IL-1beta mRNA Expression Research Articles

Molecular Approach to Identify Anti-inflammatory Potential of Stevioside in HFD-induced Type 2 Diabetic Rats: Evidence From in Vivo Study

Stevioside is a natural sweetener derived from the leaves of the Stevia rebaudiana plant. It has gained popularity as a sugar substitute due to its intense sweetness without adding calories or affecting blood sugar levels, making it a suitable option for people with diabetes or those looking to reduce their sugar intake. Studies have shown that stevioside has glucose lowering effects. Previous studies have shown that it has significant role in skeletal muscle but its role on expression of inflammatory signaling molecules in adipose tissue against high diet and sucrose-induced type-2 diabetes in experimental rats is yet to be done. The current research was undertaken to investigate if stevioside could also exert its antidiabetic effects by circumventing adipocyte induced inflammation, a key driving factor for insulin resistance in obese individuals. Effective dose of stevioside (20 mg/kg b.wt) was administered orally for 45 days to high fat diet and sucrose induced type-2 diabetic rats. Interestingly, stevioside treatment restores the elevated serum levels of proinflammatory cytokines including tumor necrosis factor-α (TNF-α) and sterol regulatory element binding protein-1c (SREBP-1c) and enhances Peroxisome Proliferator–activated receptor-γ (PPAR-γ) in adipocytes of diabetic rats. The gene expression of IR, GLUT4 and PPAR-γ mRNA were also significantly activated in stevioside treated groups but reduced IL-1 beta, IlL-6, IKKB, TNF-alpha and NFkB mRNA expression in diabetic adipose tissue. More importantly, stevioside acts very effectively as metformin to circumvent inflammation and insulin resistance in diabetic rats. Our results clearly show that stevioside inhibits obesity induced insulin resistance by ameliorating the inflammatory events and upregulating insulin signalling molecules. Keywords: Stevioside, HFD-T2DM, pro inflammation, insulin signalling; adipose tissue, obesity; signaling pathways; Therapautics.

Treatment of Ischemic Stroke by Atorvastatin-Loaded PEGylated Liposome.

There is insufficient evidence on the effect of nanoparticles, particularly liposomes loaded with a statin, on acute ischemic stroke. We investigated the impact of atorvastatin-loaded PEG (polyethylene glycol) conjugated liposomes (LipoStatin) on the outcomes in rats with cerebral ischemia-reperfusion. PEGylated liposome loaded with atorvastatin was developed as a nanoparticle to specifically accumulate in an ischemic region and release the drug to ameliorate the harmful effects of the stroke. LipoStatin was administered to rats with transient middle cerebral artery occlusion through the tail vein immediately after reperfusion (LipoStatin group). LipoStatin efficiently accumulated at the cerebral ischemic injury site of the rat. The LipoStatin group showed a significantly reduced infarct volume (p < 0.01) in brain micro-MR imaging and improved neurological function recovery compared to the control group (p < 0.05). In addition, markedly improved brain metabolism using fluorine-18 fluorodeoxyglucose micro-PET/CT imaging was demonstrated in the LipoStatin group compared with the control group (p < 0.01). Mechanistically, as a result of evaluation through IL-1 beta, TNF-alpha, ICAM-1, and Iba-1 mRNA expression levels at 5days after cerebral ischemia, LipoStatin showed significant anti-inflammatory effects. Protein expression of occludin, JAM-A, Caveolin-1, and eNOS by western blot at 3days and fluorescent images at 7days showed considerable recovery of blood-brain barrier breakdown and endothelial dysfunction. PEGylated LipoStatin can be more effectively delivered to the ischemic brain and may have significant neuroprotective effects. Thus, PEGylated LipoStatin can be further developed as a promising targeted therapy for ischemic stroke and other major vascular diseases.

Screening of infection conditions for brain microvascular endothelial cells infected by Streptococcus suis

Streptococcus suis is a pathogen that causes swine meningitis, sepsis, and other diseases. There are 34 serotypes, of which type 2 is the most pathogenic. During the infection process of Streptococcus suis, several major virulence factors are involved and play a different roles. Streptococcal meningitis is caused by the bacteria’s ability to cross the blood-brain barrier and enter the central nervous system. Therefore, studying the interaction between Streptococcus suis and cerebral microvascular endothelial cells will help reveal meningitis's pathogenic mechanism. When studying the interaction between bacteria and cells, the number of infected bacteria and the time of infection are very important. In this study, Streptococcus suis serotype two was made into bacteria liquid and counted. Then bacteria were used to infect mouse brain microvascular endothelial cells with different multiplicity of infection (1,10,100 and 200). Cells were harvested at six h, nine h,12h,18h, and 24h after infection. The total RNAs of harvested cells were extracted, and the concentration of RNA was detected. The OD260/OD280 was between 1.8~2.4, OD260/OD230 was 1.5~2.4, and the concentration was greater than 100ng/µL. Total RNAs were reverse transcribed to cDNAs used to perform quantitative PCR to detect the mRNA expression of IL-18, IL-1beta, IL-6, and IL-10. The results showed that each MOI group's mRNA expression is higher than the control group with different infection times. When the multiplicity of infection is at 1, each group's relative expression of cytokines reaches a peak at 18hrs after infection. When the multiplicity of infection is at 10, each group's relative expression of cytokines reaches a peak at 12hrs after infection. When the multiplicity of infection is at 100, the relative expression of cytokines reaches a peak at 12hrs after infection. When the multiplicity of infection is at 200, the relative expressions of each cytokine reach a peak at 6hrs after infection. Based on the mRNA relative expression of each cytokine under different conditions, the optimal multiplicity of infection was 100, and the optimal infection time was 12h. The result provides a basis for the study of the pathogenic mechanism of meningitis.

Regulation of Cytokine and Growth Factor Gene Expression in Human Bone Marrow Stromal Cells Transformed With Simian Virus 40

Marrow stromal cells are thought to regulate hematopoiesis by producing colony-stimulating factors (CSFs) and other cytokines, either constitutively or in response to mediators such as interleukin-1 alpha (IL-1 alpha) or tumor necrosis factor-alpha (TNF alpha). The mechanisms by which these inflammatory cytokines induce CSF expression in stromal cells are not fully defined. In this study, we used human marrow stromal cells transformed by simian virus 40 (SV-MSCs) to study growth factor and cytokine gene regulation in response to IL-1 alpha and TNF alpha. IL-1 alpha induced significant and prolonged increases in steady-state mRNA levels for interleukin-6 (IL-6), interleukin-1 beta (IL-1 beta), granulocyte-macrophage CSF (GM-CSF), and, to a lesser extent, granulocyte-CSF (G-CSF); this induction was not dependent on new protein synthesis. Nuclear run-on analyses showed that IL-1 alpha transcriptionally activated the genes for IL-6, GM-CSF, and IL-1 beta, while TNF alpha transcriptionally induced expression of IL-6 and IL-1 beta. Furthermore, mRNA for IL-6 and IL-1 beta was dramatically superinduced by the combination of cycloheximide and TNF alpha. When SV-MSCs were cultured in semisolid medium, they formed colonies of blast-like cells that, when replated on plastic, resumed adherent growth. These "colony-derived" cell lines, unlike the parental SV-MSCs from which they were derived, constitutively expressed colony-stimulating activity and mRNA for GM-CSF, G-CSF, IL-6, and IL-1 beta. In this report, we show that the expression of IL-6 and IL-1 beta mRNA in the colony-derived cell lines was due, at least in part, to constitutive transcriptional activation of these genes (similar to the findings in IL-1 alpha- and/or TNF alpha-stimulated parental SV-MSCs). However, in contrast to the transcriptional activation of the GM-CSF gene seen in cytokine-induced parental SV-MSCs, GM-CSF transcripts accumulated in the colony-derived cell lines by a posttranscriptional mechanism.

Abstract 772: MicroRNA regulatory networks: Concomitant in situ detection of microRNA, mRNA and protein in cancer samples

Abstract The canonical mode of action of microRNAs involves the binding to the 3'UTR of mRNAs thereby mediating repression of protein synthesis. MicroRNAs comprise an interesting new group of cancer biomarkers and may also be therapeutic targets in cancer and other diseases using antisense oligos (ASO). The visualization of potential molecular dynamics between a microRNA, its target mRNA and the effect of protein expression can be facilitated by combined detection in tissue sections. MicroRNAs, as well as ASOs, can be detected in tissue sections or cultured cells by in situ hybridization (ISH) using LNA probes. We have developed a novel ISH assay for microRNA detection with improved sensitivity and signal-to-noise that can be used with any LNA probe on both FFPE and frozen sections. The assay is based on double-labeled LNA™ probes and detection can be either fluorescence or chromogenic staining. We have combined the fluorescence assay with immunofluorescence, for double and triple staining. We also combined the microRNA ISH assay with mRNA in situ hybridization using the highly specific RNAscope probes. We show a variety of combinations, including examples of combined staining of microRNA-17, IL-1beta mRNA or TNF-alpha mRNA, and cytokeratin proteins in automated triple staining procedures of colon cancer samples. The focal expression of IL-1beta mRNA or TNF-alpha mRNA and the loss of microRNA-17 at the invasive front involving inflammatory cells and budding cancer cells is discussed. Citation Format: Boye Schnack Nielsen. MicroRNA regulatory networks: Concomitant in situ detection of microRNA, mRNA and protein in cancer samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 772.

Influence of dietary eicosapentaenoic acid on the cardiac mechanical and mitochondrial functions during early sepsis

Sepsis triggers the failure of cardiac mitochondria and this deleterious effect could be prevented by omega-3 polyunsaturated fatty acids (PUFA). To evaluate the influence of eicosapentaenoic acid (C20: 5 omega-3 or EPA) on the mechanical and mitochondrial functions of the heart during early sepsis. Four month-old female Wistar rats were subdivided in 4 groups ( n = 8 per group) according to: (i) the 4-week diet given (deficient in omega-3 PUFA [DEF] or enriched with EPA); (ii) the surgery performed (caecal ligation at 1 cm of the apex of the heart and puncture in order to induce sepsis [CLP] or sham). The cardiac mechanical function was studied in vivo by NMR 48 h after the surgery. Immediately after, part of the myocardium was used in order to extract mitochondria and another part was frozen in order to subsequently analyse the fatty acid composition of cardiac phospholipids. The EPA groups display a large decrease in the omega-6/omega-3 PUFA ratio of cardiac phospholipids. Sepsis increases the mRNA expression of IL-1beta in the DEF group but not in the EPA one. The mechanical function is altered neither by the diets nor by the surgeries. The DEF/CLP animals have a deficient oxidative phosphorylation compared to the DEF/Sham rats: reduced mitochondrial oxygen consumption (−42%, P < 0.001) and ATP production (−37%, P < 0.05). The sepsis-induced mitochondrial damages in this group is due to an increase reactive oxygen species (ROS) release (+130 and +63% for the ROS/respiration and ROS/ATP ratios, P < 0.01 and P < 0.05, respectively). The dietary EPA prevents these damages ( P < 0.05). A sufficient EPA intake favours the upholding of the mitochondrial function during early sepsis, which could delay the subsequent development of heart failure. We plan to verify this beneficial effect of EPA in Humans affected by acute endocarditis.

Abstract 614: TRAIL Deletion in Mice Promotes Vascular Insulin Resistance

TNF-related apoptosis-inducing ligand (TRAIL) is protective in cardiovascular disease and diabetes; whether it regulates insulin resistance and inflammation in the vessel wall is unknown. Wildtype and Trail -/- mice were placed on a high fat diet (HFD) for 12 w. Plasma chemistries were assessed; glucose and insulin tolerance tests performed. Histological GLUT4 expression in skeletal muscle was assessed. Aortic inflammatory marker expression and downstream insulin signaling was also measured; myography was performed. In response to a HFD for 12 w, wildtype mice had increased aortic IL-1beta, MCP-1, IL-6, TNF-alpha and TRAIL mRNA, with reduced circulating TRAIL levels. Compared to 12 w HFD-fed wildtype mice, Trail -/- mice had significantly increased plasma glucose, insulin, and cholesterol levels. Insulin intolerance was worse with TRAIL deletion, with reduced GLUT4 expression and glucose uptake in skeletal muscle. In response to insulin, Trail -/- aortas displayed impaired vasorelaxation and reduced aortic p-Akt expression. Importantly, aortic insulin resistance was associated with &gt; 20-fold increased aortic mRNA expression for IL-1beta, IL-6, and TNF-alpha. We show that TRAIL protects against insulin resistance and type-2 diabetes, in part by regulating downstream insulin signals and inflammation in the vessel wall. These findings suggest that TRAIL may offer a novel therapeutic solution to combat inflammation and insulin resistance in diabetic vascular diseases.

Increase of Th17 Cell Phenotype in Kidney Transplant Recipients with Chronic Allograft Dysfunction.

This study was performed to determine the association of Th17 cell phenotype with chronic allograft dysfunction in kidney transplant recipients (KTRs). We compared the expression of Th17 cell phenotype in KTRs with chronic allograft dysfunction group (CAD, n = 52) with four control groups (long-term stable KTRs (LTS, n = 67), early stable KTRs (ES, n = 28), end stage renal disease (ESRD, n = 45), and healthy control (HC, n = 26). We also performed in vitro study using human proximal renal tubular epithelial cell line (HPRTEpiC) to evaluate the effect of IL-17 on human renal tubular epithelial cells. The CAD group showed increased percentage of Th17 cells out of CD4+ T cells and also increased proportion of IL-17 producing cells out of effector memory T cells or out of CCR4+CCR6+/CD4+ T cells compared to the LTS group and other control groups. Also, the serum level of IL-17, IL-33, and RAGE, and the expression of IL-1beta, RAGE, and HMGB1 mRNA showed an increase in the CAD group compared to the LTS group. In vitro study revealed that IL-17 increased production of IL-6 and IL-8 and up-regulated profibrotic gene expression such as ACTA-2 and CTGF in HPRTEpiC in a dose-dependent manner, which suggests that IL-17 has a role in the development of renal tubular cell injury. The results of our study may suggest that increase of Th17 cell phenotype could be a marker for the chronic allograft injury; hence there is a need to develop diagnostic and therapeutic tools targeting the Th17 cells pathway.

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Objectives Preeclampsia (PE) is considered as a major cause of both maternal and fetal morbidity and mortality. Plasma of preeclampsia women has elevated levels of molecular structures associated with stress and cell death, such as hyaluronan (HA), which bind to receptors present in cells of innate immunity and activate an intracellular complex called inflammasome. This study investigated the state of activation both endogenous and induced by HA in monocytes of these patients, by identifying the presence of NLRP1/NLRP3 and its association with IL-1beta, IL-18 and TNF-alpha. Methods Monocytes (5 × 10 5 cell/mL) were obtained from 15 preeclamptic (PE), 15 normotensive (NT) pregnant women and 10 non-pregnant (NP) women. The presence of inflammasome in these cells was evaluated by gene expression of NLRP3, NLRP1, caspase-1, IL-1beta, IL-18 and TNF-alpha by quantitative real-time PCR (RT-qPCR) (endogenous expression) or after in vitro activation with HA (stimulated expression). Differences among groups were analyzed by non-parametric tests with significance level set at 5%. Results In the absence of monocytes stimulation mRNA expression of NLRP3, IL-1beta and TNF-alpha was significantly higher in PE group than in NT and NP groups. mRNA expression of caspase-1 was increased in PE group compared with NT group, while IL-18 mRNA expression was lower in PE than NT and NP groups. No alteration was observed in NLRP1 expression. HA stimulation led to an increase in NLRP3, IL-1beta and TNF-alpha mRNA expression in monocytes from PE group than in NT and NP groups. Conclusions The basal up-regulation of NLRP3, caspase-1, IL-1beta and TNF-alpha mRNA expression in monocytes from preeclamptic women confirms the activated profile of these cells in PE. The higher gene expression of inflammasome by HA stimulation suggests that this component of extracellular matrix may contribute to inflammasome generation in monocytes from preeclamptic women. Financial support – FAPESP 2013/00534-5 and 2012/24697-8 Disclosures M. Romao: None. M.L. Matias: None. I.C. Weel: None. C.J. Fernandes: None. V.T. Borges: None. J.P. Araujo Jr: None. J.C. Peracoli: None. M.T. Peracoli: None.

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Objectives Preeclampsia (PE) is characterized by a state of abnormal activation of the innate immune system. This pathology is associated with hyperuricemia, elevated serum levels of inflammatory cytokines and oxidative stress.Uric acid crystals can activate the inflammasome, a multi-protein complex important for processing and releasing of interleukin-1beta (IL-1beta) and IL-18. This study investigated the endogenous or monosodium urate-induced activation of monocytes from pregnant women with PE by NLRP1/NLRP3 inflammasome identification in these cells as well as association of this complex with IL-1beta, IL-18 and TNF-alpha expression by these cells. Methods Monocytes (5 × 105 cell/mL) were obtained from 20 preeclamptic, 15 normotensive (NT) pregnant women, in the last trimester of pregnancy, and 10 non-pregnant (NP) women. The presence of inflammasome was evaluated by gene expression of NLRP3, NLRP1, caspase-1, IL-1beta, IL-18 and TNF-alpha by quantitative real-time PCR (RT-qPCR) (endogenous expression) or after in vitro stimulation with monosodium urate (MSU) (stimulated expression). Results were analyzed by non-parametric tests with significance level set at 5%. Results In the absence of stimulation, the basal mRNA expression of NLRP3, IL-1beta and TNF-alpha in monocytes was significantly higher in PE than in NT and NP groups. Gene expression of caspase-1 was increased while IL-18 expression was lower in PE compared with NT and NP groups. No alteration was observed in NLRP1 expression. An increase in NLRP3 mRNA was detected after preeclamptic patients monocytes stimulation with MSU, while no alteration was detected in NT and NP groups. Conclusions The basal up-regulation of NLRP3, caspase-1, IL-1beta and TNF-alpha mRNA expression in monocytes from preeclamptic women confirms the activated profile of these cells by the disease process. NLRP3 gene expression in monocytes from women with PE stimulated with MSU suggests that hyperuricemia contributes to the inflammasome involvement in PE pathogenesis. Financial support – FAPESP 2012/21287-3 and 2012/24697-8. Disclosures M.L. Matias: None. M. Romao: None. I.C. Weel: None. P.R. Nunes: None. V.T. Borges: None. L.G. de Oliveira: None. J.P. Araujo Jr.: None. J.C. Peracoli: None. M.T. Peracoli: None.

Inhibition of the hyaluronan oligosaccharides inflammatory response: reduction of adenosine 2A receptor activation by EPAC and PKA.

The aim of this study was to investigate the involvement of exchange proteins directly activated by cyclic adenosine (ADO) monophosphate (EPAC) in 4-mer hyaluronan (HA) oligosaccharide-induced inflammatory response in mouse normal synovial fibroblasts (NSF). Treatment of NSF with 4-mer HA increased Toll-like receptor-4, TNF-alpha and IL-1beta mRNA expression and of the related proteins, as well as nuclear factor kappaB (NF-kB) activation. Addition to NSF, previously stimulated with 4-mer HA oligosaccharides, of ADO significantly reduced NF-kB activation, TNF-alpha and IL-1beta expression. The pre-treatment of NSF with cyclic ADO monophosphate and/or PKA and/or EPAC-specific inhibitors significantly inhibited the anti-inflammatory effect exerted by ADO. In particular, the EPAC inhibitor reduced the ADO effect to a major extent than the PKA inhibitor. These results mean that both PKA and EPAC pathways are involved in ADO-induced NF-kB inhibition although EPAC seems to be more involved than PKA.

Transcription networks responsible for early regulation of Salmonella-induced inflammation in the jejunum of pigs

BackgroundThe aim of this study was to identify transcription factors/regulators that play a crucial role in steering the (innate) immune response shortly (within a few hours) after the first contact of the intestinal mucosa with an inflammatory mediator, and to test whether the processes regulated by these factors/regulators can be modulated by chemical substances of natural origin.MethodsWe experimentally induced inflammation by perfusion of surgically applied jejunal loops with Salmonella enterica subspecies enterica serovar Typhimurium DT104 in three pigs. Segments of mock and Salmonella treated loops were dissected after 2, 4 and 8 hours of perfusion. IL8 and IL1-beta mRNA expression levels were measured in mucosal scrapings of all segments. Furthermore, intra-animal microarray comparisons (isogenic) between Salmonella and mock treated segments after 8 hours, and inter-animal comparisons between similar Salmonella-treated loops of each pig at 2 and 4 hours, were performed.ResultsIL-1beta and IL8 mRNA levels, and intra-animal microarray comparisons at 8 hours between Salmonella and mock treated segments showed that the response-time and type of response to Salmonella was different in all three pigs. This plasticity allowed us to extract a comprehensive set of differentially expressed genes from inter-animal comparisons at 2 and 4 hours. Pathway analysis indicated that many of these genes play a role in induction and/or tempering the inflammatory response in the intestine. Among them a set of transcription factors/regulators known to be involved in regulation of inflammation, but also factors/regulators for which involvement was not expected. Nine out of twenty compounds of natural origin, which according to literature had the potential to modulate the activity of these factors/regulators, were able to stimulate or inhibit a Salmonella-induced mRNA response of inflammatory-reporter genes IL8 and/or nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha in cultured intestinal porcine epithelial cells.ConclusionsWe describe a set of transcription factors/regulators possibly involved in regulation of “very early” immune mechanism which determines the inflammatory status of the intestine later on. In addition, we show that these mechanisms may be modulated by chemical substances of natural origin.

Exogenous Ghrelin Administration Can Ameliorate Indomethacin-Induced Small Intestinal Damage in Mice

Purpose: The arrival of video capsule endoscopy and balloon enteroscopy revealed that non-steroidal anti-inflammatory drugs (NSAIDs) can cause more frequent and more serious complications than formerly believed in the small intestine. However, safe and effective treatment is not available for NSAIDs-induced small intestinal damage. As a possible candidate, we focused on ghrelin, an orexigenic peptide secreted mainly from stomach. Although previous reports revealed that this peptide might show gastroprotection, the effect of ghrelin on NSAIDs-induced small intestinal damage was still vague. In order to examine its therapeutic potential for NSAIDs-induced small intestinal damage, we analyzed the influence of exogenous ghrelin administration on small intestinal protection using experimental mouse model. Methods: Indomethacin or vehicle was subcutaneously administrated to C57BL/6J mice, and ghrelin or vehicle was intraperitoneally administrated before 30 minutes and 2 hours afterwards (vehicle/vehicle group, vehicle/ghrelin group, indomethacin/vehicle group, and indomethacin/ghrelin group). Small intestinal injuries were assessed by macroscopical and microscopical alterations, myeloperoxidase (MPO) activity, prostaglandin E2 (PG E2) and expression of mRNA for cox-1, cox-2, iNOS, TNF-alpha, IL-1beta, and IL-6. Results: Vehicle/vehicle group and vehicle/ghrelin group did not show small intestinal alterations. Indomethacin/ghrelin group significantly modified macroscopical and microscopical scores, and also attenuated NSAIDs-induced changes in MPO activity, PG E2 level and mRNA expression compared those in indomethacin/vehicle group. Conclusion: Ghrelin shows a strong protective efficacy against indomethacin-induced small intestinal damage in mice. Also considering the previous reports on gastroprotective role, ghrelin can prevent NSAIDs-induced mucosal damage through the whole digestive tract, which will lead to a new treatment approach toward the increase of ghrelin expression.

Regulating effect of Ginkgo biloba extract 50 on hippocampal inflammation-related cytokines in senile rats

To investigate the regulating effect of Ginkgo biloba extract 50 (GBE50) on pre-inflammatory factors interleukin-1 beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha) and anti-inflammatory factors interleukin-4 (IL-4), interleukin-10 (IL-10) of hippocampus in senile rats, in order to explore the protective mechanism of GBE50 on central nervous system of senile animals. SD rats were randomly divided into four groups: the normal group, the model group, the GBE50 group and the EGB761 group. Rats were intraperitoneally injected with 100 mg x kg(-1) D-galactose every day for 42 days to establish the senile rat model. At the 21st day, the GBE50 group and the EGB761 group were orally administered with 60 mg x kg(-1) for 21 days. IL-1beta mRNA and TNF-alpha mRNA expressions were detected by real-time fluorescence quantitative PCR assay, IL-1beta and TNF-alpha protein expressions were detected by immunohistochemistry, IL-4 and IL-10 protein contents were detected by ELISA. D-galactose caused imbalance between pre-inflammatory factors and anti-inflammatory factors of hippocampus in senile rats, GBE50 and EGB761 reduced IL-1beta mRNA expression (P < 0.05) and TNF-alpha and IL-1beta protein level (P < 0.01) and up-regulated IL-10 protein content (P < 0.01, P < 0.05). The mechanism of GBE50 in protecting central nervous system is probably related to its effect in mitigating inflammatory of central nervous system.

Bee venom inhibits 12-O-tetradecanoyl-phorbol-13-acetate (TPA) induced mouse skin inflammation

Bee venom (BV) has been used to treat diseases such as arthritis, rheumatism and pain in many countries. Although BV has recently been used as an additive of functional cosmetics, there is no evidence of the effects of BV on skin inflammation or disease. Here, we show that BV patch attachment or intra-peritoneal injection inhibited in vivo acute inflammation induced by 12-O-tetradecanoyl-phorbol-13-acetate (TPA) using the mouse ear model of inflammation. When we examined the TNF-alpha and IL-1 beta mRNA expression in BV treated ear samples by real time PCR, even though BV treatment had no significant effects, it inhibited TNF-alpha and IL-1 beta mRNA expression when compared to samples treated with TPA alone. BV also inhibited edema (based on ear weight) and inhibited neutrophil infiltration (based on myeloperoxidase (MPO) activity measured as a neutrophil marker). Taken together, these results showed that BV exerted an anti-inflammatory effect in vivo and was beneficial in an animal model of skin inflammation. These results suggest that BV might be beneficial as a topical agent for the treatment of skin inflammation.

Acute pyelonephritis and renal scarring are caused by dysfunctional innate immunity in mCxcr2 heterozygous mice

The CXCR1 receptor and chemokine CXCL8 (IL-8) support neutrophil-dependent clearance of uropathogenic Escherichia coli from the urinary tract. CXCR1 is reduced in children prone to pyelonephritis, and heterozygous hCXCR1 polymorphisms are more common in this patient group than in healthy individuals, strongly suggesting a disease association. Since murine CXCR2 (mCXCR2) is functionally similar to human CXCR1, we determined effects of gene heterozygosity on the susceptibility to urinary tract infection by infecting heterozygous (mCxcr2+/−) mice with uropathogenic Escherichia coli. Clearance of infection and tissue damage were assessed as a function of innate immunity in comparison to that in knockout (mCxcr2−/−) and wild-type (mCxcr2+/+) mice. Acute sepsis-associated mortality was increased and bacterial clearance drastically impaired in heterozygous compared to wild-type mice. Chemokine and neutrophil responses were delayed along with evidence of neutrophil retention and unresolved kidney inflammation 1 month after infection. This was accompanied by epithelial proliferation and subepithelial fibrosis. The heterozygous phenotype was intermediate, between knockout and wild-type mice, but specific immune cell infiltrates that accompany chronic infection in knockout mice were not found. Hence, the known heterozygous CXCR1 polymorphisms may predispose patients to acute pyelonephritis and urosepsis.

Administration of obestatin accelerates the healing of chronic gastric ulcers in rats

SummaryBackgroundPrevious studies have shown that administration of obestatin exhibits a protective effect in the pancreas, attenuating the development of acute pancreatitis. The aim of the present study was to investigate the influence of obestatin administration on the healing of chronic gastric ulcers.Material/MethodsChronic gastric ulcers were induced in rats by 100% acetic acid applied to the serosal surface of the gastric wall. Obestatin was given twice a day intraperitoneally at the dose of 4, 8 or 16 nmol/kg/dose for 6 days. Six days after induction of ulcers, rats were anesthetized and the stomach was exposed for measurement of gastric blood flow and ulcer area. Biopsy samples from the gastric mucosa were taken for determination of mucosal DNA synthesis and for measurement of gastric expression of mRNA for interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α).ResultsInduction of gastric ulcers alone increased mucosal blood flow and tissue expression of mRNA for TNF-α and IL-1β, whereas gastric mucosal DNA synthesis was reduced. In rats with gastric ulcers, administration of obestatin increased gastric mucosal blood flow, accelerated the healing rate of these ulcers and partly reversed the gastric ulcer-induced reduction in gastric mucosal DNA synthesis. These results were associated with a reduction in gastric mucosal expression of pro-inflammatory IL-1β and TNF-α.ConclusionsTreatment with obestatin increases gastric mucosal blood flow and cell proliferation, leading to acceleration of healing of gastric ulcers. These effects are associated with a reduction in mucosal expression of pro-inflammatory IL-1β and TNF-α.

Histamine regulates relevant murine dendritic cell functions via H4 receptor

Histamine, produced by dendritic cells (DCs) or by other cells of the immune system, may have significant impact on DC activities. We investigated the influence of histamine and histamine H4 receptor (H4R) on some relevant functions of DCs. Histamine significantly decreased the antigen presentation capacity of splenic DCs, and this effect was reversed by a H4R antagonist. Furthermore, enhanced antigen presentation was detected in H4R-/- DCs. Prolonged histamine treatment during DC differentiation stimulated migration, albeit the increase was not significant. H4R-deficient DCs possessed significantly lower migration capacity than their wild-type counterparts. Monitoring in vivo and in vitro DC cytokine production revealed that a H4R agonist in combination with LPS, increased IL-1 beta mRNA expression, and a H4R antagonist reversed this effect. In H4R-deficient mice we detected decreased mRNA expression of some DC-derived cytokines including IFN-gamma and IL-10. Upon CFA stimulation, genotype-dependent differences were found in the expression of IL-6 and IFN-gamma. Our data suggest that H4R plays a crucial role in variety of functions of murine DCs.

Realgar is active ingredient of Angong Niuhuang pill in protection against LPS-induced neuroinflammation

To determine the effects of Angong Niuhuang pill (AGNHW) on lipopolysaccharide (LPS)-induced neuroinflammation and further to investigate the role of realgar and cinnabar on AGNHW-mediated neuroprotection. Primary rat midbrain neuron-glia cultures were used as an in vitro model to examine the effects of AGNHW on LPS-induced dopamine (DA) neuronal damage. Cultures were divided randomly into five groups: control, LPS, LPS plus AGNHW, LPS plus realgar and LPS plus cinnabar. Dopaminergic neurotoxicity was measured by [3H] DA uptake assay. The production of intracellular reactive oxygen species (ROS) was quantified via the DCFH-DA probe. Real-time RT-PCR was applied to detect the mRNA expression of pro-inflammatory factors. Then the protein levels of these factors were determined by ELISA and western blot assay. Compared with the control group, LPS apparently decreased DA uptake capacity (P < 0.05); induced the production of intracellular ROS (P < 0.05); enhanced the mRNA expression of TNF-alpha, iNOS, IL-13 and COX-2 (P < 0.05) and the release of TNF-alpha, IL-1beta and PGE2 in the supernatant of cultures (P < 0.05); and also increased the level of iNOS protein (P < 0.05). Compared with the LPS group, AGNHW and realgar significantly inhibited LPS-induced reduction of DA uptake (P < 0.05); attenuated the production of intracellular ROS (P < 0.05) and the mRNA expression of TNF-alpha, iNOS, IL-1beta and COX-2 (P < 0.05) and the release of TNF-alpha, IL-1beta and PGE2 (P < 0.05) and the level of iNOS protein (P < 0.05). However, there was no significant difference between LPS group and LPS plus cinnabar group. AGNHW is effective in protecting against LPS-induced neuroinflammation, and realgar is one of active components for AGNHW to produce anti-inflammatory effects.

Greater expression of inflammatory cytokines, adrenomedullin, and natriuretic peptide receptor-C in epicardial adipose tissue in coronary artery disease

Background Growing evidence suggests that the epicardial adipose tissue may have local interactions with coronary arteries. In addition, vasoactive peptides such as adrenomedullin and natriuretic peptide has an interaction with adipose tissue. In this study, we investigated the relationship between adipokines, adipocytokines, and vasoactive peptides expressed in epicardial adipose tissue and subcutaneous adipose tissue in patients with and without coronary artery disease (CAD). Methods We studied 20 patients with CAD and 14 patients without CAD. We obtained blood samples and epicardial and subcutaneous adipose tissue at open-heart surgery. We measured serum cytokine levels and used real-time polymerase chain reaction (PCR) to measure mRNA levels of various molecules in epicardial and subcutaneous tissue and investigated the relation between mRNA levels and clinical parameters. Results The mRNA levels of IL-6, IL-1beta, MCP-1, and TNF-alpha were significantly higher in epicardial adipose tissue than in subcutaneous adipose tissue. Interestingly, the mRNA levels of IL-6, IL-1beta, MCP-1, natriuretic peptide receptor-C (NPR-C), adrenomedullin, and leptin in epicardial adipose tissue were higher in patients with CAD than those without CAD. In contrast, mRNA levels of adiponectin, PPAR-gamma, and NPR-A were similar in the two groups. In subcutaneous tissue, mRNA expressions of IL-6, IL-1beta, MCP-1, NPR-C, adrenomedullin, and leptin were modestly higher in patients with CAD than in those without CAD. There were no differences in plasma cytokine levels between the two groups. Conclusion The mRNA levels of inflammatory cytokines, adipokines, neurohumoral factors and their receptors appear to be increased in epicardial adipose tissue independent of plasma levels of these molecules. Further studies are necessary to elucidate the pathophysiological role of these molecules in CAD.