Lipoxin A4 prevents high glucose-induced inflammatory response in cardiac fibroblast through FOXO1 inhibition.

Cardiac cells respond to various pathophysiological stimuli, synthesizing inflammatory molecules that allow tissue repair and proper functioning of the heart; however, perpetuation of the inflammatory response can lead to cardiac fibrosis and heart dysfunction. High concentration of glucose (HG) induces an inflammatory and fibrotic response in the heart. Cardiac fibroblasts (CFs) are resident cells of the heart that respond to deleterious stimuli, increasing the synthesis and secretion of both fibrotic and proinflammatory molecules. The molecular mechanisms that regulate inflammation in CFs are unknown, thus, it is important to find new targets that allow improving treatments for HG-induced cardiac dysfunction. NFκB is the master regulator of inflammation, while FoxO1 is a new participant in the inflammatory response, including inflammation induced by HG; however, its role in the inflammatory response of CFs is unknown. The inflammation resolution is essential for an effective tissue repair and recovery of the organ function. Lipoxin A4 (LXA4) is an anti-inflammatory agent with cytoprotective effects, while its cardioprotective effects have not been fully studied. Thus, in this study, we analyze the role of p65/NFκB, and FoxO1 in CFs inflammation induced by HG, evaluating the anti-inflammatory properties of LXA4. Our results demonstrated that HG induces the inflammatory response in CFs, using an in vitro and ex vivo model, while FoxO1 inhibition and silencing prevented HG effects. Additionally, LXA4 inhibited the activation of FoxO1 and p65/NFκB, and inflammation of CFs induced by HG. Therefore, our results suggest that FoxO1 and LXA4 could be novel drug targets for the treatment of HG-induced inflammatory and fibrotic disorders in the heart.

Development of synthetic lipoxin-A4 mimetics (sLXms): New avenues in the treatment of cardio-metabolic diseases.

Resolution of inflammation is a complex, dynamic process consisting of several distinct processes, including inhibition of endothelial activation and leukocyte trafficking; promotion of inflammatory cell apoptosis and subsequent non-phlogistic scavenging and degradation; augmentation of pathogen phagocytosis; modulation of stromal cell phenotype coupled to the promotion of tissue regeneration and repair. Among these tightly regulated processes, the clearance and degradation of apoptotic cells without eliciting an inflammatory response is a crucial allostatic mechanism vital to developmental processes, host defence, and the effective resolution of inflammation. These efferocytic and subsequent effero-metabolism processes can be carried out by professional and non-professional phagocytes. Defective removal or inadequate processing of apoptotic cells leads to persistent unresolved inflammation, which may promote insidious pathologies including scarring, fibrosis, and eventual organ failure. In this manuscript, the well-established role of endothelial activation and leukocyte extravasation, as classical vascular targets of the 'inflammation pharmacology', will be briefly reviewed. The main focus of this work is to bring attention to a less explored aspect of the 'resolution pharmacology', aimed at tackling defective efferocytosis and inefficient effero-metabolism, as key targeted mechanisms to prevent or pre-empt vascular complications in cardio-metabolic diseases. Despite the use of gold standard lipid-lowering drugs or glucose-lowering drugs, none of them are able to tackle the so called residual inflammatory risk and/or the metabolic memory. In this review, the development of synthetic mimetics of endogenous mediators of inflammation is highlighted. Such molecules finely tune key components across the whole inflammatory process, amongst various other novel therapeutic paradigms that have emerged over the past decade, including anti-inflammatory therapy. More specifically, FPR2-agonists in general, and Lipoxin analogues in particular, greatly enhance the reprogramming and cross-talk between classical and non-classical innate immune cells, thus inducing both termination of the pro-inflammatory state as well as promoting the subsequent resolving phase, which represent pivotal mechanisms in inflammatory cardio-metabolic diseases.

Lipoxin A4 attenuates MSU-crystal-induced NLRP3 inflammasome activation through suppressing Nrf2 thereby increasing TXNRD2.

Gout is a common inflammatory disease. The activation of NLRP3 inflammasome induced by monosodium urate (MSU) crystals has a critical role in gout, and its prevention is beneficial for patients. Lipoxin A4 (LXA4) is an endogenous lipoxygenase-derived eicosanoid mediator with powerful anti-inflammatory properties. However, whether LXA4 can suppress NLRP3 inflammasome activation induced by MSU crystals remains unclear. This study aimed to investigate the protective effect of LXA4 on MSU-crystal-induced NLRP3 inflammasome activation and its underlying molecular mechanisms. We found that LXA4 inhibited MSU-crystal-induced NLRP3 inflammasome activation, interleukin (IL)-1ß maturation, and pyroptosis. More specifically, LXA4 suppressed the assembly of the NLRP3 inflammasome, including oligomerization and speck formation of ASC, and ASC-NLRP3 interaction. Furthermore, LXA4 suppressed oxidative stress, the upstream events for NLRP3 inflammasome activation, as evidenced by the fact that LXA4 eliminated total reactive oxygen species (ROS) generation and alleviated nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation and mitochondrial dysfunction. However, LXA4 also depressed the Nrf2 activation, a critical molecule in the antioxidant pathway, and then exerted an inhibitory impact on Klf9 expression and promotional impact on TXNRD2 expression, two molecules located downstream of Nrf2 in sequence. Knockdown of TXNRD2 reversed the LXA4-induced depression of ROS and NLRP3 inflammasome. Moreover, LXA4 alleviated joint inflammation and decreased the production of cleaved caspase-1 and matured IL-1ß in gouty arthritis rats. Taken together, our findings demonstrate that LXA4 can attenuate MSU-crystal-induced NLRP3 inflammasome activation, probably through suppressing Nrf2 activation to increase TXNRD2 expression. The present study highlights the potential of LXA4 as an attractive new gout treatment candidate.

Impact of High-Molecular-Weight Hyaluronic Acid on Gene Expression in Rabbit Achilles Tenocytes In Vitro.

(1) Background: Surgical tendon repair often leads to adhesion formation, leading to joint stiffness and a reduced range of motion. Tubular implants set around sutured tendons might help to reduce peritendinous adhesions. The lubricant hyaluronic acid (HA) is a viable option for optimizing such tubes with the goal of further enhancing the anti-adhesive effect. As the implant degrades over time and diffusion is presumed, the impact of HA on tendon cells is important to know. (2) Methods: A culture medium of rabbit Achilles tenocytes was supplemented with high-molecular-weight (HMW) HA and the growth curves of the cells were assessed. Additionally, after 3, 7 and 14 days, the gene expression of several markers was analyzed for matrix assembly, tendon differentiation, fibrosis, proliferation, matrix remodeling, pro-inflammation and resolution. (3) Results: The addition of HA decreased matrix marker genes, downregulated the fibrosis marker α-SMA for a short time and slightly increased the matrix-remodeling gene MMP-2. Of the pro-inflammatory marker genes, only IL-6 was significantly upregulated. IL-6 has to be kept in check, although IL-6 is also needed for a proper initial inflammation and efficient resolution. (4) Conclusions: The observed effects in vitro support the intended anti-adhesion effect and therefore, the use of HMW HA is promising as a biodegradable implant for tendon repair.

The 15-Epilipoxin-A4 Pathway with Prophylactic Aspirin in Preventing Preeclampsia: A Longitudinal Cohort Study.

INTRODUCTION: The benefit of aspirin in preventing preeclampsia is increasingly recognized; however, its mechanism of action remains unclear. Nonobstetric studies have described an anti-inflammatory effect of aspirin through the 15-epilipoxin-A4 pathway (aspirin-triggered lipoxin [ATL]). However, the anti-inflammatory mechanism of aspirin in the prevention of preeclampsia remains unknown. OBJECTIVE/HYPOTHESIS: To examine (1) the difference in longitudinal endogenous lipoxin-A4 (En-Lipoxin-A4) concentration in low-risk (LR) and high-risk (HR) pregnancies, and (2) the effect of aspirin on endogenous ATL concentration and the associated effect on cytokine profile of HR women. METHODS: Plasma from 220 HR women was collected at 12, 16, 20, 24, 28, 32, and 36 weeks of gestation. Adherence to aspirin was biochemically verified. Plasma En-Lipoxin-A4 and ATL concentrations were analyzed using liquid chromatography mass spectrometry, and cytokines, interleukin (IL)-10, tumor necrosis factor-α, interferon-γ, IL-8, and IL-1ß, with the high-sensitivity multibead Luminex® assay. RESULTS: HR women have up to 70% lower plasma concentration of En-Lipoxin-A4 (P < 0.001) than LR women. HR women with adequate aspirin adherence (HR-AA) (n = 82) had higher plasma concentration of ATL (P < .001), lower concentration of IL-8 from 16 to 36 weeks of gestation (P < .001), and increased IL-10 concentration from 16 to 28 weeks of gestation (P = .03) compared with high-risk women who were not on aspirin (HR-NA). HR-AA who did not develop preeclampsia had higher plasma En-lipoxin-A4 (P < .001), ATL (P = .02), and IL-10 concentrations (P < .001) with lower IL-8 concentration (P = .004) than HR women who developed preeclampsia. DISCUSSION: Plasma concentration of En-Lipoxin-A4 is lower in HR women than in LR controls. Adequate adherence with aspirin results in an increase in ATL and IL-10 with reduced IL-8 plasma concentration. This study suggests a potential anti-inflammatory role of aspirin through the ATL pathway with prophylactic aspirin in HR pregnant women.

Lipoxin A4 attenuates uric acid-activated, NADPH oxidase-dependent oxidative stress by interfering with translocation of p47phox in human umbilical vein endothelial cells.

LipoxinA4 (LXA4) is a well-known key mediator of endogenous anti-inflammation and of the resolution of inflammation. Considerable oxidative stress occurs during inflammation due to the generation of reactive oxidative species (ROS). Moreover, high levels of uric acid (UA) contribute to endothelial cell dysfunction, which can promote disease-related morbidity, and NADPH oxidase-derived ROS are crucial regulatory factors in these responses. However, LXA4 also has the potential to reduce oxidative stress. The aim of the present study was to examine whether LXA4 could suppress UA-induced oxidative stress in human umbilical vein endothelial cells (HUVECs) and to investigate its mechanisms of action in vitro. HUVECs were incubated with or without LXA4, followed by the addition of UA. ROS levels were then measured using 2,7-dichlorodihydrofluorescein diacetate and lucigenin-enhanced chemiluminescence was used to evaluate NADPH oxidase activity. p47phox or p22phox small interfering (si)RNA were transfected into HUVECs and protein levels of p47phox were detected using western blot analysis. LXA4 significantly inhibited UA-induced generation of ROS to the same extent as the NADPH oxidase inhibitor, diphenyleneiodonium chloride. Notably, transfection of p47phox siRNA attenuated the generation of ROS and the activation of NADPH oxidase. Cells transfected with p22phox siRNA demonstrated a significant reduction in the expression of p47phox on the membrane. Further experiments demonstrated that LXA4 interfered with the transfer of p47phox from the cytoplasm to the cell membrane. These findings suggested that LXA4 inhibited the release of NADPH oxidase derived ROS in HUVECs stimulated by UA. A potential mechanism of action underlying this effect could be LXA4-mediated suppression of NADPH oxidase activity, leading to inhibition of p47phox translocation from the cytoplasm to the cell membrane.

15-Epi-LXA4 and MaR1 counter inflammation in stromal cells from patients with Achilles tendinopathy and rupture.

Resolution of inflammation is poorly understood in Achilles tendon disorders. Herein, we investigated the bioactive lipid mediator profiles of tendon-derived stromal cells isolated from patients with Achilles tendinopathy (AT) or Achilles rupture (AR) under baseline and IL-1ß-stimulated conditions. We also determined whether incubating these cells with 2 of the mediators produced by tendon-derived stromal cells, 15-epi-Lipoxin A4 (15-epi-LXA4) or maresin (MaR)-1, moderated their proinflammatory phenotype. Under baseline conditions, AT cells showed concurrent increased levels of proinflammatory eicosanoids and proresolving mediators compared with AR cells. IL-1ß treatment induced profound prostaglandin E2 release in AR compared with AT cells. Incubation of IL-1ß treated AT and AR tendon-derived stromal cells in 15-epi-LXA4 or MaR1 reduced proinflammatory eicosanoids and potentiated the release of proresolving mediators. These mediators also induced specialized proresolving mediator (SPM) biosynthetic enzymes arachidonate lipoxygenase (ALOX) 12 and ALOX15 and up-regulated the proresolving receptor ALX compared with vehicle-treated cells. Incubation in 15-epi-LXA4 or MaR1 also moderated the proinflammatory phenotype of AT and AR cells, regulating podoplanin, CD90, signal transducer and activator of transcription (STAT)-1, IL-6, IFN regulatory factor (IRF) 5, and TLR4 and suppressed c-Jun N-terminal kinase 1/2/3, Lyn, STAT-3, and STAT-6 phosphokinase signaling. In summary, we identify proresolving mediators that are active in AT and AR and propose SPMs, including 15-epi-LXA4 or MaR1, as a potential strategy to counterregulate inflammatory processes in these cells.-Dakin, S. G., Colas, R. A., Newton, J., Gwilym, S., Jones, N., Reid, H. A. B., Wood, S., Appleton, L., Wheway, K., Watkins, B., Dalli, J., Carr, A. J. 15-Epi-LXA4 and MaR1 counter inflammation in stromal cells from patients with Achilles tendinopathy and rupture.

LipoxinA4 attenuates acute pancreatitis-associated acute lung injury by regulating AQP-5 and MMP-9 expression, anti-apoptosis and PKC/SSeCKS-mediated F-actin activation.

An essential component of acute pancreatitis(AP)-induced acute lung injury(ALI) is the inflammation that is part of the body's systemic inflammatory response to a variety of systemic stimuli. Lipoxins(LXs) are considered important endogenous lipids that mediate the resolution of inflammation. In previous studies, we found that Lipoxin A4 (LXA4) reduced AP-induced pulmonary oedema and TNF-α production in lung. However, the underlying mechanism remains unclear. Due to the above studies, we investigated the aquaporin, matrix metalloprotein, apoptosis and PKC/SSeCKS signal pathway in cellular and animal models of AP-associated lung injury following LXA4 intervention. In this study, we first proved LXA4 could effectively promote F-actin reconstruction and regulate its expression in pulmonary microvascular endothelial cells both in vivo and vitro via suppressing PKC/SSeCKS signalling pathway. Next, we found that LXA4 attenuated cell growth inhibition and apoptosis in lung tissues of AP-ALI mice and HPMECs. Additionally, we demonstrated that LXA4 could regulate the expression of AQP-5 and MMP-9 to stabilize the permeability of pulmonary microvascular endothelial cell. In summary, our results suggest that the anti-inflammatory eff ;ects of LXA4 may be due to the inhibition of both the PKC/SSeCKS pathway and apoptosis to reduce alveolar fluid exudation and to the regulation of AQP-5 and MMP-9 expression to maintain the clearance of alveolar fluid. Thus, LXA4 is capable of exerting protective eff ;ects on AP-induced ALI.

Avaliação clínica do efeito de diferentes medicações intracanais nos níveis de resolvinas e lipoxinas periapicais: novas perspectivas endodônticas / Clinical evaluation of the effect of different intracanal medications on levels of periapical resolvins and lipoxins: new endodontic perspectives

RESUMO O objetivo do estudo foi avaliar o efeito da terapia endodôntica utilizando NaOCl 2,5% e medicações intracanais a base de hidróxido de cálcio e de da Nacetil cisteína (NAC) na desinfecção dos canais radiculares, na redução de endotoxinas e na estimulação de liberação dos mediadores lipídicos Resolvina E1, D2 (RvE1, RvD2) e Lipxina A4 (LxA4). Foram selecionados quarenta dentes unirradiculares, com infecção endodôntica primária e periodontite apical. Os dentes foram aleatoriamente divididos em 3 grupos de acordo com a medicação intracanal a ser utilizada: G1: soro fisiológico + Ca(OH)2 (n=14),G2: N-Acetil Cisteína (n=13), G3: clorexidina (CLX) + Ca(OH)2 (n=14). Amostras bacterianas e de endotoxinas foram coletadas do canal radicular, após abertura coronária, após preparo dos canais com limas reciprocantes (Reciproc) selecionadas de acordo com o diâmetro do canal radicular e a solução irrigante (NaOCl 2,5%) e após medicação intracanal. A análise da atividade antimicrobiana foi através da contagem de unidades formadoras de colônias (UFC/mL) de microrganismos remanescentes no canal radicular e quantificação de endotoxinas (EU/mL) através do teste Limulus Amebocyte Lysate ­ LAL. Após o preparo biomecânico (PBM) os dentes foram preenchidos com as medicações intracanais de acordo com os grupos por quatorze dias. O fluído intersticial foi coletado após preparo dos canais e após 14 dias de medicação e a quantificação dos mediadores lipídicos (RvE1, RvD2, LxA4) foi realizada através do teste imunoenzimático (ELISA). Os valores obtidos foram tabulados, analisados pelo software GraphPad Prism 6.01 e submetidos aos testes de normalidade de Kolmogorov-Smirnov e Lilliefors e em seguida aos testes Kruskal-Wallis e Dunn e Anova one-way e Tukey (P <0.05). Para carga microbiana, o PBM reduziu em média 96% dos microrganismos aeróbios e 92,5% dos anaeróbios em todos os grupos (p<0,05). A utilização de medicação intracanal à base de Ca(OH)2 manteve os níveis atingidos com o PBM (p>0,05), porém no grupo NAC houve aumento de bactérias. Os níveis de endotoxinas diminuíram após o preparo dos canais e o uso das medicações reduziu o nível de endotoxinas porém sem diferença estatística em relação à redução alcançada com o preparo biomecânico. Quanto aos mediadores lipídicos, somente no grupo NAC foi observado aumento de Rv D2. Concluiu-se que o PBM reduziu significantemente o número de bactérias e a quantidade de endotoxinas do canal, e as medicações à base de Ca(OH)2 foram eficientes na eliminação de microrganismos do canal, entretanto NAC não foi eficaz para a redução da carga microbiana; As três medicações avaliadas foram capazes de atuar no LPS bacteriano. Somente a NAC foi capaz de influenciar positivamente esse resultado com o aumento de RvD2 pós 14 dias da medicação. Os mediadores lipídicos têm grande potencial para uso nos diversos tipos de tratamento endodôntico(AU)

ABSTRACT The aim of this study was to evaluate the effect of endodontic therapy using 2.5% NaOCl and intracane medications based on calcium hydroxide and Nacetyl cysteine (NAC) on root canal disinfection, reduction of endotoxins and stimulation of release of the lipid mediators Resolvin E1, D2 (RvE1, RvD2) and Lipoxin A4 (LxA4). Forty uniradicular teeth were selected, with primary endodontic infection and apical periodontitis. The teeth were randomly divided into 3 groups according to the intracanal medication to be used: G1: saline + Ca (OH) 2 (n = 14), G2: N-Acetyl Cysteine (n = 13), G3: chlorhexidine CLX) + Ca (OH) 2 (n = 14). Bacterial and endotoxin samples were collected from the root canal after coronary opening after preparation of the channels with reciprocal files (Reciproc) selected according to the root canal diameter and the irrigating solution (NaOCl 2.5%) and after intracanal medication. The analysis of the antimicrobial activity was by count of colony forming units (CFU / mL) of microorganisms remaining in the root canal and quantification of endotoxins (EU / mL) using the Limulus Amebocyte Lysate - LAL test. After biomechanical preparation (PBM) the teeth were filled with intracanal medications according to the groups for fourteen days. The interstitial fluid was collected after preparation of the channels and after 14 days of medication and quantification of the lipid mediators (RvE1, RvD2, LxA4) was performed through the enzymelinked immunosorbent assay (ELISA). The values obtained were tabulated, analyzed by GraphPad Prism 6.01 software and submitted to Kolmogorov Smirnov and Lilliefors normality tests, followed by Kruskal-Wallis and Dunn and Anova one-way and Tukey tests (P <0.05). For microbial load, PBM reduced on average 96% of aerobic microorganisms and 92.5% of anaerobes in all groups (p <0.05). The use of intracanal medication based on Ca (OH) 2 maintained levels reached with PBM (p> 0.05), but in the NAC group there was an increase of bacteria. The endotoxin levels decreased after the preparation of the canals and the use of the medications reduced the endotoxin level, but without statistical difference in relation to the reduction achieved with the biomechanical preparation. As for the lipid mediators, only in the NAC group an increase of D2 Rv was observed. It was concluded that PBM significantly reduced the number of bacteria and the amount of endotoxins in the canal, and Ca (OH) 2-based medications were efficient in the elimination of microorganisms from the canal, however NAC was not effective for reducing the load microbial; The three medications evaluated were able to act on bacterial LPS. Only the NAC was able to positively influence this result with the increase of RvD2 after 14 days of the medication. Lipid mediators have great potential for use in various types of endodontic treatment(AU)

LipoxinA4 reduces lipopolysaccharide-induced expression of cyclooxygenase-2 and prostaglandin E2 in primary lung fibroblasts of rat / 中华急诊医学杂志

Objective To explore the effects of lipoxinA4 on expression of cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2) in rat primary lung fibroblast cells (LF) after lipopolysaccharide (LPS) challenge.Methods Primary lung fibroblast cells were incubated with various concentrations (0.1,1,10 μg/mL) of LPS for different lengths of time (3,6,9 h).Then primary lung fibroblast cells were still incubated in DMEM medium containing LPS in the presence or absence of lipoxinA4.After incubation,the supematant of medium was collected and the level of PGE2 was detected by using ELISA.The cells were harvested,and COX-2 protein was analyzed by Western blot.Results The model of acute inflammation in fibroblasts was well established by administering 1 μg/mL LPS in fibroblasts for 6 hours.Induction of COX-2 protein by LPS was inhibited by lipoxinA4.The levels of PGE2 in control group,LPS group and LPS + LipoxinA4 group were 55.84 pg/mL,411.73 pg/mL and 307.07 pg/mL,respectively,and there was a significantdifference between LPS group and LPS + LipoxinA4 group (P ＜0.01).Conclusion LipoxinA4 down-regulates the expression of the COX-2 induced by LPS in primary lung fibroblast cells and consequently inhibits the production of PGE2 in a dose dependent manner.

Aspirin-triggered lipoxin A4 attenuates lipopolysaccharide induced inflammatory response in primary astrocytes.

The activation of astrocytes contributes to inflammatory responses underlying brain injury and neurodegenerative diseases. Lipoxins have emerged as mediators of endogenous anti-inflammatory events. However, the involvement of aspirin-triggered-lipoxin A4 (ATL) in astrocyte-induced neuroinflammatory responses has not been investigated. Here, we examined the anti-inflammatory effects of ATL in the central nervous system using rat astrocyte cultures stimulated with lipopolysaccharide (LPS). We found that pretreatment with ATL exerted potent anti-inflammatory effects by inhibiting LPS-induced production of nitric oxide and prostaglandin E2. ATL also reduced the expression of cyclooxygenase 2 and inducible nitric oxide synthase mRNA and protein. Furthermore, ATL suppressed the LPS-induced translocation of the NF-κB p65 subunit to the nucleus and prevented LPS-induced IκBα phosphorylation in a dose-dependent manner. These findings suggest that ATL attenuates neuroinflammation by inhibiting the NF-κB signal transducer pathway in cultured cortical astrocytes.