Diminished Neutrophil Infiltration Research Articles

Aloin alleviates corneal injury in alkali burn via inhibiting neutrophil extracellular traps and promoting Nrf2.

Ocular chemical burns are a leading cause of blindness. The cornea is injured by alkali-induced oxidative disturbances and an inflammatory response. The aim of this study was to evaluate the protective effects of aloin, an antioxidant, and anti-inflammatory compound, on corneal alkali burn. Mice eyes were injured by NaOH and subsequently treated with aloin eye drop and intraperitoneal injection. Pathological characteristics of the eyes were examined, and corneal samples were collected for further analysis. Aloin diminished neutrophil infiltration and the production of proinflammatory cytokines. Aloin also attenuated apoptosis in human corneal epithelial cells (HCEs) by reducing oxidative stress through the activation of the Nrf2 pathway. Additionally, aloin suppressed the formation of neutrophil extracellular traps (NETs) and inhibited their deposition on the cornea. Moreover, aloin mitigated alkali-induced apoptosis in HCEs caused by NETs. These findings suggest that aloin has potential as an antioxidant and anti-inflammatory compound for treating corneal alkali burn by inhibiting NETs formation and promoting Nrf2.

The m6A methyltransferase METTL3 drives neuroinflammation and neurotoxicity through stabilizing BATF mRNA in microglia.

Persistent neuroinflammation and progressive neuronal loss are defining features of acute brain injury including traumatic brain injury (TBI) and cerebral stroke. Microglia, the most abundant type of brain-resident immune cells, continuously surveil the environment and play a central role in shaping the inflammatory state of the central nervous system (CNS). In the study, we discovered that the protein expression of METTL3 (a m6A methyltransferase) was upregulated in inflammatory microglia independent of increased Mettl3 gene transcription following TBI in both human and mouse subjects. Subsequently, we identified TRIP12, a HECT-domain E3 ubiquitin ligase, as a negative regulator of METTL3 protein expression by facilitating METTL3 K48-linked polyubiquitination. Importantly, selective ablation of Mettl3 inhibited microglial pathogenic activities, diminished neutrophil infiltration, rescued neuronal loss and facilitated functional recovery post-TBI. Using MeRIP-seq and CUT&Tag sequencing, we identified that METTL3 promoted the expression of Basic Leucine Zipper Transcriptional Factor ATF-Like (BATF), which in turn directly bound to a cohort of characteristic inflammatory cytokines and chemokine genes. Enhanced activities of BATF in microglia elicited TNF-dependent neurotoxicity and can also promote neutrophil recruitment through releasing CXCL2. Pharmacological inhibition of METTL3 using a BBB-penetrating drug-loaded nano-system showed satisfactory therapeutic effects in both TBI and stroke mouse models. Collectively, our findings identified METTL3-m6A-BATF axis as a potential therapeutic target for terminating detrimental neuroinflammation and progressive neuronal loss following acute brain injury. METTL3 protein is significantly up-regulated in inflammatory microglia due to the decreased proteasomal degradation mediated by TRIP12 and ERK-USP5 pathways. METTL3 stabilized BATF mRNA stability and promoted BATF expression through the m6A-IGF2BP2-dependent mechanism. Elevated expression of BATF elicits a pro-inflammatory gene program in microglia, and aggravates neuroinflammatory response including local immune responses and peripheral immune cell infiltration. Genetic deletion or pharmaceutically targeting METTL3-BATF axis suppressed microglial pro-inflammatory activities and promoted neurological recovery following TBI and stroke.

Jingfang granule alleviates Pseudomonas aeruginosa-induced acute lung inflammation through suppression of STAT3/IL-17/NF-κB pathway based on network pharmacology analysis and experimental validation

Ethnopharmacological relevancePseudomonas aeruginosa is an opportunistic bacterial pathogen which is the second leading cause of hospital-acquired pneumonia. Jingfang granule (JFG) is an herbal formula of Traditional Chinese medicine (TCM) widely used in treatment of acute respiratory tract infections in China. However, the molecular mechanisms of JFG in treatment of P. aeruginosa-induced acute pneumonia are not clear. Aim of studyThis study aimed to investigate the mechanisms underlying the effects of JFG on P. aeruginosa-induced acute inflammation using a mouse model of bacterial acute pneumonia. Materials and methodsThe chemical components and targets of JFG were retrieved from Traditional Chinese Medicine Systems Pharmacology (TCMSP) database, and the P. aeruginosa pneumonia-related targets were obtained from the disease databases, including Online Mendelian Inheritance in Man (OMIM), GeneCards and DisGeNet. The protein-protein interaction (PPI) network was constructed using STRING database. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID). Molecular docking was performed using AutoDockTools 1.5.6. Further in vivo experiments employed a mouse model of P. aeruginosa acute pneumonia to verify the target proteins and signaling pathways affected by JFG, which were predicted by the network pharmacology analysis. ResultsA total of 218 active components and 257 targets of JFG were retrieved from TCMSP database. Moreover, 99 intersectant targets were obtained between the 257 JFG targets and 694 disease targets. Among the intersectant targets, STAT3, IL-6, AKT1, TNF, MAPK1, MAPK3 and EGFR were identified to be the key therapeutic targets through PPI network analysis, and STAT3 was in the center of the network, which is a key regulator of IL-17 expression. KEGG pathway enrichment analysis suggested that IL-17 signaling pathway was one of the crucial inflammatory pathways affected by JFG in treatment of P. aeruginosa pneumonia. Furthermore, the in vivo experiments demonstrated that the JFG-treated mice displayed reduced proinflammatory cytokine production (IL-17, IL-1β, IL-6 and TNF), diminished neutrophil infiltration and decreased mortality, compared with the non-drug-treated mice during P. aeruginosa lung infection. Moreover, the expression or phosphorylation levels of the key regulators in STAT3/IL-17/NF-κB axis including STAT3, ERK1/2 (MAPK3/1), AKT, NF-κB p65 and RORγt were significantly reduced in the lung tissues of the JFG-treated mice. ConclusionJFG was effective in treatment of P. aeruginosa acute lung infection, which reduced inflammatory responses through suppressing STAT3/IL-17/NF-κB pathway.

Neuropeptide W Exhibits Preventive and Therapeutic Effects on Acetic Acid-Induced Colitis via Modulation of the Cyclooxygenase Enzyme System.

The novel peptide neuropeptide W (NPW) was originally shown to function in the control of feeding behavior and energy homeostasis. The aim of this study was to elucidate the putative preventive and therapeutic effects of NPW on colitis-associated oxidative injury and the underlying mechanisms for its action. Sprague-Dawley rats in the acute colitis groups received NPW (0.5, 1 or 5µg/kg/day) injections prior to induction of colitis with acetic acid, while the chronic colitis groups were treated after the induction of colitis. In both acute and chronic colitis (CC) groups, treatments were continued for 5days and the rats were decapitated at the 24th hour of the last injections and colon tissues were collected for assessments. NPW pretreatment given for 5days before colitis induction, as well as treating rats with NPW during the 5-day course of CC, abolished colonic lipid peroxidation. NPW treatment prevented colitis-induced reduction in blood flow, diminished neutrophil infiltration, and pro-inflammatory cytokine responses. NPW pretreatment only at the higher dose reduced colonic edema and microscopic score and preserved colonic glutathione stores. Elevations in cyclooxygenase (COX) enzyme activity and COX-1 protein level during the acute phase of colitis as well as reduction in COX-2 were all reversed with NPW pretreatment. In contrast, NPW treatment was effective in reducing the elevated COX-2 concentration during the chronic phase. NPW alleviates acetic acid-induced oxidative colonic injury in rats through the upregulation of colonic blood flow as well as the inhibition of COX-2 protein expression and pro-inflammatory cytokine production.

In vivo Anti-inflammatory Activity of Lipidated Peptidomimetics Pam-(Lys-βNspe)6-NH2 and Lau-(Lys-βNspe)6-NH2 Against PMA-Induced Acute Inflammation

Host Defense Peptides (HDPs) are key components of innate immunity that exert antimicrobial, antibiofilm, and immunomodulatory activities in all higher organisms. Synthetic peptidomimetic analogs were designed to retain the desirable pharmacological properties of HDPs while having improved stability toward enzymatic degradation, providing enhanced potential for therapeutic applications. Lipidated peptide/β-peptoid hybrids [e.g., Pam-(Lys-βNspe)6-NH2 (PM1) and Lau-(Lys-βNspe)6-NH2 (PM2)] are proteolytically stable HDP mimetics displaying anti-inflammatory activity and formyl peptide receptor 2 antagonism in human and mouse immune cells in vitro. Here PM1 and PM2 were investigated for their in vivo anti-inflammatory activity in a phorbol 12-myristate 13-acetate (PMA)-induced acute mouse ear inflammation model. Topical administration of PM1 or PM2 led to attenuated PMA-induced ear edema, reduced local production of the pro-inflammatory chemokines MCP-1 and CXCL-1 as well as the cytokine IL-6. In addition, diminished neutrophil infiltration into PMA-inflamed ear tissue and suppressed local release of reactive oxygen and nitrogen species were observed upon treatment. The obtained results show that these two peptidomimetics exhibit anti-inflammatory effects comparable to that of the non-steroidal anti-inflammatory drug indomethacin, and hence possess a potential for treatment of inflammatory skin conditions.

Reduced Neutrophil Extracellular Trap (NET) Formation During Systemic Inflammation in Mice With Menkes Disease and Wilson Disease: Copper Requirement for NET Release.

Neutrophil extracellular traps (NETs) contribute to pathological disorders, and their release was directly linked to numerous diseases. With intravital microscopy (IVM), we showed previously that NETs also contribute to the pathology of systemic inflammation and are strongly deposited in liver sinusoids. Over a decade since NET discovery, still not much is known about the metabolic or microenvironmental aspects of their formation. Copper is a vital trace element essential for many biological processes, albeit its excess is potentially cytotoxic; thus, copper levels are tightly controlled by factors such as copper transporting ATPases, ATP7A, and ATP7B. By employing IVM, we studied the impact of copper on NET formation during endotoxemia in liver vasculature on two mice models of copper excess or deficiency, Wilson (ATP7B mutants) and Menkes (ATP7A mutants) diseases, respectively. Here, we show that respective ATP7 mutations lead to diminished NET release during systemic inflammation despite unaltered intrinsic capacity of neutrophils to cast NETs as tested ex vivo. In Menkes disease mice, the in vivo effect is mostly due to diminished neutrophil infiltration of the liver as unmutated mice with a subchronic copper deficiency release even more NETs than their controls during endotoxemia, whereas in Wilson disease mice, excess copper directly diminishes the capacity to release NETs, and this was further confirmed by ex vivo studies on isolated neutrophils co-cultured with exogenous copper and a copper-chelating agent. Taken together, the study extends our understanding on how microenvironmental factors affect NET release by showing that copper is not a prerequisite for NET release but its excess affects the trap casting by neutrophils.

Attenuation of neutrophil-mediated liver injury in mice by drug-free E-selectin binding polymer

Inflammation with neutrophils infiltration is a prominent feature of alcohol-related liver disease (ARLD) and contributes to the severity of liver injury. Although an array of potential treatments has been studied, the standard treatment regimen is controversial and can induce severe side effects and infection-related complications. E-selectin, a cytokine inducible cell adhesion molecule, mediates the initial interaction of leucocytes with endothelial cells, and facilitates their further adhesion and extravasation into inflamed tissues. Given the important role of E-selectin in leukocytes trafficking, we hypothesized that a synthetic polymer presenting multiple copies of E-selectin binding peptide in a polyvalent manner (P-Esbp) may block the “roads” that facilitate neutrophil infiltration, inhibit the recruitment of neutrophils to the inflamed sites and reduce the extent of liver injury. We now demonstrate in vitro that P-Esbp reduced the recruitment of neutrophils (collected from blood of donors) on activated human umbilical vein endothelial cells (HUVEC) under flow conditions. Pre-treatment of mice with P-Esbp prior to alcohol binge attenuated alcohol-induced serum transaminase (ALT, AST) elevation, reduced pro-inflammatory cytokines (TNFα and IL-1ẞ) and chemokines (MIP-2/CXCL2 and MCP-1/CCL2) in National Institute on Alcohol Abuse and Alcoholism (NIAAA) model. Also, the up-regulation of neutrophil marker Ly6G and the number of MPO positive cells in the injured tissue was significantly reduced by the treatment, indicating diminished neutrophil infiltration. Moreover, as a result of P-Esbp treatment, E-selectin expression in the liver (mRNA and protein level) was downregulated, suggesting a potential to decrease ongoing local inflammatory response. Overall, our findings highlight the anti-inflammatory properties of the “drug-free” P-Esbp and its therapeutic potential to attenuate an excessive inflammation where infiltrating neutrophils can damage tissues and organs.

Caspase-11-dependent IL-1α release boosts Th17 immunity against Paracoccidioides brasiliensis

The granulomatous lesion resulting from infection with the fungus Paracoccidioides brasiliensis is characterized by a compact aggregate of mature cells, surrounded by a fibroblast- and collagen-rich content. Granuloma formation requires signaling elicited by inflammatory molecules such as members of the interleukin-1 family. Two members of this family have been thoroughly studied, namely IL-1α and IL-1β. In this study, we addressed the mechanisms underlying IL-1α secretion and its functional role on the host resistance to fungal infection. We found that, the expression of caspase-11 triggered by P. brasiliensis infection of macrophages depends on IFN-β production, because its inhibition reduced procaspase-11 levels. Curiously, caspase-11 deficiency did not impair IL-1β production, however caspase-11 was required for a rapid pore-mediated cell lysis. The plasma membrane rupture facilitated the release of IL-1α, which was necessary to induce NO production and restrict fungal replication. Furthermore, P. brasiliensis-infected macrophages required IL-1α to produce optimal levels of IL-6, a major component of Th17 lymphocyte differentiation. Indeed, IL-1α deficiency accounted for a significant reduction of Th17 lymphocytes in lungs of infected mice, correlating with diminished neutrophil infiltration in the lungs. Strikingly, we identified that IL-1α directly reprograms the transcriptional profile of Th17-committed lymphocytes, increasing cellular proliferation, as for boosting IL-17 production by these cells. Beyond neutrophil chemotaxis in vivo, IL-17 also amplified IL-1α production by infected macrophages in vitro, endorsing a critical amplification loop of the inflammatory response. Therefore, our data suggest that the IFN-β/caspase-11/IL-1α pathway shapes a protective antifungal Th17 immunity, revealing a molecular mechanism underlying the cross-talk between innate and adaptive immunity.

Vitamin B6 reduces oxidative stress in lungs and liver in experimental sepsis.

Sepsis is a life-threatening organ dysfunction induced by a disrupted host response to infecting pathogens. Inflammation and oxidative stress are intrinsically related to sepsis progression and organ failure. Vitamin B6 is an important cellular cofactor for metabolic processes and has anti-inflammatory and antioxidant properties. We aimed at evaluating the effect of vit B6 on inflammation and oxidative stress markers in the liver and lung of rats subjected to a relevant animal model of polymicrobial sepsis. Adult male Wistar rats were submitted to cecal ligation and perforation model and immediately after sepsis induction, vit B6 was administered as a single dose (600 mg/kg, subcutaneous). Twenty-four hours later, the lung and liver were harvest for neutrophil infiltration, oxidative markers to lipids and protein and antioxidant activity of endogenous enzyme. Vitamin B6 diminished neutrophil infiltration in both organs, oxidative markers in the liver and restored catalase activity levels in the lung of septic animals. Vitamin B6 exerts anti-inflammatory and antioxidant effects in peripheral organs after polymicrobial sepsis.

Dermal γδ T Cells Do Not Freely Re-Circulate Out of Skin and Produce IL-17 to Promote Neutrophil Infiltration during Primary Contact Hypersensitivity.

The role of mouse dermal γδ T cells in inflammatory skin disorders and host defense has been studied extensively. It is known that dendritic epidermal T cells (DETC) have a monomorphic γδ T cell receptor (TCR) and reside in murine epidermis from birth. We asked if dermal γδ cells freely re-circulated out of skin, or behaved more like dermal resident memory T cells (TRM) in mice. We found that, unlike epidermal γδ T cells (DETC), dermal γδ cells are not homogeneous with regard to TCR, express the tissue resident T cell markers CD69 and CD103, bear skin homing receptors, and produce IL-17 and IL-22. We created GFP+: GFP− parabiotic mice and found that dermal γδ T cells re-circulate very slowly—more rapidly than authentic αβ TCR TRM, but more slowly than the recently described dermal αβ TCR T migratory memory cells (TMM). Mice lacking the TCR δ gene (δ-/-) had a significant reduction of 2,4-dinitrofluorobenzene (DNFB)-induced contact hypersensitivity (CHS). We created mice deficient in dermal γδ T cells but not DETC, and these mice also showed a markedly reduced CHS response after DNFB challenge. The infiltration of effector T cells during CHS was not reduced in dermal γδ T cell-deficient mice; however, infiltration of Gr-1+CD11b+ neutrophils, as well as ear swelling, was reduced significantly. We next depleted Gr-1+ neutrophils in vivo, and demonstrated that neutrophils are required for ear swelling, the accepted metric for a CHS response. Depletion of IL-17-producing dermal Vγ4+ cells and neutralization of IL-17 in vivo, respectively, also led to a significantly reduced CHS response and diminished neutrophil infiltration. Our findings here suggest that dermal γδ T cells have an intermediate phenotype of T cell residence, and play an important role in primary CHS through producing IL-17 to promote neutrophil infiltration.

Cathelicidin Augments Epithelial Receptivity and Pathogenesis in ExperimentalEscherichia coliCystitis

Cathelicidin is a proposed defender against infection of the urinary tract via its antimicrobial properties, but its activity has not been delineated in a dedicated cystitis model. Female C57Bl/6 mice, wild type or deficient in cathelin-related antimicrobial peptide (CRAMP; an ortholog of the sole human cathelicidin, LL-37), were infected transurethrally with the cystitis-derived uropathogenic Escherichia coli (UPEC) strain UTI89. Infection course was evaluated by bladder titers, intracellular bacterial community quantification, and histological analysis. Immune responses and resolution were characterized through cytokine profiling, microscopy, and quantitation of epithelial recovery from exfoliation. CRAMP-deficient mice exhibited significantly lower bladder bacterial loads and fewer intracellular bacterial communities during acute cystitis. Although differences in bacterial titers were evident as early as 1 hour after infection, CRAMP-deficient mice showed no baseline alterations in immune activation, uroepithelial structure, apical expression of uroplakins (which serve as bacterial receptors), or intracellular bacterial growth rate. CRAMP-deficient hosts demonstrated less intense cytokine responses, diminished neutrophil infiltration, and accelerated uroepithelial recovery. Mice lacking the antimicrobial peptide cathelicidin experienced less severe infection than wild-type mice in a well-established model of cystitis. Although CRAMP exhibits in vitro antibacterial activity against UPEC, it may enhance UPEC infection in the bladder by promoting epithelial receptivity and local inflammation.

HMG-CoA reductase inhibition prior reperfusion improves reparative fibrosis post-myocardial infarction in a preclinical experimental model

BackgroundStudies in patients support a beneficial effect of statin treatment early after acute coronary syndrome and/or prior percutaneous coronary intervention. However, statin effect during total occlusion remains unknown. ObjectivesTo investigate whether infusion of activated simvastatin during ischemia and prior reperfusion and oral administration thereafter confers cardioprotection and improves cardiac healing in a preclinical model of myocardial infarction. MethodsPigs (n=24) fed a 10day Western-type diet underwent a 90min coronary-balloon occlusion (MI) being randomized to a single intravenous infusion of active β-hydroxy acid derivative of simvastatin (β-OH-S; 0.3mg/kg) 15min prior to reperfusion or vehicle. Animals were either sacrificed 2.5h post-reperfusion or kept under the same regime±simvastatin (p.o., 20mg/day) for 3weeks. Jeopardized and remote myocardium was obtained for molecular/histological studies. Echocardiography was assessed. Resultsβ-OH-S infusion prior to reperfusion reduced coronary and cardiac oxidative DNA-damage, diminished neutrophil infiltration at the site of ischemia, preserved mitochondrial membrane potential and reduced apoptosis in the ischemic myocardium (lower mRNA levels of Fas, casp8, p53, and casp3 and mitochondrial-p-Bcl2; and reduced TUNEL and active caspase-3; p<0.05 vs. vehicle/control). This treatment regime attenuated reperfusion-related arrhythmias and stunning leading to a 40% increased myocardial salvage (p<0.05 vs. vehicle/control). 3weeks post-MI simvastatin-treated animals showed P-PKCε increase, lower intramyocardial lipotoxicity, TβRII/Smad2/3 signaling restoration and subsequent myofibroblast differentiation and collagen-fibril formation in the evolving scar (p<0.05 vs. control). Simvastatin suppressed cardiac RhoA mobilization and triggered Akt/eNOS signaling. ConclusionsAcute HMG-CoA-reductase inhibition during total ischemia and prior reperfusion limits reperfusion injury and prolonged oral simvastatin treatment thereafter improves cardiac healing post-MI.

Analgesic and Anti-Inflammatory Bioactivities of Eburicoic Acid and Dehydroeburicoic Acid Isolated from Antrodia camphorata on the Inflammatory Mediator Expression in Mice

Eburicoic acid (TR1) and dehydroeburicoic acid (TR2), an active ingredient from Antrodia camphorata (AC) solid-state culture, were evaluated for analgesic and anti-inflammatory effects. Treatment with TR1 and TR2 significantly inhibited a number of acetic acid-induced writhing responses and formalin-induced pain in the late phase. In the anti-inflammatory test, TR1 and TR2 decreased paw edema at the fourth and fifth hour after λ-carrageenan (Carr) administration and increased the activities of catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx) in the paw edema tissue. We also demonstrated that TR1 and TR2 significantly attenuated the malondialdehyde (MDA), nitric oxide (NO), tumor necrosis factor (TNF-α), and interleukin-1β (IL-1β) levels in either edema paw or serum at the fifth hour after Carr injection. Western blotting revealed that TR1 and TR2 decreased Carr-induced inducible nitric oxide synthase (iNOS) and cycloxyclase (COX-2) expressions at the fifth hour in paw edema. Treatment with TR1 and TR2 also diminished neutrophil infiltration into the paw edema at the fifth hour. The present study suggests that the anti-inflammatory mechanisms of TR1 and TR2 might be related to the decrease of inflammatory cytokines and an increase of antioxidant enzyme activity.

P2Y12 Null Mice Are Protected Against Sepsis-Induced Lung Injury

AbstractAbstract 615The P2Y12 receptor is expressed on the platelet surface and mediates ADP-induced aggregation, but it has recently been shown to be expressed also in other cells of the immune system. Furthermore, the P2Y12 receptor gene variants correlate with pulmonary inflammation and asthma, and P2Y12 receptor deficiency or platelet depletion abrogated dust mite-induced airway inflammation, suggesting an important role for the P2Y12 receptor in pulmonary inflammation. Excessive neutrophil infiltration into the lungs is a hallmark of acute lung injury. The mechanisms that trigger this infiltration are poorly understood but may be the result of the development of a pro-inflammatory phenotype in the lung. Platelets are increasingly recognized for their ability to modulate immune responses through their interaction with neutrophils, dendritic cells, and T lymphocytes. These studies suggest an important regulatory role for the P2Y12 receptor and activated platelets in regulating neutrophil influx into the lung.Using a mouse model of intra-abdominal sepsis and acute lung injury (cecal ligation and double puncture (CLP)), we examined the role of the P2Y12 receptor in neutrophil migration and lung inflammation using P2Y12 null mice. In this study wild type (WT) and P2Y12 null (KO) mice underwent Sham surgery or CLP surgery (5 animals/group). At 24 hours post surgery, blood and lung tissue samples were collected.Our results show that the sepsis-induced increase in circulating white blood cells (WBC) was attenuated in CLP KO mice, compared with CLP WT mice (circulating WBC in CLP WT: 1.62 ± 0.41×103cell/ml* compared with CLP KO: 0.8 ± 0.4×103cell/ml, *p<0.05 WT CLP versus KO CLP mice). There were no significant differences in the WBC count in WT Sham surgery animals as compared to the KO sham surgery animals (Sham WT: 0.8 ± 0.2×103cell/ml vs. Sham KO: 0.8 ± 0.1×103cell/ml). Interestingly, no significant changes in the platelet count between the groups were observed. Next we investigated the role of P2Y12 in regulating sepsis-induced elevations in plasma levels of cytokines (TNF-a, IL-10, IL-6 and MIP-1b) (Figure 1). The concentration of each cytokine was elevated during sepsis in both WT and KO animals as compared to sham surgery animals, but, the sepsis-induced increase was significantly lower in P2Y12 null mice (**p < 0.01 KO CLP model versus WT- CLP). [Display omitted] Finally we studied neutrophil infiltration in lung tissues using hematoxylin and eosin (H & E) staining and myeloperoxidase (MPO) activity in lung tissue homogenates. Analysis of H & E staining demonstrated that the increase in inflammatory cell infiltration detected in WT was diminished in KO mice, suggesting a decrease in inflammation in the lungs of P2Y12 null mice. Furthermore, the MPO concentration was significantly decreased in KO CLP lung tissue as compared to WT CLP (Figure 2), (*p < 0.05; KO CLP model versus wild type). [Display omitted] In conclusion, our data shows a decrease in plasma cytokine levels and in circulating WBC, as well as a diminished neutrophil infiltration in the lungs indicating that P2Y12 null mice are protected against sepsis-induced lung injury. This suggests a regulatory role for P2Y12 receptor during inflammation. Disclosures:No relevant conflicts of interest to declare.

Anti-Inflammatory Activities of Inotilone from Phellinus linteus through the Inhibition of MMP-9, NF-κB, and MAPK Activation In Vitro and In Vivo

Inotilone was isolated from Phellinus linteus. The anti-inflammatory effects of inotilone were studied by using lipopolysaccharide (LPS)-stimulated mouse macrophage RAW264.7 cells and λ-carrageenan (Carr)-induced hind mouse paw edema model. Inotilone was tested for its ability to reduce nitric oxide (NO) production, and the inducible nitric oxide synthase (iNOS) expression. Inotilone was tested in the inhibitor of mitogen-activated protein kinase (MAPK) [extracellular signal-regulated protein kinase (ERK), c-Jun NH2-terminal kinase (JNK), p38], and nuclear factor-κB (NF-κB), matrix-metalloproteinase (MMP)-9 protein expressions in LPS-stimulated RAW264.7 cells. When RAW264.7 macrophages were treated with inotilone together with LPS, a significant concentration-dependent inhibition of NO production was detected. Western blotting revealed that inotilone blocked the protein expression of iNOS, NF-κB, and MMP-9 in LPS-stimulated RAW264.7 macrophages, significantly. Inotilone also inhibited LPS-induced ERK, JNK, and p38 phosphorylation. In in vivo tests, inotilone decreased the paw edema at the 4th and the 5th h after Carr administration, and it increased the activities of catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx). We also demonstrated that inotilone significantly attenuated the malondialdehyde (MDA) level in the edema paw at the 5th h after Carr injection. Inotilone decreased the NO and tumor necrosis factor (TNF-α) levels on serum at the 5th h after Carr injection. Western blotting revealed that inotilone decreased Carr-induced iNOS, cyclooxygenase-2 (COX-2), NF-κB, and MMP-9 expressions at the 5th h in the edema paw. An intraperitoneal (i.p.) injection treatment with inotilone diminished neutrophil infiltration into sites of inflammation, as did indomethacin (Indo). The anti-inflammatory activities of inotilone might be related to decrease the levels of MDA, iNOS, COX-2, NF-κB, and MMP-9 and increase the activities of CAT, SOD, and GPx in the paw edema through the suppression of TNF-α and NO. This study presents the potential utilization of inotilone, as a lead for the development of anti-inflammatory drugs.

Inducible Nitric Oxide Synthase and Cyclooxygenase-2 Participate in Anti-inflammatory Activity of Imperatorin from Glehnia littoralis

In this study, we have investigated the anti-inflammatory effects of imperatorin, a compound isolated from the roots of Glehnia littoralis, using a lipopolysaccharide (LPS)-stimulated mouse macrophage (RAW264.7) in vitro and a carrageenan (Carr)-induced mouse paw edema model in vivo. When RAW264.7 macrophages were treated with imperatorin together with LPS, a significant concentration-dependent inhibition of NO production was detected. Western blotting revealed that imperatorin blocked the protein expression of iNOS and cyclooxygenase-2 (COX-2) in LPS-stimulated RAW264.7 macrophages significantly. In the anti-inflammatory test, imperatorin decreased the paw edema at 4 and 5 h after Carr administration and increased the activities of catalase, superoxide dismutase, and glutathione peroxidase in paw edema. We also demonstrated that imperatorin significantly attenuated the malondialdehyde level in the edema paw at the fifth hour after Carr injection. Imperatorin decreased the NO and tumor necrosis factor and prostaglandin E2 levels on serum at 5 h after Carr injection. Western blotting revealed that imperatorin decreased Carr-induced iNOS and COX-2 expressions at 5 h in edema paw. An intraperitoneal injection treatment with imperatorin also diminished neutrophil infiltration into sites of inflammation as did indomethacin. The results suggested that imperatorin had anti-inflammatory effects in LPS-stimulated RAW 264.7 cells and Carr-injected mice, respectively. In addition, inhibition of elevated iNOS and COX-2 protein expression as well as neutrophil infiltration of Carr-injected paws may be involved in the beneficial effects of imperatorin.

Anti-inflammatory Activities of 6β-Acetoxy-7α-hydroxyroyleanone from Taiwania cryptomerioides Hayata ex Vivo and in Vivo

Excess production of nitric oxide (NO) by inducible NO synthase (iNOS) in activated macrophages is linked to acute and chronic inflammation. Thus, it would be valuable to develop inhibitors of NO production and/or iNOS for potential therapeutic use. This study investigated the anti-inflammatory effects of 6β-acetoxy-7α-hydroxyroyleanone (AHR), a compound isolated from the bark of Taiwania cryptomerioides Hayata, using lipopolysaccharide (LPS)-stimulated mouse macrophage (RAW 264.7) ex vivo and carrageenan (Carr)-induced mouse paw edema model in vivo. When RAW 264.7 macrophages were treated with different concentrations of AHR (0, 0.31, 0.62, 1.25, and 2.50 μg/mL) together with LPS (100 ng/mL), a significant concentration-dependent inhibition of NO production was detected. Western blotting revealed that AHR blocked protein expression of iNOS and cyclooxygenase-2 (COX-2) in LPS-stimulated RAW 264.7 macrophages, significantly. In the anti-inflammatory test, AHR (1.25 and 2.50 mg/kg) decreased paw edema at 4 and 5 h after λ-carrageenan (Carr) administration and increased the activities of catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx) in the liver tissue. It was also demonstrated that AHR significantly attenuated the malondialdehyde (MDA) level in the edema paw at 5 h after Carr injection. AHR (0.62, 1.25, and 2.50 mg/kg) decreased the NO levels on both edema paw and serum at 5 h after Carr injection. Also, AHR diminished the serum tumor necrosis factor (TNF-α) at 5 h after Carr injection. Western blotting revealed that AHR (2.50 mg/kg) decreased Carr-induced iNOS and COX-2 expressions at 5 h in the edema paw. An intraperitoneal (ip) injection treatment with AHR also diminished neutrophil infiltration into sites of inflammation, as did indomethacin (Indo). The anti-inflammatory activities of AHR might be related to the decrease in the levels of MDA, iNOS, and COX-2 in the edema paw and to the increase in the activities of CAT, SOD, and GPx in the liver through the suppression of TNF-α and NO.

Antinociceptive Activities and the Mechanisms of Anti‐Inflammation of Asiatic Acid in Mice

Asiatic acid (AA), a pentacyclic triterpene compound in the medicinal plant Centella asiatica, was evaluated for antinociceptive and anti-inflammatory effects. Treatment of male ICR mice with AA significantly inhibited the numbers of acetic acid-induced writhing responses and the formalin-induced pain in the late phase. In the anti-inflammatory test, AA decreased the paw edema at the 4th and 5th h after λ-carrageenan (Carr) administration and increased the activities of catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx) in the liver tissue. AA decreased the nitric oxide (NO), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) levels on serum level at the 5th h after Carr injection. Western blotting revealed that AA decreased Carr-induced inducible nitric oxide synthase (iNOS), cyclooxygenase (COX-2), and nuclear factor-κB (NF-κB) expressions at the 5th h in the edema paw. An intraperitoneal (i.p.) injection treatment with AA also diminished neutrophil infiltration into sites of inflammation as did indomethacin (Indo). The anti-inflammatory mechanisms of AA might be related to the decrease in the level of MDA, iNOS, COX-2, and NF-κB in the edema paw via increasing the activities of CAT, SOD, and GPx in the liver.

Analgesic Effects and the Mechanisms of Anti-inflammation of Ergostatrien-3β-ol from Antrodia camphorata Submerged Whole Broth in Mice

Ergostatrien-3beta-ol (ST1), an active and major ingredient from Antrodia camphorata (AC) submerged whole broth was evaluated for the analgesic and anti-inflammatory effects. Treatment of male imprinting control region (ICR) mice with ST1 (1, 5, and 10 mg/kg) significantly inhibited the numbers of acetic-acid-induced writhing response in 10 min. Also, our result showed that ST1 (10 mg/kg) significantly inhibited the formalin-induced pain in the late phase (p < 0.001). In the anti-inflammatory test, ST1 (10 mg/kg) decreased the paw edema at 4 and 5 h after lambda-carrageenin (Carr) administration and increased the activities of catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx) in the liver tissue. We also demonstrated that ST1 significantly attenuated the malondialdehyde (MDA) level in the edema paw at 5 h after Carr injection. ST1 (1, 5, and 10 mg/kg) decreased the nitric oxide (NO) levels on both the edema paw and serum level at 5 h after Carr injection. Also, ST1 (5 and 10 mg/kg) diminished the serum tumor necrosis factor (TNF-alpha) at 5 h after Carr injection. Western blotting revealed that ST1 (10 mg/kg) decreased Carr-induced inducible nitric oxide synthase (iNOS), and cycloxyclase (COX-2) expressions at 5 h in the edema paw. An intraperitoneal (ip) injection treatment with ST1 also diminished neutrophil infiltration into sites of inflammation, as did indomethacin (Indo). The anti-inflammatory mechanisms of ST1 might be related to the decrease in the level of MDA, iNOS, and COX-2 in the edema paw via increasing the activities of CAT, SOD, and GPx in the liver through the suppression of TNF-alpha and NO.

Ccr1 deficiency reduces inflammatory remodelling and preserves left ventricular function after myocardial infarction

Myocardial necrosis triggers inflammatory changes and a complex cytokine cascade that are only incompletely understood. The chemokine receptor CCR1 mediates inflammatory recruitment in response to several ligands released by activated platelets and up-regulated after myocardial infarction (MI). Here, we assess the effect of CCR1 on remodelling after MI using Ccr1-deficient (Ccr1−/−) mice. MI was induced in Ccr1−/− or wild-type mice by proximal ligation of the left anterior descending (LAD). Mice were sacrificed and analysed at day 1, 4, 7, 14 and 21 after MI. While initial infarct areas and areas at risk did not differ between groups, infarct size increased to 20.6±8.4% of the left ventricle (LV) in wild-type mice by day 21 but remained at 11.2±1.2% of LV (P<0.05) in Ccr1−/− mice. This attenuation in infarct expansion was associated with preserved LV function, as analysed by isolated heart studies according to Langendorff. Left ventricular developed pressure was 84.5±19.8 mmHg in Ccr1−/− mice compared to 49.0±19.7 mmHg in wild-type mice (P<0.01) and coronary flow reserve was improved in Ccr1−/− mice. An altered post-infarct inflammatory pattern was observed in Ccr1−/− mice characterized by diminished neutrophil infiltration, accelerated monocyte/lymphocyte infiltration, decreased apoptosis, increased cell proliferation and earlier myofibroblast population in the infarcted tissue. In conclusion, functional impairment and structural remodelling after MI is reduced in the genetic absence of Ccr1 due to an abrogated early inflammatory recruitment of neutrophils and improved tissue healing, thus revealing a potential therapeutic target.