the development of efficient inhibitory medications. The current study investigates the inflammatory signaling pathways activated by ANG II in cultures of human monocytic U-937 cells, and the effects of specific pharmacological inhibitors of signaling intermediates on MMP-9 gene (MMP-9) expression and activity. MMP-9 expression was determined by real-time PCR and supernatants were analyzed for MMP-9 activity by ELISA and zymography methods. A multi-target ELISA kit was employed to evaluate IκB, NF-κB, JNK, p38, and STAT3 activation following treatments. Stimulation with ANG II (100 nM) significantly increased MMP-9 expression and activity, and also activated NF-κB, JNK, and p38 by 3.8-, 2.8- and 2.2-fold, respectively (P < 0.01). ANG II-induced MMP-9 expression was significantly reduced by 75 and 67%, respectively, by co-incubation of the cells with a selective inhibitor of protein kinase C (GF109203X, 5 µM) or of rho kinase (Y-27632, 15 µM), but not with inhibitors of phosphoinositide 3-kinase (wortmannin, 200 nM), tyrosine kinases (genistein, 100 µM) or of reactive oxygen species (α-tocopherol, 100 µM). Thus, protein kinase C and Rho kinase are important components of the inflammatory signaling pathways activated by ANG II to increase MMP-9 expression in monocytic cells. Both signaling molecules may constitute potential targets for effective management of inflammation.

Troglitazone-activated PPARγ inhibits LPS-induced lung alveolar type II epithelial cells injuries via TNF-α

Acute lung injury (ALI) or acute respiratory distress syndrome (ARDS) are common syndromes characterized by diffuse, acute injury to the alveolar epithelium and pulmonary vascular endothelial cells, with high mortality rate for there are no effective pharmacological therapies. Peroxisome proliferators-activated receptor γ (PPARγ), a member of the nuclear hormone receptor superfamily of ligand-activated transcription factors, is ubiquitously expressed within the lung. Recent studies have indicated PPARγ can protect lung tissue and alleviate pulmonary inflammatory injury. But no studies examined whether PPARγ agonists can protect the alveolar epithelial cells cultured in vitro. We observed the protective effect of PPARγ in LPS-induced alveolar type II epithelial cells injury. The results showed troglitazone-activated PPARγ could inhibit the production of TNF-α, one of the most important inflammatory factors, and then increased the expression of surfactant-associated protein A (SP-A) and attenuate the apoptosis of alveolar type II epithelial cells. Our results suggest that PPARγ may have a potential therapeutic effect on ALI.

Inhibition of LPS‐induced C/EBPδ by trichostatin a has a positive effect on LPS‐induced cyclooxygenase 2 expression in RAW264.7 cells

Cyclooxygenase 2 (COX-2) is an important inflammatory factor. Previous studies have indicated that COX-2 is induced with lipopolysaccharide (LPS) treatment. Here, we found that an inhibitor of histone deacetylase (HDAC), trichostatin A (TSA), cannot repress LPS-induced COX-2 but it increased the COX-2 level in RAW264.7 cells. We found no significant difference in NF-kappaB activation and ERK1/2 phosphorylation, but LPS-induced C/EBP delta expression was completely abolished after TSA treatment of LPS-treated cells. Interesting, reporter assay of C/EBP delta promoter revealed that Sp1-binding site is important. Although there was no alteration in c-Jun levels, but the phosphorylation of c-Jun at its C-terminus was increased dramatically. A DNA-associated protein assay (DAPA) and chromatin immunoprecipitation assay (ChIP) indicated that c-Jun was recruited via Sp1 to the promoter of C/EBP delta after LPS treatment; this recruitment of c-Jun was repressed by TSA. C/EBP delta inhibition by TSA resulted in increased binding of C/EBP alpha and C/EBP beta to the COX-2 promoter. Therefore, TSA has a positive effect on LPS-induced COX-2 since it decreases the C/EBP delta level by reducing c-Jun recruitment by Sp1 to the C/EBP delta promoter, resulting in increased the recruitment of C/EBP alpha and C/EBP beta to the COX-2 promoter.

S1747 Corticotropin Releasing Factor Overexpressing (CRF-OE) Mice Display Upregulation of Proinflammatory Markers in the Colon: A Possible Implication in the Influence of Chronic Stress on the Inflammatory Bowel Diseases (IBD)

Background: Stress has an impact on disease flares, with the majority of IBD patients endorsing a link between their illness and stress (Am J Gastroenterol 2006; 101:775). Chronic stress was also found to be strongly predictive of subsequent IBD relapse (Health Psychol 2002; 21:531; Gut 2008; 57:1386). CRF is a key mediator of stress response and CRF-OE mice showed chronic stress-like neuroendocrine, autonomic and immunological changes and are a valid animal model for chronic stress (Peptides 2001; 22:733). CRF deficiency reduced colonic inflammation in a mouse model of experimental colitis (Endocrinology 2008; 149:3403). Aims: 1. To characterize CRF gene expression and upregulation in the colon in transgenic mice overexpressing CRF compared with the hypothalamus. 2. To assess gene expression of proand anti-inflammatory markers in the mouse colon under CRF overexpression as a chronic stress condition. Methods: The proximal colon and hypothalamus were collected from female CRF-OE mice and their wild type (WT) littermates (Oregon Health and Science University, Portland, OR; 22-28 g, 5-6/group). Gene expression of CRF in the proximal colon and hypothalamus (positive control) and proinflammatory markers (MCP-1, MIP-1α, IL-1β, TNF-α, iNos, Cox2), early growth response gene-1 (Egr-1), an important inflammatory transcription factor and antiinflammatory markers (IL-6, TGF-β) in the proximal colon were assessed using reverse transcription polymerase chain reaction (RT-PCR) and real time quantitative PCR. Results: CRF transcript was significantly elevated by 2.8 fold in hypothalamus and also in the proximal colon by 2.2 fold in CRF-OE mice compared to WT mice. The gene expression of proinflammatory markers and Egr-1 was significantly upregulated in the proximal colon of CRF-OE mice while antiinflammatory markers detected in this study were unchanged (table 1). Conclusion: These data show that the CRF gene is expressed and upregulated in the colon like in the hypothalamus in the CRF-OE mice and established new evidence that chronic stress promotes proinflammation in the mouse colon which may play an important proinflammatory role in IBS patients. (Supproted by NIH grand DK057238, DK41301). Table 1. CRF overexpression induces upregulation of proinflammatory markers in mice colon (% wild type (WT) mice; *p<0.05 vs WT mice)

TNF-α/NF-κB/Snail pathway in cancer cell migration and invasion

Tumour necrosis factor-alpha (TNF-α) is an important inflammatory factor that acts as a master switch in establishing an intricate link between inflammation and cancer. A wide variety of evidence has pointed to a critical role of TNF-α in tumour proliferation, migration, invasion and angiogenesis. The function of TNF-α as a key regulator of the tumour microenvironment is well recognised. We will emphasise the contribution of TNF-α and the nuclear factor-κB pathway on tumour cell invasion and metastasis. Understanding the mechanisms underlying inflammation-mediated metastasis will reveal new therapeutic targets for cancer prevention and treatment.

Trichostatin A Inhibits LPS‐induced Cyclooxygenase 2 Expression Through Inhibition of C/EBPδ Gene Expression in RAW264.7 Cells

Cyclooxygenase 2 (COX‐2) is an important inflammatory factor. Previous studies have indicated that COX‐2 could be induced under LPS treatment, and could be inhibited by trichostatin A (TSA). Our previous research reveals that C/EBPδ induced by LPS is through the activation of the pathway of ERK/JNK1/2/p38 and NF‐κB, followed by the recruitment of c‐Rel and c‐Jun to the C/EBPδ promoter. Herein we found trichostatin A (TSA) could inhibit LPS‐induced Cox‐2 gene expression is due to the inhibition of C/EBPδ. Furthermore, the inhibition of C/EBPδ was through preventing c‐Jun recruitment by Sp1 to the C/EBPδ promoter, but not due to the repression of ERK or NF‐κB pathway. In addition, we also found that the interaction of Sp1 and c‐Jun was inhibited under TSA treatment. At last, it is interesting that c‐terminus of c‐Jun was phosphorylated under TSA treatment. To detect the level of PP2B under TSA treatment found that the level of PP2B was reduced. Taken together, inhibition of LPS‐induced COX‐2 by TSA was due to repression of C/EBPδ, and C/EBPδ down‐regulation was through the inhibition of PP2B led the highly phosphorylation at c‐terminus of to repress the interaction of c‐Jun and Sp1.