Regulation of circADAMTS6-miR-324-5p-PIK3R3 ceRNA pathway may be a novel mechanism of IL-1ß-induced osteoarthritic chondrocytes.

INTRODUCTION: A disintegrin and metallopeptidase with thrombospondin type 1 motif 6 (ADAMTS6)-derived circular RNA (circADAMTS6; hsa_circ_0008667) is a novel regulator in interleukin (IL)-1ß-induced apoptosis of human chondrocytes (HCs). Here, we planned to probe into its role and mechanism underlying HCs injury in osteoarthritis. MATERIALS AND METHODS: Real time-quantitative PCR and immunoblotting estimated the abundance of RNA and protein, respectively. Cell proliferation and apoptosis were measured by WST-8, EdU, fluorescein isothiocyanate, and caspase3/7 activity assays. Levels of inflammatory cytokines (IL-6 and tumor necrosis factor-α), apoptosis-related proteins (Bcl-2 and Bcl-2-associated X protein), extracellular matrix (ECM)-related proteins (matrix metalloproteinase-13 and collagen type II alpha-1), and PI3K/AKT/mTOR signaling pathway-related proteins (AKT, mTOR, phosphorylated-AKT, and phosphorylated-mTOR) were evaluated by enzyme-linked immunosorbent assays and immunoblotting. Target relationship was confirmed by dual-luciferase reporter, Argonaute-2 immunoprecipitation and RNA pull-down assays. RESULTS: Abundances of circADAMTS6 and phosphoinositide-3-kinase regulatory subunit 3 (PIK3R3) were underexpressed, and microRNA (miR)-324-5p was elevated in human osteoarthritic tissues and IL-1ß-induced HCs. Overexpressing circADAMTS6 and inhibiting miR-324-5p enhanced proliferation and ECM synthesis, but suppressed apoptosis and inflammatory response in IL-1ß-challenged HCs. Besides, silencing circADAMTS6 caused similar effects of IL-1ß stress on HCs. Mechanically, there was a direct interaction between miR-324-5p and circADAMTS6 or PIK3R3, and IL-1ß-induced activation of PI3K/AKT/mTOR signaling pathway was suppressed by circADAMTS6 overexpression and miR-324-5p silencing. Furthermore, counteractive effects of miR-324-5p upregulation on circADAMTS6 overexpression and PIK3R3 knockdown on miR-324-5p silencing were observed. CONCLUSION: CircADAMTS6-miR-324-5p-PIK3R3 axis might participate in IL-1ß-induced HCs dysfunction via competing endogenous RNA mechanism and the PI3K/AKT/mTOR signaling pathway.

Cyanidin attenuates the apoptosis of rat nucleus pulposus cells and the degeneration of intervertebral disc via the JAK2/STAT3 signal pathway in vitro and in vivo.

CONTEXT: Cyanidin has been shown to have therapeutic potential in osteoarthritis. However, it is unclear whether cyanidin prevents the progression of intervertebral disc degeneration (IVDD). OBJECTIVE: This study evaluates the effects of cyanidin on IVDD in vitro and in vivo. MATERIALS AND METHODS: Nucleus pulposus cells (NPCs) isolated from lumbar IVD of 4-week-old male Sprague-Dawley (SD) rats were exposed to 20 ng/mL IL-1ß, and then treated with different doses (0-120 µM) of cyanidin for 24 h. SD rats were classified into three groups (n = 8) and treated as follows: control (normal saline), IVDD (vehicle), IVDD + cyanidin (50 mg/kg). Cyanidin was administered intraperitoneally for 8 weeks. RESULTS: The IC50 of cyanidin for NPCs was 94.78 µM, and cyanidin had no toxicity at concentrations up to 500 mg/kg in SD rats. Cyanidin inhibited the apoptosis of NPCs induced by IL-1ß (12.73 ± 0.61% vs. 18.54 ± 0.60%), promoted collagen II (0.82-fold) and aggrecan (0.81-fold) expression, while reducing MMP-13 (1.02-fold) and ADAMTS-5 (1.40-fold) expression. Cyanidin increased the formation of autophagosomes in IL-1ß-induced NPCs, and promoted LC3II/LC3I (0.83-fold) and beclin-1 (0.85-fold) expression, which could be reversed by chloroquine. Cyanidin inhibited the phosphorylation of JAK2 (0.47-fold) and STAT3 (0.53-fold) in IL-1ß-induced NPCs. The effects of cyanidin could be enhanced by AG490. Furthermore, cyanidin mitigated disc degeneration in IVDD rats in vivo. DISCUSSION AND CONCLUSIONS: Cyanidin improved the function of NPCs in IVDD by regulating the JAK2/STAT3 pathway, which may provide a novel alternative strategy for IVDD. The mechanism of cyanidin improving IVDD still needs further work for in-depth investigation.

Acute Inflammation Alters Brain Energy Metabolism in Mice and Humans: Role in Suppressed Spontaneous Activity, Impaired Cognition, and Delirium.

Systemic infection triggers a spectrum of metabolic and behavioral changes, collectively termed sickness behavior, which while adaptive, can affect mood and cognition. In vulnerable individuals, acute illness can also produce profound, maladaptive, cognitive dysfunction including delirium, but our understanding of delirium pathophysiology remains limited. Here, we used bacterial lipopolysaccharide (LPS) in female C57BL/6J mice and acute hip fracture in humans to address whether disrupted energy metabolism contributes to inflammation-induced behavioral and cognitive changes. LPS (250 µg/kg) induced hypoglycemia, which was mimicked by interleukin (IL)-1ß (25 µg/kg) but not prevented in IL-1RI-/- mice, nor by IL-1 receptor antagonist (IL-1RA; 10 mg/kg). LPS suppression of locomotor activity correlated with blood glucose concentrations, was mitigated by exogenous glucose (2 g/kg), and was exacerbated by 2-deoxyglucose (2-DG) glycolytic inhibition, despite preventing IL-1ß synthesis. Using the ME7 model of chronic neurodegeneration in female mice, to examine vulnerability of the diseased brain to acute stressors, we showed that LPS (100 µg/kg) produced acute cognitive dysfunction, selectively in those animals. These acute cognitive impairments were mimicked by insulin (11.5 IU/kg) and mitigated by glucose, demonstrating that acutely reduced glucose metabolism impairs cognition selectively in the vulnerable brain. To test whether these acute changes might predict altered carbohydrate metabolism during delirium, we assessed glycolytic metabolite levels in CSF in humans during inflammatory trauma-induced delirium. Hip fracture patients showed elevated CSF lactate and pyruvate during delirium, consistent with acutely altered brain energy metabolism. Collectively, the data suggest that disruption of energy metabolism drives behavioral and cognitive consequences of acute systemic inflammation.SIGNIFICANCE STATEMENT Acute systemic inflammation alters behavior and produces disproportionate effects, such as delirium, in vulnerable individuals. Delirium has serious short and long-term sequelae but mechanisms remain unclear. Here, we show that both LPS and interleukin (IL)-1ß trigger hypoglycemia, reduce CSF glucose, and suppress spontaneous activity. Exogenous glucose mitigates these outcomes. Equivalent hypoglycemia, induced by lipopolysaccharide (LPS) or insulin, was sufficient to trigger cognitive impairment selectively in animals with existing neurodegeneration and glucose also mitigated those impairments. Patient CSF from inflammatory trauma-induced delirium also shows altered brain carbohydrate metabolism. The data suggest that the degenerating brain is exquisitely sensitive to acute behavioral and cognitive consequences of disrupted energy metabolism. Thus "bioenergetic stress" drives systemic inflammation-induced dysfunction. Elucidating this may offer routes to mitigating delirium.

Investigation of synovial fluid lubricants and inflammatory cytokines in the horse: a comparison of recombinant equine interleukin 1 beta-induced synovitis and joint lavage models.

BACKGROUND: Lameness is a debilitating condition in equine athletes that leads to more performance limitation and loss of use than any other medical condition. There are a limited number of non-terminal experimental models that can be used to study early inflammatory and synovial fluid biophysical changes that occur in the equine joint. Here, we compare the well-established carpal IL-1ß-induced synovitis model to a tarsal intra-articular lavage model, focusing on serial changes in synovial fluid inflammatory cytokines/chemokines and the synovial fluid lubricating molecules lubricin/proteoglycan 4 and hyaluronic acid. The objectives of this study were to evaluate clinical signs; synovial membrane and synovial fluid inflammation; and synovial fluid lubricants and biophysical properties in response to carpal IL-1ß synovitis and tarsal intra-articular lavage. RESULTS: Hyaluronic acid (HA) concentrations, especially high molecular weight HA, and synovial fluid viscosity decreased after both synovitis and lavage interventions. Synovial fluid lubricin concentrations increased 17-20-fold for both synovitis and lavage models, with similar changes in both affected and contralateral joints, suggesting that repeated arthrocentesis alone resulted in elevated synovial fluid lubricin concentrations. Synovitis resulted in a more severe inflammatory response based on clinical signs (temperature, heart rate, respiratory rate, lameness and joint effusion) and clinicopathological and biochemical parameters (white blood cell count, total protein, prostaglandin E2, sulfated glycosaminoglycans, tumor necrosis factor-α and CC chemokine ligands - 2, - 3, - 5 and - 11) as compared to lavage. CONCLUSIONS: Synovial fluid lubricin increased in response to IL-1ß synovitis and joint lavage but also as a result of repeated arthrocentesis. Frequent repeated arthrocentesis is associated with inflammatory changes, including increased sulfated glycosaminoglycan concentrations and decreased hyaluronic acid concentrations. Synovitis results in more significant inflammatory changes than joint lavage. Our data suggests that synovial fluid lubricin, TNF-α, CCL2, CCL3, CCL5, CCL11 and sGAG may be useful biomarkers for synovitis and post-lavage joint inflammation. Caution should be exercised when performing repeated arthrocentesis clinically or in experimental studies due to the inflammatory response and loss of HA and synovial fluid viscosity.

Ultra-Low Dose Cytokines in Rheumatoid Arthritis, Three Birds with One Stone as the Rationale of the 2LARTH® Micro-Immunotherapy Treatment.

Tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) are two cytokines involved in the perpetuation of the chronic inflammation state characterizing rheumatoid arthritis (RA). Significant advances in the treatment of this pathology have been made over the past ten years, partially through the development of anti-TNF and anti-IL-1 therapies. However, major side effects still persist and new alternative therapies should be considered. The formulation of the micro-immunotherapy medicine (MIM) 2LARTH® uses ultra-low doses (ULD) of TNF-α, IL-1ß, and IL-2, in association with other immune factors, to gently restore the body's homeostasis. The first part of this review aims at delineating the pivotal roles played by IL-1ß and TNF-α in RA physiopathology, leading to the development of anti-TNF and anti-IL-1 therapeutic agents. In a second part, an emphasis will be made on explaining the rationale of using multiple therapeutic targets, including both IL-1ß and TNF-α in 2LARTH® medicine. Particular attention will be paid to the ULD of those two main pro-inflammatory factors in order to counteract their overexpression through the lens of their molecular implication in RA pathogenesis.

Effects of adipokine administration to the hypothalamic preoptic area on body temperature in rats.

The effects of adipokine administration to the hypothalamic preoptic area (POA), which is one of the body temperature (BT) regulation centers in the central nervous system, on BT were investigated in male Wistar rats. BT was measured in conscious rats using telemetry. Insulin-like growth factor-1 (IGF-1), interleukin-1ß (IL-1ß), monocyte chemoattractant protein-1 and lipocalin-2 produced hyperthermia, and the effects induced by IL-1ß (25 ng) and IGF-1 (5 µg) were sustainable and remarkable. IL-6 did not show any significant effect. The IGF-1-induced effect was inhibited by pretreatment with IGF binding protein 3 (IGFBP3) or NVP-AEW541 (NVP, a selective inhibitor of type 1 IGF receptor tyrosine kinase, IGF1R TK). NVP-induced inhibition was observed only in the early phase of IGF-1-induced hyperthermia. In addition, IGF-1 increased the IL-1ß concentration in the microdialysate of POA perfusion, but did not increase the IL-1ß concentration in the plasma or the PGE2 concentration in the microdialysate. These findings suggested that IGF-1 produced hyperthermia, which was mediated, at least a part, through an increased IL-1ß concentration after activation of IGF1R TK in the POA, and the IGF-IGFBP system possibly participates in BT homeostasis in the POA.

LncRNA RMRP knockdown promotes proliferation and inhibits apoptosis in osteoarthritis chondrocytes by miR-206/CDK9 axis.

The etiology of osteoarthritis (OA) has been discussed widely, but the molecular mechanisms beneath OA aggravation have not yet been investigated in detail. This study focused on the role of lncRNA RMRP (RMRP) on OA progression. We found that the expression of RMRP was significantly increased in cartilage tissues of patients with OA. CCK-8 and colony formation assays showed that RMRP knockdown promoted proliferation of chondrocytes treated with IL-1ß. Flow cytometry and caspase-3 activity analysis indicated that RMRP silence inhibited apoptosis of chondrocytes treated with IL-1ß. Moreover, luciferase reporter, RNA pull-down and RIP assays showed that RMRP competing with miR-206. Additionally, CDK9 acted as a direct target of miR-206. Moreover, rescue assays indicated that miR-206 inhibitor or pcDNA-CDK9 reversed the effects of RMRP suppression on the proliferation and apoptosis of chondrocytes. Taken together, our results indicated that RMRP knockdown could promote proliferation and inhibit apoptosis in OA chondrocytes via the miR-206/CDK9 axis.

Induction of Adaptive Immunity Leads to Nigrostriatal Disease Progression in MPTP Mouse Model of Parkinson's Disease.

Although the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model is the most widely used animal model for Parkinson's disease (PD), it is known that nigrostriatal pathologies do not persist in the acute MPTP mouse model. This study highlights the importance of adaptive immunity in driving persistent and progressive disease in acute MPTP-intoxicated mice. Although marked infiltration of T cells into the nigra was found on 1 d of MPTP insult, T cell infiltration decreased afterward, becoming normal on 30 d of insult. Interestingly, twice-weekly supplementation of RANTES and eotaxin, chemokines that are involved in T cell trafficking, drove continuous T cell infiltration to the nigra and incessant glial inflammation. Supplementation of RANTES and eotaxin was also associated with the induction of nigral α-synuclein pathology, persistent loss of dopaminergic neurons and striatal neurotransmitters, and continuous impairment of motor functions in MPTP-intoxicated mice. In contrast, supplementation of TNF-α and IL-1ß, widely studied proinflammatory cytokines, did not induce persistent disease in MPTP-insulted mice. Our results suggest that induction of adaptive immunity by RANTES and eotaxin could hold the key for driving persistent nigrostriatal pathologies in the MPTP mouse model, and that targeting these factors may halt disease progression in PD patients.

Inflammasome and Fas-Mediated IL-1ß Contributes to Th17/Th1 Cell Induction in Pathogenic Bacterial Infection In Vivo.

CD4+ Th cells play crucial roles in orchestrating immune responses against pathogenic microbes, after differentiating into effector subsets. Recent research has revealed the importance of IFN-γ and IL-17 double-producing CD4+ Th cells, termed Th17/Th1 cells, in the induction of autoimmune and inflammatory diseases. In addition, Th17/Th1 cells are involved in the regulation of infection caused by the intracellular bacterium Mycobacterium tuberculosis in humans. However, the precise mechanism of Th17/Th1 induction during pathogen infection is unclear. In this study, we showed that the inflammasome and Fas-dependent IL-1ß induces Th17/Th1 cells in mice, in response to infection with the pathogenic intracellular bacterium Listeria monocytogenes In the spleens of infected wild-type mice, Th17/Th1 cells were induced, and expressed T-bet and Rorγt. In Pycard-/- mice, which lack the adaptor molecule of the inflammasome (apoptosis-associated speck-like protein containing a caspase recruitment domain), Th17/Th1 induction was abolished. In addition, the Fas-mediated IL-1ß production was required for Th17/Th1 induction during bacterial infection: Th17/Th1 induction was abolished in Fas-/- mice, whereas supplementation with recombinant IL-1ß restored Th17/Th1 induction via IL-1 receptor 1 (IL-1R1), and rescued the mortality of Fas-/- mice infected with Listeria IL-1R1, but not apoptosis-associated speck-like protein containing a caspase recruitment domain or Fas on T cells, was required for Th17/Th1 induction, indicating that IL-1ß stimulates IL-1R1 on T cells for Th17/Th1 induction. These results indicate that IL-1ß, produced by the inflammasome and Fas-dependent mechanisms, contributes cooperatively to the Th17/Th1 induction during bacterial infection. This study provides a deeper understanding of the molecular mechanisms underlying Th17/Th1 induction during pathogenic microbial infections in vivo.

Protein kinase C-ε contributes to a chronic inhibitory effect of IL-1ß on voltage-gated sodium channels in mice with febrile seizure.

This study aimed to understand the role of Interleukin-1ß in mouse febrile seizures. To investigate the chronic effects of raised Interleukin-1ß on seizures, the sodium currents of hippocampal neurons were recorded by whole-cell voltage clamp. Interleukin-1ß inhibited sodium currents in mouse hippocampal neurons and verified that protein kinase C epsilon contributed to the effect of Interleukin-1ß exposure. The inhibitory effect was also identified in neurons from a protein kinase C epsilon null mutant mouse. Action potentials were recorded using a ramp depolarizing current. Peak spike depolarization was significantly reduced by Interleukin-1ß treatment, and was abolished following the administration of a protein kinase C epsilon inhibitor, εV1-2. However, neither Interleukin-1ß nor εV1-2 had any significant effect on spike threshold. Interleukin-1ß reduced the amplitude of action potentials due to its inhibitory effect on sodium channels. This is hypothesised to decrease the release of presynaptic transmitters of neuroexcitability, thus exerting a neuroprotective role in excitotoxicity. To ascertain the role of protein kinase C epsilon on febrile seizures in vivo, a heated water-bath model was used to identify susceptible mice. It was found that protein kinase C epsilon reduced susceptibility to, and frequency of, febrile seizure onset. This may be related to the neuroprotective effect of Interleukin-1ß on hippocampal neurons.

Blood-borne interleukin-1ß acts on the subfornical organ to upregulate the sympathoexcitatory milieu of the hypothalamic paraventricular nucleus.

We previously reported that microinjection of the proinflammatory cytokine interleukin-1ß (IL-1ß) into the subfornical organ (SFO) elicits a pressor response accompanied by increases in inflammation and renin-angiotensin system (RAS) activity in the SFO and hypothalamic paraventricular nucleus (PVN). The present study sought to determine whether blood-borne IL-1ß induces similar neurochemical changes in the SFO and PVN and, if so, whether increased inflammation and RAS activity at the SFO level orchestrate the sympathoexcitatory response to circulating IL-1ß. In urethane-anesthetized male Sprague-Dawley rats, intravenous injection of IL-1ß (500 ng) increased blood pressure, heart rate, renal sympathetic nerve activity, and mRNA for angiotensin-converting enzyme, angiotensin II type 1a receptor, cyclooxygenase-2, tumor necrosis factor-α, and IL-1ß, as well as the tumor necrosis factor-α p55 receptor and the IL-1 receptor, in the SFO and PVN. Pretreatment with SFO microinjections of the angiotensin II type 1a receptor blocker losartan (1 µg), the angiotensin-converting enzyme inhibitor captopril (1 µg), or the cyclooxygenase-2 inhibitor NS-398 (2 µg) attenuated expression of these excitatory mediators in the SFO and downstream in the PVN and the IL-1ß-induced pressor responses. An SFO lesion minimized the IL-1ß-induced expression of inflammatory and RAS components as well as c-Fos, an indicator of neuronal excitation, in the PVN. These studies demonstrate that circulating IL-1ß, which increases in cardiovascular disorders such as hypertension and heart failure, acts on the SFO to increase inflammation and RAS activity in the SFO and PVN and that intervening in these neurochemical processes in the SFO can significantly reduce the sympathetic response.

Developmental prefrontal mRNA expression of D2 dopamine receptor splice variants and working memory impairments in rats after early life Interleukin-1ß elevation.

Long (D2L) and Short (D2S) isoforms of D2 dopamine receptor differ in their biochemical and physiological properties, which could affect functioning of prefrontal cortex. Contribution of distinct D2 dopamine receptor isoforms to cognitive dysfunctions and its developmental regulation are currently not fully elucidated. In the present study, we evaluated developmental mRNA expression of D2S/D2L dopamine receptor isoforms within the rat medial prefrontal cortex (mPFC) in the model of neurodevelopmental cognitive dysfunction. Working memory performance (Y-maze spontaneous alternations) and D2S/D2L mRNA expression in the mPFC (by qRT-PCR) were evaluated in juvenile (P27), adolescent (P42-47) and adult (P75-90) rats after chronic early life treatment with proinflammatory cytokine interleukin (IL)-1ß (1 µg/kg i.p. daily P15-21). It was shown that IL-1ß elevation during the 3rd week of life leads to working memory deficit originating in juvenile animals and persisting into adulthood. D2S mRNA expression was strongly downregulated during adolescence, and such downregulation was exaggerated in animals injected with IL-1ß during P15-21. Early life IL-1ß administrations influenced developmental changes in the D2S/D2L mRNA ratio. This measure was found to be decreased in adolescent and adult control (intact and vehicle-treated) rats compared to juvenile control, while in the case of IL-1ß-treated animals, the decrease in D2S/D2L ratio was observed only in adulthood but not in adolescence compared to juvenile rats. During the adolescence, D2S mRNA expression was downregulated and D2S/D2L ratio was upregulated in the mPFC of rats treated with IL-1ß during the 3rd week of life compared to controls. Based on these data we conclude that changes in the developmental expression of D2 dopamine receptor splice variants within mPFC may underlie long-lasting cognitive deficit associated with neonatal pathology.

Nebulization of Cyclic Arginine-Glycine-(D)-Aspartic Acid-Peptide Grafted and Drug Encapsulated Liposomes for Inhibition of Acute Lung Injury.

PURPOSE: Acute lung injury (ALI) is a fatal syndrome in critically ill patients. It is characterized by lung edema and inflammation. Numerous pro-inflammatory mediators are released into alveoli. Among them, interleukin-1beta (IL-1ß) causes an increase in solute permeability across the alveolar-capillary barrier leading to edema. It activates key effector cells (alveolar epithelial and endothelial cells) releasing inflammatory chemokines and cytokines. The purpose of the study was to demonstrate that nebulized liposomes inhibit ALI in vivo. METHODS: In vivo ALI model was simulated through intra-tracheal instillation of IL-1ß solution (100 µg/mL in PBS, pH 7.2, 200 µL) in male Sprague-Dawley rats. Various formulations were tested in ALI induced rats. These formulations include plain liposomes (PL), methylprednisolone sodium succinate solution (MPS solution), cRGD-peptide grafted liposomes (LcRGD) and methylprednisolone sodium succinate encapsulated and cRGD-peptide grafted liposomes (MPS-LcRGD). Formulations were nebulized in vivo in rats using micro-pump nebulizer. RESULTS: Liposome formulations exhibited higher levels of drug concentration in lungs. The physicochemical parameters demonstrated that the liposome formulations were stable. On the basis of aerodynamic droplet-size, nebulized formulations were estimated to deposit in different regions of respiratory tract, especially alveolar region, Among the formulations, MPS-LcRGD caused significant reduction of edema, neutrophil infiltration and inflammation biochemical marker levels. CONCLUSION: From the results, it can be inferred that nebulization of targeted liposomes had facilitated spatial and temporal modulation of drug delivery resulting in alleviation of ALI.

Dietary eicosapentaenoic acid normalizes hippocampal omega-3 and 6 polyunsaturated fatty acid profile, attenuates glial activation and regulates BDNF function in a rodent model of neuroinflammation induced by central interleukin-1ß administration.

PURPOSE: Interleukin (IL)-1ß can activate glial cells to trigger neuroinflammation and neurodegeneration. Lower omega (n)-3 polyunsaturated fatty acids (PUFAs) and lower n-3/n-6 PUFA ratios occur in the brain of patients with Alzheimer's disease (AD). We have previously reported that an n-3 PUFA, eicosapentaenoic acid (EPA), can improve memory and attenuate neurodegeneration-like changes in animal models of AD. However, whether and how EPA modulates glial cell activity and functions remains unclear. The aim of this study was to test the hypothesis that EPA may attenuate neuroinflammation by inhibiting microglial activation and microglia-produced proinflammatory cytokines, and by enhancing the expression of astrocytes-produced neurotrophins and their receptors. METHODS: Male Long-Evans rats were fed either palm oil supplemented diet or EPA supplemented diet for 42 days. On day 36 of diet feeding, rats received an intracerebroventricular injection of IL-1ß or saline for 7 days. The glial activation, the expression of amyloid precursor protein (APP), calcium-dependent phospholipase (cPL) A2, brain-derived neurotrophic factor (BDNF) and its receptor, and PUFA profile in the hippocampus were analyzed. RESULTS: IL-1ß elevated biomarkers of microglial CD11b and astrocyte GFAP expression, increased the expression of APP, tumor-necrosis factor (TNF)-α, but reduced BDNF and its receptor (TrKB). IL-1ß also lowered n-3 EPA and docosapentaenoic acid concentrations but increased n-6 PUFAs and cPLA2 activity in the hippocampus. EPA supplement normalized the n-3 and n-6 PUFA profiles and cPLA2 levels, inhibited glial activation, reduced APP and TNF-α expression, as well as up-regulated BDNF and TrKB. CONCLUSION: Supplementation with EPA appear to have potential effects on improving glial over-activation, n3/n6 imbalance and BDNF down-regulation, which contribute to anti-inflammatory and may provide beneficial effects on inflammation-associated disease such as AD.

Effects of tumour necrosis factor-alpha and interleukin-1 beta on in vitro development of bovine secondary follicles.

The objective of this study was to determine the effects of TNF-α and IL-1ß on development and survival of bovine secondary follicle culture in vitro for 18 days. Secondary follicles (~0.2 mm) were isolated from ovarian cortex and individually cultured at 38.5°C, with 5% CO2 in air, for 18 days, in TCM-199+ alone (cultured control) or supplemented with 10 ng/ml IL-1ß, 10 ng/ml TNF-α or both TNF-α and IL-1ß. The effects of these treatments on growth, follicular survival, antrum formation, viability, ultrastructure and mRNA levels for GDF-9, c-MOS, H1foo and Cyclin B1 were evaluated. The results showed that addition of TNF-α to culture medium increased follicular diameter and rate of antrum formation, whereas that of IL-1ß and a mixture of IL-1ß and TNF-α did not do so. Ultrastructural analysis showed that, among the tested cytokine treatments, follicles cultured in the presence of TNF-α had the best-preserved oocytes and granulosa cells. The presence of TNF-α, IL-1ß or both did not influence the expression of mRNAs analysed. In conclusion, in contrast to IL-1ß, TNF-α promotes growth of and antrum formation in in vitro cultured bovine secondary follicles, while their ultrastructure and viability were maintained.

Synthesis, chemical behavior, structure elucidation and iNOS inhibitory activity of 1-substituted 3-methylsulfanyl-5,6,7,8-tetrahydro-1H-[1,2,4]triazolo[1,2-a]pyridazines.

Novel slim and shapely sp3-rich nitrogen containing heterocyclic ring systems are sought-after platforms for the expansion of molecular diversity in lead discovery. The present work describes the synthesis and characterization of a series of derivatives of hitherto unknown 3-methylsulfanyl-5,6,7,8-tetrahydro-1H-[1,2,4]triazolo[1,2-a]pyridazines 2. This approach was guided by a computational study, aiming at an optimization of previously reported [1,2,4]triazolo[1,2-a]pyridazine-1-thiones 1 known to inhibit the inducible nitric oxide synthase (iNOS). The title compounds are accessible by methylation of compounds 1 under mild conditions. The products were biologically evaluated by the same cell-based assay as applied for previous products of type 1 using RINm5F cells, which were stimulated to produce NO on the influence of proinflammatory cytokines IL-1ß and IFN-γ. Compounds 2 did not display the anticipated improved iNOS inhibitory activity in the selected assay but contribute to SAR in the field. In addition, an unprecedented formation of side-products 3 via oxidation has been investigated. The novel scaffolds represent attractive starting points for the construction of diverse molecules which differ considerably from known compounds based on flat and lipophilic aromatic scaffolds.

[The effects of interleukin-1 beta and gamma-quantum braking radiation on mesenchymal progenitor cells].

In murine bone-marrow stromal microenvironment cells and in human multipotent mesenchymal stromal cells (MMSCs), proinflammatory cytokine interleukin-1 beta (IL-1ß) serves as a growth factor. In murine bone tissue, IL-1ß expression increases in vivo after irradiation. Here, we have presented our evaluation of the effects of exogenous IL-1ß on the expression of NF-kB transcription factors in human MMSCs and stromal layer cells of murine long-term bone marrow cultures (LTBMCs). The cytokine signaling pathway was also activated in murine LTBMC by braking electron radiation in doses of 3-12 Gy. The level of expression of genes that code for IL-1ß, IL-1ß type-I receptor and NF-kB and IKK protein families have been studied at different time points post exposure. In both human and murine stromal cells, exogenous IL-1ß led to an increase in the level of expression of its own gene, while levels of expression of NF-kB and IKK gene families were not substantially changed. Nevertheless, in human cells, a significant correlation between levels of expression of IL-1ß and all NF-kB family genes was detected. It points to a similarity in IL-1ß signal pathways in mesenchymal and hematopoietic cells, where the posttranslational modifications of NF-kB transcription factors play a major role. The irradiation of murine LTBMC resulted in a transient increase in the expression of genes that code NF-kB transcription factors and IL-1ß. These results indicate an important role of Rel, Rela, Relb, and Nfkb2 genes in the induction of IL-1ß signal pathway in murine stromal cells. An increase in IL-1ß expression after the irradiation of stromal cells may be related to both the induction of inflammation due to massive cell death and to a profound stimulation of the expression of this proinflammatory cytokine expression.

Interleukin-1ß increases neuronal death in the hippocampal dentate gyrus associated with status epilepticus in the developing rat. / La interleucina-1ß aumenta la muerte neuronal en el giro dentado del hipocampo asociada al estado epiléptico en la rata en desarrollo.

BACKGROUND: Interleukin-1ß (IL-1ß) increases necrotic neuronal cell death in the CA1 area after induced status epilepticus (SE) in developing rats. However, it remains uncertain whether IL-1ß has a similar effect on the hippocampal dentate gyrus (DG). In this study, we analysed the effects of IL-1ß on 14-day-old Wistar rats experiencing DG neuronal death induced by SE. METHODS: SE was induced with lithium-pilocarpine. Six hours after SE onset, a group of pups was injected with IL-1ß (at 0, 0.3, 3, 30, or 300ng/µL) in the right ventricle; another group was injected with IL-1ß receptor (IL-1R1) antagonist (IL-1Ra, at 30ng/µL) of IL-1RI antagonist (IL-1Ra) alone, and additional group with 30ng/µL of IL-1Ra plus 3ng/µL of IL-1ß. Twenty-four hours after SE onset, neuronal cell death in the dentate gyrus of the dorsal hippocampus was assessed using haematoxylin-eosin staining. Dead cells showed eosinophilic cytoplasm and condensed and fragmented nuclei. RESULTS: We observed an increased number of eosinophilic cells in the hippocampal DG ipsilateral to the site of injection of 3ng/µL and 300ng/µL of IL-1ß in comparison with the vehicle group. A similar effect was observed in the hippocampal DG contralateral to the site of injection of 3ng/µL of IL-1ß. Administration of both of IL-1ß and IL-1Ra failed to prevent an increase in the number of eosinophilic cells. CONCLUSION: Our data suggest that IL-1ß increases apoptotic neuronal cell death caused by SE in the hippocampal GD, which is a mechanism independent of IL-1RI activation.

Short term exposure of beta cells to low concentrations of interleukin-1ß improves insulin secretion through focal adhesion and actin remodeling and regulation of gene expression.

Type 2 diabetes involves defective insulin secretion with islet inflammation governed in part by IL-1ß. Prolonged exposure of islets to high concentrations of IL-1ß (>24 h, 20 ng/ml) impairs beta cell function and survival. Conversely, exposure to lower concentrations of IL-1ß for >24 h improves these same parameters. The impact on insulin secretion of shorter exposure times to IL-1ß and the underlying molecular mechanisms are poorly understood and were the focus of this study. Treatment of rat primary beta cells, as well as rat or human whole islets, with 0.1 ng/ml IL-1ß for 2 h increased glucose-stimulated (but not basal) insulin secretion, whereas 20 ng/ml was without effect. Similar differential effects of IL-1ß depending on concentration were observed after 15 min of KCl stimulation but were prevented by diazoxide. Studies on sorted rat beta cells indicated that the enhancement of stimulated secretion by 0.1 ng/ml IL-1ß was mediated by the NF-κB pathway and c-JUN/JNK pathway acting in parallel to elicit focal adhesion remodeling and the phosphorylation of paxillin independently of upstream regulation by focal adhesion kinase. Because the beneficial effect of IL-1ß was dependent in part upon transcription, gene expression was analyzed by RNAseq. There were 18 genes regulated uniquely by 0.1 but not 20 ng/ml IL-1ß, which are mostly involved in transcription and apoptosis. These results indicate that 2 h of exposure of beta cells to a low but not a high concentration of IL-1ß enhances glucose-stimulated insulin secretion through focal adhesion and actin remodeling, as well as modulation of gene expression.

An altered peripheral IL6 response in major depressive disorder.

Major depressive disorder (MDD) is one of the most prevalent major psychiatric disorders with a lifetime prevalence of 17%. Recent evidence suggests MDD is not only a brain dysfunction, but a systemic disease affecting the whole body. Central and peripheral inflammatory changes seem to be a centerpiece of MDD pathology: a subset of patients show elevated blood cytokine and chemokine levels that partially normalize with symptom improvement over the course of anti-depressant treatment. As this inflammatory process in MDD is poorly understood, we hypothesized that the peripheral tissues of MDD patients will respond differently to inflammatory stimuli, resulting in an aberrant transcriptional response to elevated pro-inflammatory cytokines. To test this, we used MDD patient- and control-derived dermal fibroblast cultures to investigate their response to an acute treatment with IL6, IL1ß, TNFα, or vehicle. Following RNA isolation and subsequent cDNA synthesis, quantitative PCR was used to determine the relative expression level of several families of inflammation-responsive genes. Our results showed comparable expression of the tested genes between MDD patients and controls at baseline. In contrast, MDD patient fibroblasts had a diminished transcriptional response to IL6 in all the gene sets tested (oxidative stress response, mitochondrial function, and lipid metabolism). We also found a significant increase in baseline and IL6 stimulated transcript levels of the IL6 receptor gene. This IL6 receptor transcript increase in MDD fibroblasts was accompanied by an IL6 stimulated increase in induction of SOCS3, which dampens IL6 receptor signaling. Altogether our results demonstrate that there is an altered transcriptional response to IL6 in MDD, which may represent one of the molecular mechanisms contributing to disease pathophysiology. Ultimately we hope that these studies will lead to validation of novel MDD drug targets focused on normalizing the altered IL6 response in patients.