Dysregulation of neuroprotective lipoxin pathway in astrocytes in response to cytokines and ocular hypertension​.

Glaucoma leads to vision loss due to retinal ganglion cell death. Astrocyte reactivity contributes to neurodegeneration. Our recent study found that lipoxin B4 (LXB4), produced by retinal astrocytes, has direct neuroprotective actions on retinal ganglion cells. In this study, we aimed to investigate how the autacoid LXB4 influences astrocyte reactivity in the retina under inflammatory cytokine-induced activation and during ocular hypertension. The protective activity of LXB4 was investigated in vivo using the mouse silicone-oil model of chronic ocular hypertension. By employing a range of analytical techniques, including bulk RNA-seq, RNAscope in-situ hybridization, qPCR, and lipidomic analyses, we discovered the formation of lipoxins and expression of the lipoxin pathway in rodents (including the retina and optic nerve), primates (optic nerve), and human brain astrocytes, indicating the presence of this neuroprotective pathway across various species. Findings in the mouse retina identified significant dysregulation of the lipoxin pathway in response to chronic ocular hypertension, leading to an increase in 5-lipoxygenase (5-LOX) activity and a decrease in 15-LOX activity. This dysregulation was coincident with a marked upregulation of astrocyte reactivity. Reactive human brain astrocytes also showed a significant increase in 5-LOX. Treatment with LXB4 amplified the lipoxin biosynthetic pathway by restoring and amplifying the generation of another member of the lipoxin family, LXA4, and mitigated astrocyte reactivity in mouse retinas and human brain astrocytes. In conclusion, the lipoxin pathway is functionally expressed in rodents, primates, and human astrocytes, and is a resident neuroprotective pathway that is downregulated in reactive astrocytes. Novel cellular targets for LXB4's neuroprotective action are inhibition of astrocyte reactivity and restoration of lipoxin generation. Amplifying the lipoxin pathway is a potential target to disrupt or prevent astrocyte reactivity in neurodegenerative diseases, including retinal ganglion cell death in glaucoma.

LXA4 protected mice from renal ischemia/reperfusion injury by promoting IRG1/Nrf2 and IRAK-M-TRAF6 signal pathways.

Excessive inflammatory response and increased oxidative stress play an essential role in the pathophysiology of ischemia/reperfusion (I/R)-induced acute kidney injury (IRI-AKI). Emerging evidence suggests that lipoxin A4 (LXA4), as an endogenous negative regulator in inflammation, can ameliorate several I/R injuries. However, the mechanisms and effects of LXA4 on IRI-AKI remain unknown. In this study, A bilateral renal I/R mouse model was used to evaluate the role of LXA4 in wild-type, IRG1 knockout, and IRAK-M knockout mice. Our results showed that LXA4, as well as 5-LOX and ALXR, were quickly induced, and subsequently decreased by renal I/R. LXA4 pretreatment improved renal I/R-induced renal function impairment and renal damage and inhibited inflammatory responses and oxidative stresses in mice kidneys. Notably, LXA4 inhibited I/R-induced the activation of TLR4 signal pathway including decreased phosphorylation of TAK1, p36, and p65, but did not affect TLR4 and p-IRAK-1. The analysis of transcriptomic sequencing data and immunoblotting suggested that innate immune signal molecules interleukin-1 receptor-associated kinase-M (IRAK-M) and immunoresponsive gene 1 (IRG1) might be the key targets of LXA4. Further, the knockout of IRG1 or IRAK-M abolished the beneficial effects of LXA4 on IRI-AKI. In addition, IRG1 deficiency reversed the up-regulation of IRAK-M by LXA4, while IRAK-M knockout had no impact on the IRG1 expression, indicating that IRAK-M is a downstream molecule of IRG1. Mechanistically, we found that LXA4-promoted IRG1-itaconate not only enhanced Nrf2 activation and increased HO-1 and NQO1, but also upregulated IRAK-M, which interacted with TRAF6 by competing with IRAK-1, resulting in deactivation of TLR4 downstream signal in IRI-AKI. These data suggested that LXA4 protected against IRI-AKI via promoting IRG1/Itaconate-Nrf2 and IRAK-M-TRAF6 signaling pathways, providing the rationale for a novel strategy for preventing and treating IRI-AKI.

Lipoxin A4 levels predict site-specific clinical improvements post scaling and root planing and correlate negatively with periodontal pathogens in severe periodontitis.

BACKGROUND: Serving as a stop signal of inflammation, the role of lipoxin A4 (LXA4) in periodontitis remains to be clarified. This study is aimed to examine the changes in LXA4 levels in gingival crevicular fluid (GCF) after scaling and root planing (SRP) and to determine the relationship between LXA4 levels and treatment outcomes and periodontal pathogens in severe periodontitis. METHODS: A total of 74 GCF samples were collected from 21 severe periodontitis participants at the deepest affected sites. These sites were re-sampled at 1, 3, and 6 months after SRP. Besides, GCF samples were also collected from 25 periodontally healthy participants. Clinical parameters including probing depth (PD) and clinical attachment level (CAL) in periodontitis group were recorded. LXA4 levels and periodontal pathogens in the GCF were analyzed by ELISA and PCR, respectively. Correlations between GCF LXA4 levels and treatment effect and periodontal pathogens were assessed. RESULTS: LXA4 levels in GCF significantly increased after SRP (p < 0.05), but remained lower than those observed in healthy individuals (p < 0.05). Sites with lower baseline LXA4 concentrations were more likely to experience greater improvements in PD at 6 months post-SRP (area under the curve [AUC] = 0.792), and the improvements were positively correlated with the increase of LXA4 at these sites post-treatment (p < 0.05). Furthermore, more elevated LXA4 levels were observed in sites that became negative for Prevotella intermedia or Tannerella forsythia after SRP. CONCLUSION: Baseline LXA4 in GCF has the potential to predict the site-specific response of severe periodontal lesions to SRP. The increase of LXA4 levels after treatment was positively correlated with clinical improvements and negatively correlated with the presence of Prevotella intermedia or Tannerella forsythia.

Lipoxins A4 and B4 inhibit glial cell activation via CXCR3 signaling in acute retinal neuroinflammation.

Lipoxins are small lipids that are potent endogenous mediators of systemic inflammation resolution in a variety of diseases. We previously reported that Lipoxins A4 and B4 (LXA4 and LXB4) have protective activities against neurodegenerative injury. Yet, lipoxin activities and downstream signaling in neuroinflammatory processes are not well understood. Here, we utilized a model of posterior uveitis induced by lipopolysaccharide endotoxin (LPS), which results in rapid retinal neuroinflammation primarily characterized by activation of resident macroglia (astrocytes and Müller glia), and microglia. Using this model, we observed that each lipoxin reduces acute inner retinal inflammation by affecting endogenous glial responses in a cascading sequence beginning with astrocytes and then microglia, depending on the timing of exposure; prophylactic or therapeutic. Subsequent analyses of retinal cytokines and chemokines revealed inhibition of both CXCL9 (MIG) and CXCL10 (IP10) by each lipoxin, compared to controls, following LPS injection. CXCL9 and CXCL10 are common ligands for the CXCR3 chemokine receptor, which is prominently expressed in inner retinal astrocytes and ganglion cells. We found that CXCR3 inhibition reduces LPS-induced neuroinflammation, while CXCR3 agonism alone induces astrocyte reactivity. Together, these data uncover a novel lipoxin-CXCR3 pathway to promote distinct anti-inflammatory and proresolution cascades in endogenous retinal glia.

Resolvin and lipoxin metabolism network regulated by Hyssopus Cuspidatus Boriss extract in asthmatic mice.

Resolvin (Rv) and lipoxin (Lx) play important regulative roles in the development of several inflammation-related diseases. The dysregulation of their metabolic network is believed to be closely related to the occurrence and development of asthma. The Hyssopus Cuspidatus Boriss extract (SXCF) has long been used as a treatment for asthma, while the mechanism of anti-inflammatory and anti-asthma action targeting Rv and Lx has not been thoroughly investigated. In this study, we aimed to investigate the effects of SXCF on Rv, Lx in ovalbumin (OVA)-sensitized asthmatic mice. The changes of Rv, Lx before and after drug administration were analyzed based on high sensitivity chromatography-multiple response monitoring (UHPLC-MRM) analysis and multivariate statistics. The pathology exploration included behavioral changes of mice, IgE in serum, cytokines in BALF, and lung tissue sections stained with H&E. It was found that SXCF significantly modulated the metabolic disturbance of Rv, Lx due to asthma. Its modulation effect was significantly better than that of dexamethasone and rosmarinic acid which is the first-line clinical medicine and the main component of Hyssopus Cuspidatus Boriss, respectively. SXCF is demonstrated to be a potential anti-asthmatic drug with significant disease-modifying effects on OVA-induced asthma. The modulation of Rv and Lx is a possible underlying mechanism of the SXCF effects.

BML-111, the agonist of lipoxin A4, suppresses epithelial-mesenchymal transition and migration of MCF-7 cells via regulating the lipoxygenase pathway.

Introduction: Aberrant epithelial-mesenchymal transition (EMT) and migration frequently occur during tumour progression. BML-111, an analogue of lipoxin A4, has been implicated in inflammation in cancer research. Methods: 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, western blot, Reverse Transcription Polymerase Chain Reaction (RT-PCR), transwell assay, immunofluorescence, and immunohistochemistry were conducted in this study. Results: In vitro experiments revealed that BML-111 inhibited EMT and migration in CoCl2-stimulated MCF-7 cells. These effects were achieved by inhibiting MMP-2 and MMP-9, which are downregulated by 5-lipoxygenase (5-LOX). Moreover, BML-111 inhibited EMT and migration of breast cancer cells in BALB/c nude mice inoculated with MCF-7 cells. Conclusion: Our results suggest that BML-111 may be a potential therapeutic drug for breast cancer and that blocking the 5-LOX pathway could be a possible approach for mining effective drug targets.

Lipoxin-mediated signaling: ALX/FPR2 interaction and beyond.

In the aftermath of tissue injury or infection, an efficient resolution mechanism is crucial to allow tissue healing and preserve appropriate organ functioning. Pro-resolving bioactive lipids prevent uncontrolled inflammation and its consequences. Among these mediators, lipoxins were the first described and their pro-resolving actions have been mainly described in immune cells. They exert their actions mostly through formyl-peptide receptor 2 (ALX/FPR2 receptor), a G-protein-coupled receptor whose biological function is tremendously complex, primarily due to its capacity to mediate variable cellular responses. Moreover, lipoxins can also interact with alternative receptors like the cytoplasmic aryl hydrocarbon receptor, the cysteinyl-leukotrienes receptors or GPR32, triggering different intracellular signaling pathways. The available information about this complex response mediated by lipoxins is addressed in this review, going over the different mechanisms used by these molecules to stop the inflammatory reaction and avoid the development of dysregulated and chronic pathologies.

Sirtuin6 and Lipoxin A4 levels are decreased in severe periodontitis.

OBJECTIVE: Sirtuin6 plays an important role in the regulation of inflammation, homeostasis, and apoptosis, and it has anti-inflammatory effects on several diseases. Lipoxin A4 is a pro-resolving lipid mediator of inflammation and inhibits hypoxia-induced apoptosis and oxidative stress. Considering that Lipoxin A4 and Sirtuin6 have protective effects on inflammatory diseases, the aim of this study is to determine the possible roles of these molecules on periodontitis inflammation in saliva and serum and to reveal the relationship of these data with clinical periodontal parameters. MATERIAL AND METHODS: A total of 20 stage III/grade B periodontitis and 20 periodontally healthy subjects were included in this cross-sectional study (all never smokers and systemically healthy). Clinical periodontal parameters (plaque index, probing pocket depth, bleeding on probing, clinical attachment loss) were recorded. Saliva and serum levels of Sirtuin6 and Lipoxin A4 were analyzed by enzyme-linked immunosorbent assay. RESULTS: Serum Sirtuin6 and saliva Lipoxin A4 levels were significantly lower in the periodontitis group than the control group (respectively, p = 0.0098, p = 0.0008). There were negative correlations between all periodontal clinical parameters and saliva Lipoxin A4 level (p < 0.05) and between probing pocket depth, clinical attachment loss, and serum and saliva Sirtuin6 levels (respectively, r = - 0.465 and r = - 0.473, p < 0.05). CONCLUSIONS: Decreased levels of serum Sirtuin6 and saliva Lipoxin A4 in periodontitis patients and their correlation with clinical periodontal parameters suggest that serum Sirtuin6 and saliva Lipoxin A4 may be related with periodontal inflammation. CLINICAL RELEVANCE: Scientific rationale for the study: Sirtuin6 is one of seven members of the family of NAD + dependent protein that played an important role in the regulation of inflammation, energy metabolism, homeostasis, and apoptosis. Sirtuin6 is associated with the pathogenesis of several diseases. Lipoxin A4 is a lipid mediator that inhibits hypoxia-induced apoptosis and oxidative stress, and it has an active role in the resolution of periodontal inflammation. No studies that investigated the potential role Sirtuin6 and its relationship with inflammation resolution and apoptosis mechanisms in severe periodontitis patients. PRINCIPAL FINDINGS: the serum Sirtuin6 and saliva Lipoxin A4 levels were significantly lower and negatively correlated with clinical periodontal parameters in the patients with periodontitis than the healthy controls. PRACTICAL IMPLICATIONS: this study shows that serum Sirtuin6 and saliva Lipoxin A4 may be candidate biomarkers related with periodontal inflammation and estimating to periodontal status. CLINICAL TRIAL REGISTRATION: NCT05417061.

The biosynthetic pathways of the protectins.

Evidence for the biosynthetic pathways of the specialized pro-resolving mediator (SPM) protectin D1 (PD1) and its biochemical further local metabolism were presented during the 8th European Workshop on Lipid Mediators, organized June 29th-July 1st, 2022, in Stockholm, Sweden. Herein, we provide an extended and detailed discussion of these topics. PD1, one of 43 SPMs reported so far, exhibits very potent pro-resolution and anti-inflammatory bioactions. Many research groups worldwide have confirmed these and other interesting bioactions. The protectins constitute, together with the lipoxins, resolvins, and maresins, the four individual SPM families, which have received a great interest in basic biomedical research and drug discovery efforts.

Promising Anti-Inflammatory Tools: Biomedical Efficacy of Lipoxins and Their Synthetic Pathways.

Lipoxins (LXs) have attracted widespread attention as a class of anti-inflammatory lipid mediators that are produced endogenously by the organism. LXs are arachidonic acid (ARA) derivatives that include four different structures: lipoxin A4 (LXA4), lipoxin B4 (LXB4), and the aspirin-induced differential isomers 15-epi-LXA4 and 15-epi-LXB4. Because of their unique biological activity of reducing inflammation in the body, LXs have great potential for neuroprotection, anti-inflammatory treatment of COVID-19, and other related diseases. The synthesis of LXs in vivo is achieved through the action of lipoxygenase (LO). As a kind of important enzyme, LO plays a major role in the physiological processes of living organisms in mammals and functions in some bacteria and fungi. This suggests new options for the synthesis of LXs in vitro. Meanwhile, there are other chemical and biochemical methods to synthesize LXs. In this review, the recent progress on physiological activity and synthetic pathways of LXs is summarized, and new insights into the synthesis of LXs in vitro are provided.

A general synthesis of aromatic and heteroaromatic lipoxin B4 analogues.

Lipoxins are an important class of pro-resolving mediators that play a crucial role in the resolution of inflammation. Thus, the synthesis of more chemically and metabolically stable synthetic lipoxin analogues is an area of significant interest. Whereas synthetic analogues of lipoxin A4 (LXA4) have been well studied, analogues of lipoxin B4 (LXB4) have been the focus of considerably less attention. Herein we report the asymmetric synthesis of a focused library of LXB4 mimetics in which the triene core of the molecule has been replaced with different aromatic and heteroaromatic rings. The synthesis of each of these analogues was achieved by a general strategy in which the key steps were a Suzuki cross coupling between a common upper chain fragment and an aromatic lower chain, followed by a stereoselective ketone reduction.

The effect of low-dose aspirin on aspirin triggered lipoxin, interleukin 1 beta, and prostaglandin E2 levels in periapical fluid: a double-blind randomized clinical trial.

BACKGROUND: The role of pro-resolving mediators in inflammation is a new concern in research. The effect of low-dose aspirin on production of a special kind of these mediators named aspirin triggered lipoxin (ATL) has been studied on different tissues. This randomized clinical trial evaluated the effect of low-dose aspirin on ATL and pro-inflammatory mediators' level in periapical fluid of necrotic teeth with large lesions. METHODS: Twenty-four patients with necrotic pulp and periapical lesion were randomly assigned to low-dose aspirin and placebo groups. In the first appointment, canals were shaped up to F3 size and #40 K-file and cleaned with 10 milliliters 2.5% sodium hypochlorite and 17% Ethylenediaminetetraacetic acid. Periapical fluid was sampled by a paper cone. The tooth was temporized without any intracanal medication. Tablets were administered for 7 days, then the teeth were re-opened and the sampling were repeated. Interleukin-1 beta (IL-1ß), prostaglandin E2 (PGE2) and ATL were analyzed by enzyme-linked immunosorbent assay. Data were analyzed with paired t-test using SPSS statistical software, version 21 (α = 0.05). RESULTS: A significant reduction in PGE2 and IL-1ß was noted in the aspirin-treated group while an increase in ATL was observed (P < 0.001). There was no significant difference in the mediator scores before and after in the placebo-treated group (P > 0.05). CONCLUSION: Low-dose aspirin can influence the inflammatory process by reducing pro-inflammatory mediators such as PGE2 and IL-1ß, as well as increasing the pro-resolving mediators such as ATL. TRIAL REGISTRATION: IRCT20191211045702N1.

Lipoxin A4 promotes antibiotic and monocyte bacterial killing in established Pseudomonas aeruginosa biofilm formed under hydrodynamic conditions.

Pseudomonas aeruginosa is a gram-negative, opportunistic bacteria commonly found in wounds and in lungs of immunocompromised patients. These bacteria commonly form biofilms which encapsulate the bacteria, making it difficult for antibiotics or immune cells to reach the bacterial cells. We previously reported that Lipoxin A4 (LxA4 ), a Specialized Pro-resolving Mediator, has direct effects on P. aeruginosa where it reduced biofilm formation and promoted ciprofloxacin antibiotic efficacy in a static biofilm-forming system. In the current studies, we examined the actions of LxA4 on established biofilms formed in a biofilm reactor under dynamic conditions with constant flow and shear stress. These conditions allow for biofilm growth with nutrient replenishment and for examination of bacteria within the biofilm structure. We show that LxA4 helped ciprofloxacin reduction of live/dead ratio of bacteria within the biofilm. THP-1 monocytes interacted with the biofilm to increase the number of viable bacteria within the biofilm as well as TNF-α production in the biofilm milieu, suggesting that monocyte interaction with bacterial biofilm exacerbates the inflammatory state. Pre-treatment of the THP-1 monocytes with LxA4 abolished the increase in biofilm bacteria and reduced TNF-α production. The effect of decreased biofilm bacteria was associated with increased LxA4 -induced monocyte adherence to biofilm but not increased bacteria killing suggesting that the mechanism for the reduced biofilm bacteria was due to LxA4 -mediated increase in adherence to biofilm. These results suggest that LxA4 can help antibiotic efficacy and promote monocyte activity against established P. aeruginosa biofilm formed under hydrodynamic conditions.

Modulation of the activation of endothelial nitric oxide synthase and nitrosative stress biomarkers by aspirin triggered lipoxins: A possible mechanism of action of aspirin in the antiphospholipid syndrome.

PROBLEM: Antiphospholipid syndrome (APS) is characterized by the clinical manifestation of vascular thrombosis (VT) or pregnancy morbidity (PM) and antiphospholipid antibodies (aPL) that can modify the nitric oxide production. Low-dose aspirin is used in the prevention and treatment of diverse alterations of pregnancy. One of the mechanisms of action of aspirin is to induce the production of aspirin-triggered-lipoxins (ATL). The aim of this study was to evaluate the modulatory effect of ATL over the activation of endothelial nitric oxide synthase (eNOS) and nitrosative stress biomarkers induced by aPL. METHODS: We used polyclonal IgG and sera from women with aPL and PM/VT or VT only, and from women with PM only and positive for non-criteria aPL (SN-OAPS). In these sera, biomarkers of nitrosative stress (nitrites and nitrotyrosine) were measured. The protein expression of nitrotyrosine and the phosphorylation of eNOS (at Ser1177) were estimated in human umbilical vein endothelial cells (HUVECs) stimulated with polyclonal IgG with or without ATL. RESULTS: Women with SN-OAPS showed increased circulating levels of nitrites and nitrotyrosine. Likewise, polyclonal IgG from either SN-OAPS or VT patients stimulated nitrotyrosine expression in HUVECs. ATL decreased the nitrotyrosine expression induced by polyclonal IgG from the SN-OAPS group. ATL also recovered the reduced eNOS phosphorylation at Ser1177 in HUVECs stimulated with polyclonal IgG from women with PM/VT or SN-OAPS. CONCLUSIONS: Increased nitrosative stress present in serum of women with SN-OAPS is associated with IgG-mediated impaired endothelial NO synthesis in endothelial cells. ATL prevent these cellular changes.

Lipoxin receptor agonist and inhibition of LTA4 hydrolase prevent tight junction disruption caused by P. aeruginosa filtrate in airway epithelial cells.

Airway diseases can disrupt tight junction proteins, compromising the epithelial barrier and making it more permeable to pathogens. In people with pulmonary disease who are prone to infection with Pseudomonas aeruginosa, pro-inflammatory leukotrienes are increased and anti-inflammatory lipoxins are decreased. Upregulation of lipoxins is effective in counteracting inflammation and infection. However, whether combining a lipoxin receptor agonist with a specific leukotriene A4 hydrolase (LTA4H) inhibitor could enhance these protective effects has not to our knowledge been investigated. Therefore, we explored the effect of lipoxin receptor agonist BML-111 and JNJ26993135 a specific LTA4H inhibitor that prevents the production of pro-inflammatory LTB4 on tight junction proteins disrupted by P. aeruginosa filtrate (PAF) in human airway epithelial cell lines H441 and 16HBE-14o. Pre-treatment with BML-111 prevented an increase in epithelial permeability induced by PAF and conserved ZO-1 and claudin-1 at the cell junctions. JNJ26993135 similarly prevented the increased permeability induced by PAF, restored ZO-1 and E-cadherin and reduced IL-8 but not IL-6. Cells pre-treated with BML-111 plus JNJ26993135 restored TEER and permeability, ZO-1 and claudin-1 to the cell junctions. Taken together, these data indicate that the combination of a lipoxin receptor agonist with a LTA4H inhibitor could provide a more potent therapy.

A novel protective role of lipoxin in inhibiting diabetic vascular calcification via YAP signalling: Health prevention and regulation.

Vascular calcification is highly prevalent in diabetes patients, with detrimental consequences and no effective prevention and treatment strategies are currently available. Though the protective effect of lipoxin (LX) against vascular diseases has been demonstrated, its effect on diabetic vascular calcification remains unknown. AGEs dose-dependently induced calcification and the expression of osteogenesis-related markers, coupled with the activation of yes-associated protein (YAP). Mechanistically, YAP activation enhanced the AGE-induced osteogenic phenotype and calcification, but inhibition of YAP signalling alleviated this response. Further, an in vivo diabetic mouse model was established using a combination of a high-fat diet and multiple formulations of low-dose streptozotocin. Consistent with the in vitro results, diabetes promoted YAP expression and its subcellular localization in the nucleus in the arterial tunica media. The results demonstrate that LX attenuates the trans-differentiation and calcification of VSMCs in diabetes mellitus via YAP signalling, suggesting LX to be a potent therapeutic for preventing diabetic vascular calcification.

Therapeutic activity of lipoxin A4 in TiO2-induced arthritis in mice: NF-κB and Nrf2 in synovial fluid leukocytes and neuronal TRPV1 mechanisms.

Background: Lipoxin A4 (LXA4) has anti-inflammatory and pro-resolutive roles in inflammation. We evaluated the effects and mechanisms of action of LXA4 in titanium dioxide (TiO2) arthritis, a model of prosthesis-induced joint inflammation and pain. Methods: Mice were stimulated with TiO2 (3mg) in the knee joint followed by LXA4 (0.1, 1, or 10ng/animal) or vehicle (ethanol 3.2% in saline) administration. Pain-like behavior, inflammation, and dosages were performed to assess the effects of LXA4 in vivo. Results: LXA4 reduced mechanical and thermal hyperalgesia, histopathological damage, edema, and recruitment of leukocytes without liver, kidney, or stomach toxicity. LXA4 reduced leukocyte migration and modulated cytokine production. These effects were explained by reduced nuclear factor kappa B (NFκB) activation in recruited macrophages. LXA4 improved antioxidant parameters [reduced glutathione (GSH) and 2,2-azino-bis 3-ethylbenzothiazoline-6-sulfonate (ABTS) levels, nuclear factor erythroid 2-related factor 2 (Nrf2) mRNA and Nrf2 protein expression], reducing reactive oxygen species (ROS) fluorescent detection induced by TiO2 in synovial fluid leukocytes. We observed an increase of lipoxin receptor (ALX/FPR2) in transient receptor potential cation channel subfamily V member 1 (TRPV1)+ DRG nociceptive neurons upon TiO2 inflammation. LXA4 reduced TiO2-induced TRPV1 mRNA expression and protein detection, as well TRPV1 co-staining with p-NFκB, indicating reduction of neuronal activation. LXA4 down-modulated neuronal activation and response to capsaicin (a TRPV1 agonist) and AITC [a transient receptor potential ankyrin 1 (TRPA1) agonist] of DRG neurons. Conclusion: LXA4 might target recruited leukocytes and primary afferent nociceptive neurons to exert analgesic and anti-inflammatory activities in a model resembling what is observed in patients with prosthesis inflammation.

Lipoxins and their relationship with inflammation-associated diseases. A systematic review.

AIM: To determine the relationship of lipoxin levels with inflammation and disease development in adults and children. METHODS: We conducted a systematic review. The search strategy included Medline, Ovid, EMBASE, LILACS, The Cochrane Central Register of Controlled Trials, and Open Gray. We included Clinical trials, cohort studies, case-control studies, and cross-sectional studies. Animal experiments were excluded. RESULTS: We included fourteen studies in this review, nine consistently showing decreased lipoxin levels and anti-inflammatory markers or increased pro-inflammatory markers in cardiovascular disease, metabolic syndrome, Alzheimer's disease, periodontitis, or autism. Five studies showed increased lipoxin levels and pro-inflammatory markers in pre-eclampsia, asthma, and coronary disease. On the other hand, one showed increased lipoxin levels and decreased pro-inflammatory marker levels. CONCLUSIONS: Decreases in lipoxins are associated with developing pathologies such as cardiovascular and neurological diseases, indicating that lipoxins protect against these pathologies. However, in other pathologies, such as asthma, pre-eclampsia, and periodontitis, which are associated with chronic inflammation despite increased levels of LXA4, the increase in inflammation suggests a possible failure of this regulatory pathway. Therefore, further studies are necessary to evaluate the role of LXA4 in the pathogenesis of inflammatory diseases.

Effect of the Lipoxin Receptor Agonist BML-111 on Cigarette Smoke Extract-Induced Macrophage Polarization and Inflammation in RAW264.7 Cells.

Background: Macrophages are known to play a crucial role in the chronic inflammation associated with Chronic Obstructive Pulmonary Disease (COPD). BML-111, acting as a lipoxin A4 (LXA4) receptor agonist, has shown to be effective in protecting against COPD. However, the precise mechanism by which BML-111 exerts its protective effect remains unclear. Methods: In order to establish a cell model of inflammation, cigarette smoke extract (CSE) was used on the RAW264.7 cell line. Afterwards, an Enzyme-linked immunosorbent assay (ELISA) kit was employed to measure concentrations of tumor necrosis factor-α (TNF-α), interleukin-1beta (IL-1ß), interleukin-18 (IL-18), and interleukin-10 (IL-10) in the cell supernatants of the RAW264.7 cells.In this study, we examined the markers of macrophage polarization using two methods: quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot analysis. Additionally, we detected the expression of Notch-1 and Hes-1 through Western blotting. Results: BML-111 effectively suppressed the expression of pro-inflammatory cytokines TNF-α, IL-1ß, and IL-18, as well as inflammasome factors NLRP3 and Caspase-1, while simultaneously up-regulating the expression of the anti-inflammatory cytokine IL-10 induced by CSE. Moreover, BML-111 reduced the expression of iNOS, which is associated with M1 macrophage polarization, and increased the expression of Arg-1, which is associated with M2 phenotype. Additionally, BML-111 downregulated the expression of Hes-1 and the ratio of activated Notch-1 to Notch-1 induced by CSE. The effect of BML-111 on inflammation and macrophage polarization was reversed upon administration of the Notch-1 signaling pathway agonist Jagged1. Conclusion: BML-111 has the potential to suppress inflammation and modulate M1/M2 macrophage polarization in RAW264.7 cells. The underlying mechanism may involve the Notch-1 signaling pathway.

Aspirin modulates production of pro-inflammatory and pro-resolving mediators in endothelial cells.

Endothelial cells synthesize biochemical signals to coordinate a response to insults, resolve inflammation and restore barrier integrity. Vascular cells release a variety of vasoactive bioactive lipid metabolites during the inflammatory response and produce pro-resolving mediators (e.g., Lipoxin A4, LXA4) in cooperation with leukocytes and platelets to bring a halt to inflammation. Aspirin, used in a variety of cardiovascular and pro-thrombotic disorders (e.g., atherosclerosis, angina, preeclampsia), potently inhibits proinflammatory eicosanoid formation. Moreover, aspirin stimulates the synthesis of pro-resolving lipid mediators (SPM), so-called Aspirin-Triggered Lipoxins (ATL). We demonstrate that cytokines stimulated a time- and dose-dependent increase in PGI2 (6-ketoPGF1α) and PGE2 formation that is blocked by aspirin. Eicosanoid production was caused by cytokine-induced expression of cyclooxygenase-2 (COX-2). We also detected increased production of pro-resolving LXA4 in cytokine-stimulated endothelial cells. The R-enantiomer of LXA4, 15-epi-LXA4, was enhanced by aspirin, but only in the presence of cytokine challenge, indicating dependence on COX-2 expression. In contrast to previous reports, we detected arachidonate 5-lipoxygenase (ALOX5) mRNA expression and its cognate protein (5-lipoxygenase, 5-LOX), suggesting that endothelial cells possess the enzymatic machinery necessary to synthesize both pro-inflammatory and pro-resolving lipid mediators independent of added leukocytes or platelets. Finally, we observed that, endothelial cells produced LTB4 in the absence of leukocytes. These results indicate that endothelial cells produce both pro-inflammatory and pro-resolving lipid mediators in the absence of other cell types and aspirin exerts pleiotropic actions influencing both COX and LOX pathways.