Lipoxin A4 Treatment Research Articles

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

BackgroundServing 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.MethodsA 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.ResultsLXA4 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.ConclusionBaseline 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.

Lipoxin A4 (LXA4) Reduces Alkali-Induced Corneal Inflammation and Neovascularization and Upregulates a Repair Transcriptome.

To investigate the anti-inflammatory and anti-angiogenic effects of the bioactive lipid mediator LXA4 on a rat model of severe corneal alkali injury. To induce a corneal alkali injury in the right eyes of anesthetized Sprague Dawley rats. They were injured with a Φ 4 mm filter paper disc soaked in 1 N NaOH placed on the center of the cornea. After injury, the rats were treated topically with LXA4 (65 ng/20 μL) or vehicle three times a day for 14 days. Corneal opacity, neovascularization (NV), and hyphema were recorded and evaluated in a blind manner. Pro-inflammatory cytokine expression and genes involved in cornel repair were assayed by RNA sequencing and capillary Western blot. Cornea cell infiltration and monocytes isolated from the blood were analyzed by immunofluorescence and by flow cytometry. Topical treatment with LXA4 for two weeks significantly reduced corneal opacity, NV, and hyphema compared to the vehicle treatment. RNA-seq and Western blot results showed that LXA4 decreased the gene and protein expression of pro-inflammatory cytokines interleukin (IL)-1β and IL-6 and pro-angiogenic mediators matrix metalloproteinase (MMP)-9 and vascular endothelial growth factor (VEGFA). It also induces genes involved in keratinization and ErbB signaling and downregulates immune pathways to stimulate wound healing. Flow cytometry and immunohistochemistry showed significantly less infiltration of neutrophils in the corneas treated with LXA4 compared to vehicle treatment. It also revealed that LXA4 treatment increases the proportion of type 2 macrophages (M2) compared to M1 in blood-isolated monocytes. LXA4 decreases corneal inflammation and NV induced by a strong alkali burn. Its mechanism of action includes inhibition of inflammatory leukocyte infiltration, reduction in cytokine release, suppression of angiogenic factors, and promotion of corneal repair gene expression and macrophage polarization in blood from alkali burn corneas. LXA4 has potential as a therapeutic candidate for severe corneal chemical injuries.

Combined Application of Exosomes and FPR2 Agonist LXA4 in Controlling Fetal Membrane Inflammation and Promoting Fetal Membrane Tissue Repair.

Preterm premature rupture of membranes (pPROM) is a common pregnancy disease closely related to inflammation. The formyl peptide receptor 2 (FPR2), a member of the G protein-coupled receptor family involved in defense responses, inflammation, and disturbances in glucose and lipid metabolism, is associated with pregnancy diseases. Lipoxin A4 (LXA4) can activate FPR2 and inhibit the inflammatory signals. Exosomes derived from mesenchymal stem cells are good materials for anti-inflammatory and tissue repair. This study aims to investigate the anti-inflammatory and tissue repair effects of the combined application of exosomes derived from human umbilical cord mesenchymal stem cells and FPR2 agonist LXA4. In this study, LPS was used to establish the inflammation model of pregnant mice and HTR8 cells, and LXA4 and exosome treatment were carried out to observe the fetal membranes' tissue repair. The scanning and transmission electron microscopy of fetal membrane tissue indicated that the structure of pPROM tissue was disordered, and the cell gap was significantly increased. The results of the inflammatory mice model suggested that LPS can cause damage to the fetal membrane structure. LXA4 combined with exosome treatment can inhibit the production of MMP2 and MMP9, and promote neovascularization by inhibiting the p38 MAPK/Nuclear factor kB p65 (NFkB) pathway in the inflammation model of HTR8 cells and pregnant mice, thus helping to control inflammation and tissue repair.

Abstract P130: Specialized Pro-resolving Mediator, Lipoxin A4, Attenuates Age-dependent Vascular Damage

Chronic, low-grade inflammation that develops with advanced age is known as inflammaging. This phenomenon is associated with large artery stiffness in age-dependent hypertension. Specialized pro-resolving mediators (SPMs), including Lipoxin A4 (LXA4) and Resolvin D1 (RD1), are derived from essential fatty acids, and they act as immunoresolvents to reduce inflammation and increase the clearance of pathogens and dying cells. However, LXA4, but not RD1, acts on formyl peptide receptor 2, which is known to modulate actin filaments and resolve inflammation. Therefore, we hypothesized that Specialized pro-resolving mediators, would restore mechanical stress and distensibility in aorta from older rats. To test this hypothesis, thoracic aortas from male Wistar rats (3 and 6 months old) were cut into 2 mm rings and incubated in the presence or absence of RD1 or LXA4; both 10 nM for 24 hours. Stress-strain curves were calculated via Tissue Puller (560TP-II, DMT). The aortic rings from 6-month-old rats presented with increased stress when compared to aortas of the 3-month-old (Maximal stress: 2749 ± 190 mN/mm 2 vs 4207 ± 242 mN/mm 2 , respectively; p=0.0015). Interestingly, LXA4 reduced stress in aorta from 6-month-old rats (Maximal stress: 2514 ± 552 mN/mm 2 , p=0.0282). No changes were observed in aorta from 3-month-old rats after LXA4 treatment. Resolvin D1 did not change vascular mechanics in aorta from 3- or 6-month-old rats. Overall, these data suggest that even modest advances in age are related with changes in vascular mechanics of large arteries and LXA4 via FPR2 can decrease inflammaging-related vascular stiffness.

Combination of resolvin E1 and lipoxin A4 promotes the resolution of pulpitis by inhibiting NF-κB activation through upregulating sirtuin 7 in dental pulp fibroblasts.

ObjectivesTo determine whether the combination of resolvin E1 (RvE1) and lipoxin A4 (LXA4) could promote resolution of pulpitis and to investigate the mechanism.Materials and MethodsPreliminary screening was first conducted in four specialized pro‐resolving mediators (SPMs). Real‐time quantitative polymerase chain reaction, western blotting, enzyme‐linked immunosorbent assay and double‐immunofluorescence labelling were employed to assess the expression of RelA, SIRT1, SIRT6, SIRT7 and pro‐inflammatory factors. Dental pulp fibroblasts (DPFs) were transfected with siRNA to assess the biological role of SIRT7. A pulpitis model was utilized to evaluate the in vivo curative effect.ResultsPreliminary results showed that RvE1 and LXA4 reduced the expression of RelA more markedly than other two SPMs. Both RvE1 and LXA4 treatment downregulated nuclear factor kappa B (NF‐κB) activation and increased the expression of SIRT1, SIRT6 and SIRT7, more so in combination than alone. Double‐immunofluorescence labelling showed that SIRT7 co‐localized with p‐p65 and Ac‐p65 in the nucleus. Inhibiting ChemR23 and ALX reversed the expression of RelA mRNA, p‐p65 and Ac‐p65 proteins, pro‐inflammatory factors, SIRT1, SIRT6 and SIRT7. Silencing SIRT7 significantly increased p‐p65 and Ac‐p65 protein levels and decreased SIRT1 and SIRT6 expression. In vivo experiments showed that combined administration of RvE1 and LXA4 promoted pulpitis markedly to resolution.ConclusionsCombination of RvE1 and LXA4 effectively inhibited NF‐κB activation by upregulating SIRT7 expression in DPFs, leading to reduced production of pro‐inflammatory factors and promotion of pulpitis resolution.

Influence of Lipoxin-A4 Treatment on Cytokine, Chemokine Genes Expression, and Phenotypic Distribution of Lymphocyte Subsets During Experimental Liver Fibrosis.

Lipoxins are anti-inflammatory, pro-resolving molecules that are secreted by immune cells such as neutrophils and macrophages. Lipoxins are a metabolite of the arachidonic acid pathway that resolve inflammation in fibrotic liver by producing several anti-inflammatory molecules. In this study, phenotypic distribution activation markers of lymphocytes in the spleen and expression levels of chemokines (chemokine (C-X-C motif) receptor 3, chemokine (C-X-C motif) ligand 10) cytokines (interferon gamma, tumor necrosis factor alpha, interleukin-6, interleukin-10) in the liver of lipoxin A4-treated fibrotic mice were investigated. Liver fibrosis was induced in BALB/c mice by thioacetamide administration. Lipoxin A4 was administered during last 2 weeks of induction. Fibrosis level was determined by using Knodell scoring. Lymphocytes were identified by flow-cytometry. Expression levels of genes were measured by quantitative real time polymerase chain reaction in liver homogenates. Lipoxin A4 treatment caused an elevation of T-lymphocyte percentage in the spleen. Interestingly, administration of lipoxin A4 significantly reduced B-lymphocyte population in spleen of fibrotic group. CD8+ cytotoxic T cell frequency significantly reduced in thioacetamide-induced mice; however, lipoxin A4 administration increased that percentage significantly. Lipoxin A4 treatment significantly reduced frequency of activated (CD8+CD69+) cytotoxic T cells. Expression levels of chemokines significantly reduced in the liver after lipoxin A4 treatment. While expression levels of interferon gamma, tumor necrosis factor alpha, and interleukin-6 significantly reduced in the liver after lipoxin A4 treatment, an anti-inflammatory cytokine interleukin-10 expression was almost at similar levels in all experimental groups. Lipoxin A4 performs its anti-inflammatory effect by reducing the frequency of activated T cells and expression levels of chemokines cytokines responsible from inflammatory immune response in the liver.

Lipoxin A4 promotes adipogenic differentiation and browning of mouse embryonic fibroblasts.

Recently, it has been irrefutably discovered that brown adipocytes dissipate energy as heat and protect against obesity. Researchers make great efforts to explore approaches for its activation. Lipoxin A4 (LXA4) has been proven to reverse adipose tissue inflammation and improve insulin resistance, but its function on brown adipocyte differentiation has been poorly understood, which therefore to be investigated in the present study. Mouse embryonic fibroblasts (MEFs) were induced and differentiated to model brown adipocytes, and treated with LXA4 at 0, 1, 5, and 10nM for 0-14 d. Afterwards, Oil Red O staining detected lipid droplets. In differentiated MEFs with or without LXA4 (10nM) treatment, western blot and quantitative real-time polymerase chain reaction (qRT-PCR) assessed adipocyte browning marker uncoupling protein 1 (UCP-1), and brown adipogenesis markers peroxisome proliferator-activated receptor gamma (PPARγ), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α), cyclooxygenase-2 (COX-2), and positive regulation domain containing 16 (PRDM16) as well as lipogenic genes of stearoyl-CoA desaturase 1 (SCD1), fatty acid synthase (FASN), glucose transporter type 4 (GLUT4), and carbohydrate response element binding protein (ChREBP). The induced differentiation of MEFs toward brown adipocytes was successful. LXA4 promoted intracellular accumulation of lipid droplets of induced cells and increased UCP-1 expression in a dose- or time-dependent manner. Under the administration of LXA4, brown adipogenesis markers and lipogenic genes were further upregulated. LXA4 made a contribution to induce differentiation of MEFs to brown adipocytes, which could be regarded a new drug target for obesity management.

Lipoxin A4 protects primary spinal cord neurons fromErastin-induced ferroptosis by activating the Akt/Nrf2/HO-1 signaling pathway.

Ferroptosis is an iron‐dependent programmed cell death, which participates in the pathogenesis of spinal cord injury (SCI). Our previous study has revealed that Lipoxin A4 (LXA4) exerts a protective role in SCI. Here, we investigated whether LXA4 can protect SCI through inhibiting neuronal ferroptosis. We treated primary spinal cord neurons with Erastin (ferroptosis activator) to induce ferroptosis. Erastin treatment reduced cell viability and enhanced cell death of primary spinal cord neurons, which was rescued by ferrostatin‐1 (ferroptosis inhibitor). Moreover, Erastin repressed glutathione peroxidase 4 (GPX4) expression and the levels of glutathione and cysteine in primary spinal cord neurons. Erastin also enhanced the expression of ferroptosis biomarkers (PTGS2 and ACSL4) and the levels of reactive oxygen species (ROS) in primary spinal cord neurons. The influence conferred by Erastin was effectively abolished by LXA4 treatment. Furthermore, LXA4 enhanced the protein expression of p‐AKT, nuclear factor (erythroid‐derived 2)‐like 2 (Nrf2) and haem‐oxygenase‐1 (HO‐1) in primary spinal cord neurons. LXA4‐mediated inhibition of ferroptosis of primary spinal cord neurons was prohibited by LY294002 (AKT inhibitor), brusatol (Nrf2 inhibitor) or zinc protoporphyrin (HO‐1 inhibitor). In conclusion, this work demonstrated that LXA4 exerted a neuroprotective effect in Erastin‐induced ferroptosis of primary spinal cord neurons by activating the Akt/Nrf2/HO‐1 signaling pathway. Thus, this work provides novel insights into the mechanisms of action of LXA4 in ferroptosis of primary spinal cord neurons and indicates that LXA4 may be a potential therapeutic agent for SCI.

Lipoxin A4 activates ALX/FPR2 to attenuate inflammation in Aspergillus fumigatus keratitis

PurposeTo explore the anti-inflammatory effect of lipoxin A4 (LXA4) in Aspergillus fumigatus (A. fumigatus) keratitis and the underlying mechanisms. MethodsIn A. fumigatus keratitis mouse models, enzyme-linked immunosorbent assay (ELISA) was used to detect the level of LXA4. Clinical scores were utilized to evaluate fungal keratitis (FK) severity. Fungal load was assessed by plate count. Immunofluorescence staining, HE staining and myeloperoxidase (MPO) assays were carried out to evaluate the neutrophil infiltration and activity. In A. fumigatus infected mouse corneas and inactivated A. fumigatus-stimulated RAW264.7 cells, quantitative real time polymerase chain reaction (qRT-PCR) and ELISA were applied to assess the expression of pro-inflammatory mediators and anti-inflammatory factors.Reactive oxygen species (ROS) was determined by 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) staining in RAW264.7 cells. ResultsLXA4 level was significantly increased in mice with A. fumigatus keratitis. In an A. fumigatus keratitis mouse model, LXA4 treatment alleviated FK severity, reduced fungal load and repressed neutrophil infiltration and activity. Additionally, LXA4 inhibited the expression of pro-inflammatory mediators including IL-1β, TNF-α, IL-6, cyclooxygenase-2 (COX-2), TLR-2, TLR-4, Dectin-1 and iNOS, and promoted the expression of anti-inflammatory factors IL-10 and Arg-1. In RAW264.7 cells, LXA4 receptor/formyl peptide receptor 2 (ALX/FPR2) blockade reversed the anti-inflammatory effect of LXA4. LXA4 suppressed inactivated A. fumigatus induced elevated ROS production in RAW264.7 cells, which was abrogated by ALX/FPR2 antagonist Boc-2. ConclusionLXA4 ameliorated inflammatory response by suppressing neutrophil infiltration, downregulating the expression of pro-inflammatory mediators and ROS production through ALX/FPR2 receptor in A. fumigatus keratitis.

Lipoxin A4 protects against paraquat‑induced acute lung injury by inhibiting the TLR4/MyD88‑mediated activation of the NF‑κB and PI3K/AKT pathways.

Paraquat (PQ) causes serious oxidative stress and inflammatory responses, particularly to the lungs. Since lipoxin A4 (LXA4) functions as an anti-inflammatory mediator, the present study aimed to explore its effects on PQ-induced acute lung injury (ALI) and to elucidate the possible underlying mechanisms. PQ was administered to male SD rats and RAW264.7 cells to establish a model of poisoning, and LXA4 was used as an intervention drug. LXA4 treatment attenuated PQ-induced lung injury, and this was accompanied by decreased tumor necrosis factor (TNF)-α and interleukin (IL)-1β secretion levels, and reduced oxidative stress damage. Additionally, LXA4 treatment inhibited the activation of the inflammation-related signaling molecules, Toll-like receptor 4 (TLR4), myeloid differentiation primary response 88 (MyD88), nuclear factor (NF)-κB p65, p-phosphoinositide 3-kinase (PI3K) and p-AKT. Furthermore, the in vitro experiments further confirmed that the beneficial effects of LXA4 on PQ-induced damage were TLR4-dependent. Hence, the present study demonstrated that LXA4 attenuated PQ-induced toxicity in lung tissue and RAW264.7 macrophages, and that this protective effect may be closely related to the mitigation of inflammatory responses, oxidative stress damage and the TLR4/MyD88-mediated activation of the PI3K/AKT/NF-κB pathway.

Lipoxin A4 alleviates lung injury in sepsis rats through p38/MAPK signaling pathway.

The aim of the study was to explore the effect of lipoxin A4 (LXA4) on lung injury in sepsis rats through the p38/mitogen-activated protein kinase (MAPK) signaling pathway. Sprague-Dawley rats were used for the study. The rat model of sepsis-induced acute lung injury was established via cecal ligation (Sepsis group, n=20). LXA4 (0.1 mg/kg) was injected at 6 h after modeling (Treatment group, n=20), and a The Control group (n=20) was also set up. The 7-day survival rate was 100% in The Control group, and LXA4 raised the survival rate of rats in the Sepsis group from 40% to 60% (P<0.01). Alveolar fluid clearance (AFC) significantly declined and the wet/dry weight (W/D) ratio of lung tissues rose remarkably in the Sepsis group compared with those in the Control group, while LXA4 restored AFC and reduced the W/D ratio of lung tissues (P<0.05), suggesting that LXA4 treatment reduces lung fluids and partially enhances AFC, thus lowering the W/D ratio of lung. The total cell count, polymorphonuclear neutrophils (PMN) percentage and concentration of tumor necrosis factor-α (TNF-α) and interleukin (IL)-6 in bronchoalveolar lavage fluid (BALF) were obviously increased in the Sepsis group compared with those in the Control group, while they were markedly decreased in the Treatment group (P<0.05). The activity of myeloperoxidase (MPO) in lung tissue homogenate was evidently higher in the Sepsis group than that in The Control group, while it was notably lower in the Treatment group than that in the Sepsis group after LXA4 treatment (P<0.05). Moreover, it was observed microscopically that the morphology of lung tissues was intact in the Control group. Finally, the results of Western blotting manifested that the p-p38/ MAPK protein expression was remarkably increased in the Sepsis group, indicating the activation of the p38/MAPK pathway, while it was remarkably decreased in the Treatment group, indicating the inhibited activity of the pathway (P<0.05). LXA4 has an anti-inflammatory effect on sepsis rats with lung injury, and such effect is related to the p38/MAPK signaling pathway.

A novel role for lipoxin A4 in driving a lymph node-eye axis that controls autoimmunity to the neuroretina.

The eicosanoid lipoxin A4 (LXA4) has emerging roles in lymphocyte-driven diseases. We identified reduced LXA4 levels in posterior segment uveitis patients and investigated the role of LXA4 in the pathogenesis of experimental autoimmune uveitis (EAU). Immunization for EAU with a retinal self-antigen caused selective downregulation of LXA4 in lymph nodes draining the site of immunization, while at the same time amplifying LXA4 in the inflamed target tissue. T cell effector function, migration and glycolytic responses were amplified in LXA4-deficient mice, which correlated with more severe pathology, whereas LXA4 treatment attenuated disease. In vivo deletion or supplementation of LXA4 identified modulation of CC-chemokine receptor 7 (CCR7) and sphingosine 1- phosphate receptor-1 (S1PR1) expression and glucose metabolism in CD4+ T cells as potential mechanisms for LXA4 regulation of T cell effector function and trafficking. Our results demonstrate the intrinsic lymph node LXA4 pathway as a significant checkpoint in the development and severity of adaptive immunity.

The prospective curative role of lipoxin A4 in induced gastric ulcer in rats: Possible involvement of mitochondrial dynamics signaling pathway.

This study purposed to examine the prospective curative role of lipoxin A4 (LXA4 ) in induced gastric ulcer in rats and explore the possible involvement of mitochondrial dynamics signaling pathway. Forty-eight male Wistar rats were divided into four groups: control, indomethacin (IND), IND + omeprazole (IND + Omez), and IND+ LXA4 groups. At the end of the experiment, the gastric pH, gastric fluid volume, total gastric acidity, ulcer index, and curative index were estimated. The gene expression of mitochondrial related protein 1 and mitofusin 2 were determined. In addition, some mitochondrial parameters include mitochondrial transmembrane potential, complex-I activity and reactive oxygen species were measured. Also, some gastric biochemical parameters, histopathological, and immunohistochemical analyses of the gastric mucosa were determined. We found that IND induced gastric ulcer, as manifested by the biochemical, histopathological, and immunohistochemical analyses. Both Omez and LXA4 treatment for 15 days alleviated the IND-induced gastric ulcer as explored by ameliorating the biochemical, histopathological, and immunohistochemical findings. We concluded that LXA4 mitigated the IND-induced gastric ulcer via improving the mitochondrial dynamic imbalance and mitochondrial dysfunction, in addition to its anti-apoptotic, anti-inflammatory, and antioxidant properties.

Lipoxin A4 ameliorates alveolar fluid clearance disturbance in lipopolysaccharide-induced lung injury via aquaporin 5 and MAPK signaling pathway.

A characteristic of acute lung injury (ALI) is the inflammatory damage of alveolar fluid transport. Lipoxins are endogenous lipids involving in the resolution of inflammation. It is found that lipoxin A4 (LXA4) has the distinct properties to improve the anti-edema and pro-resolution function in inflammation. Since aquaporins (AQPs) have essential roles in the integrity of barrier function during fluid transport, especially AQP5 in the maintaining of the epithelium permeability, the current study is aimed to evaluate the potential role of LXA4 in regulating alveolar fluid clearance (AFC) during fluid transport and the corresponding change of AQP5 in the lung. ALI was induced by the lipopolysaccharide (LPS) intraperitoneal injection, and LXA4 treatment was given 8 hours after LPS administration. We investigated changes in the capacity of AFC, pro-inflammatory cytokine concentrations in bronchoalveolar lavage fluid (BALF) and the severity of ALI. Then AQP5 expression in lung tissue and potential regulatory pathways in LPS-induced ALI was explored. LXA4 treatment was found to inhibit AFC capacity, inflammatory cytokine release, partially, alleviate ALI severity, and restored AQP5 expression partially. Additionally, we found that LXA4 played a protective role by the inhibition of the phosphorylation of p38 and JNK. In summary, our results suggest that LXA4 plays a protective role in lipopolysaccharide-induced ALI by restoring AFC capacity and upregulating AQP5 expression and inhibiting the phosphorylation of p38 and JNK. These findings suggest potential new mechanism of LXA4 as anti-inflammation therapy for the impairment of alveolar fluid transport in ALI.

Colorectal Cancer Is Associated with a Deficiency of Lipoxin A4, an Endogenous Anti-inflammatory Mediator.

Unresolved inflammation, due to insufficient production of proresolving anti-inflammatory lipid mediators, can lead to tumorigenesis. Among these mediators, lipoxin A4 (LXA4) has potent anti-carcinogenic properties, and may serve as key target for modulating inflammation-associated cancer like colorectal cancer. The purpose of present study was to clarify the roles of LXA4 in colorectal cancer. We investigated the effects and underlying mechanisms of LXA4 in colorectal cancer and its relationship with tumor-associated inflammation and immune microenvironment by employing clinical samples and mouse colorectal cancer cell line CT26-bearing tumor model as well as colorectal cancer cells. It was found that colorectal cancer is associated with dysregulation of immune microenvironment and deficiency of LXA4 that could play different roles at different stages of tumor growth: inhibiting early but promoting late tumor growth. Analysis of peripheral immune cells in subcutaneous xenograft mice model disclosed that early LXA4 treatment induced lymphocytes and inhibited neutrophils and monocytes, while late LXA4 treatment induced neutrophils but inhibited lymphocytes. Detailed analysis of tumor microenvironment revealed that early LXA4 treatment could inhibit inflammatory mediators expressions and leukocytes infiltration into tumor. Furthermore, LXA4 could suppress the expressions of p-ERK, p-P38 and NF-κB in subcutaneous xenograft. Additionally, LXA4 could inhibit the proliferation and migration of colorectal cancer cells, and, meanwhile, inhibit the proliferation and migration of colorectal cancer cells stimulated by activated macrophage-conditioned media. These findings suggest that colorectal cancer is associated with a deficiency of LXA4 that could suppress colorectal cancer via modulating tumor-associated inflammation and immune microenvironment as well as inhibiting colorectal cancer cell development.

Lipoxin A4 inhibits the invasion and migration of endometrial stromal cells by down-regulating NF-κB signaling-mediated autophagy

Objective: To investigate whether the suppressive effects of lipoxin A4 (LXA4) on endometriosis are mediated by the regulation of autophagic activity, and to further explore the actual molecular mechanism. Methods: (1) Eutopic and ectopic endometria were obtained from 13 patients with endometriosis, and 10 eutopic endometria collected from non-endometriosis patients were used as control. The expression of the autophagy-related biochemical markers [microtubule-associated protein 1 light chain 3 (LC3) and p62] were detected by western blot. Levels of LXA4 in the biopsies were measured by ELISA. (2) Primary human endometrial stromal cells (ESC) were isolated and cultured in vitro from eutopic endometria of infertility patients with endometriosis. After treatment with exogenous LXA4 or autophagy inhibitor 3-methyladenine (3-MA) or autophagy inducer rapamycin, cell migration and invasion were evaluated by transwell assay, and autophagy was detected by western blot. (3) ESC were treated with LXA4, the gene expressions of nuclear factor kappa B (NF-κB) etc. were examined by quantitative real-time PCR, and the activation of NF-κB signaling was detected by western blot. (4) ESC were incubated with 10 μmol/L NF-κB inhibitor BAY11-7080, the autophagic activation was detected by western blot. Results: (1) Autophagy-related marker, LC3-Ⅱ and LC3-Ⅱ/LC3-Ⅰ ratio, showed a significant up-regulation in ectopic lesions of endometriosis compared with eutopic endometria of affected or healthy women (all P<0.05) . However, the LXA4 level significantly decreased in ectopic tissue (P<0.05) . There was a significant negative correlation between LXA4 concentration and relative expression of LC3-Ⅱ in ectopic lesions (r= -0.780, P=0.002) . (2) 10 and 100 nmol/L exogenous LXA4 could significantly down-regulate the LC3-Ⅱ protein expression and up-regulate the p62 protein expression (all P<0.05) . LXA4 markedly inhibited the invasion and migration of ESC (P<0.05) ;while the reactivation of autophagy by rapamycin almost reversed the anti-invasion and anti-migration effects of LXA4. (3) After LXA4 treatment, the expression level of NF-κB gene significantly decreased (P<0.05) . Furthermore, the results of western blot analysis showed that the nuclear translocation of NF-κB p65 was markedly down-regulated under LXA4 treatment (P<0.05) . (4) The NF-κB inhibitor BAY11-7080 markedly suppressed the autophagic activation of LXA4 (P<0.05) . Conclusion: LXA4 could inhibit the invasion and migration of ESC by down-regulating the NF-κB signaling-mediated autophagy.

Lipoxin A4 may attenuate the progression of obesity-related glomerulopathy by inhibiting NF-κB and ERK/p38 MAPK-dependent inflammation

ObjectivesTo investigate the effects of lipoxin A4 (LXA4) on inflammatory responses in obesity-related glomerulopathy (ORG) mouse model and its potential mechanisms. MethodsMale C57BL/6 mice were randomly divided into 4 groups: normal, model, LXA4, and LXA4/Boc-2 groups (n = 8). Mice in LXA4 group were intraperitoneally injected with LXA4 (40 ng/kg) once daily for 3 days following 12 weeks of high-fat diet (HFD) feeding. LXA4 receptor antagonist, Boc-2, was administered in LXA4/Boc-2 group prior to LXA4 treatment to block the effects of LXA4. Renal morphology and function impairment were determined. Inflammation was tested by measuring serum and mRNA levels of pro-inflammatory cytokines and chemokines. HFD-induced activation of nuclear factor-kappa B (NF-κB) and phosphorylation of mitogen-activated protein kinases (MAPKs) were investigated by immunohistochemistry and western blot. ResultsHFD-feeding caused significant renal injury, pathological changes and inflammation in model group mice. LXA4 injection significantly alleviated HFD-induced effects on renal morphology and functions, as demonstrated by lower kidney index, glomerular diameter, 24 h urine protein, urinary albumin creatinine ratio and renal histomorphology. Moreover, HFD-induced accumulation of pro-inflammatory cytokines and chemokines were obviously attenuated by LXA4 administration, so did the HFD-induced activation of NF-κB and ERK/p38 MAPK pathways. However, these effects were markedly abrogated by BOC-2 pretreatment. ConclusionLXA4 significantly attenuated HFD-induced renal inflammation and injury in ORG models, and these effects may be associated with the inhibition of activation of NF-κB and ERK/p38 MAPK pathways. The findings of our study may shed light on LXA4 showed a potential therapeutic application in ORG.

Reprogramming tumor stroma using an endogenous lipid lipoxin A4 to treat pancreatic cancer

Pancreatic stellate cells (PSCs) are the precursors of cancer-associated fibroblasts (CAFs), which potentiate pancreatic tumor growth and progression. In this study, we investigated whether Lipoxin A4 (LXA4), an endogenous bioactive lipid, can inhibit the differentiation of human PSCs (hPSCs) into CAF-like myofibroblasts and thereby hPSC-induced pro-tumorigenic effects. LXA4 significantly inhibited TGF-β-mediated differentiation of hPSCs by inhibiting pSmad2/3 signalling. Furthermore, treatment with LXA4 abolished the paracrine effects (proliferation and migration of Panc-1 tumor cells) of hPSCs in vitro. These data demonstrated that LXA4 can interrupt pro-tumoral paracrine signalling of hPSCs. Furthermore, LXA4 treatment significant decreased the size and growth rate of 3D-heterospheroids comprised of hPSC and Panc-1 and these effects were exhibited due to inhibition of hPSC-induced collagen1 expression. In vivo, we examined the therapeutic efficacy of LXA4 in a co-injection (Panc-1 and hPSCs) subcutaneous tumor model. Intriguingly, LXA4 significantly abolished the tumor growth (either injected intratumor or intraperitoneally), attributed to a significant reduction in fibrosis, shown with collagen1 expression. Altogether, this study proposes LXA4 as a potent inhibitor for hPSCs which can be applied to reprogram tumor stroma in order to treat pancreatic cancer.

Lipoxin A4 protects against spinal cord injury via regulating Akt/nuclear factor (erythroid-derived 2)-like 2/heme oxygenase-1 signaling

Spinal cord injury (SCI) is a devastating physical trauma worldwide. The mechanisms of SCI are still not clear and the effective treatment is limited. Lipoxin A4 (LXA4) possesses anti-inflammatory and neuroprotective effects. The present study was designed to further evaluate the molecular mechanisms of LXA4-induced protective effects in a rat model of SCI. We found that LXA4 increased Basso, Beattie and Bresnahan (BBB) scores, increased mechanical paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) to a radiant heat, reduced the lesion volume, decreased Bax mRNA expression and increased Bcl-2 expression after SCI. The phosphorylation of Akt and protein expression of Nrf2 and HO-1 were reduced after SCI. LXA4 treatment significantly inhibited the reduction of Akt phosphorylation and Nrf2 and HO-1 protein expression. Injection of LY294002 notably inhibited the phosphorylation of Akt, and the expression of total Akt and Nrf2 and HO-1 after SCI in LXA4-treated rats. LY294002 prohibited LXA4-induced effects after SCI. shNrf2 injection markedly decreased both Nrf2 and HO-1 expression in LXA4-treated rats after SCI. ZnPP notably decreased HO-1 expression but did not markedly affect Nrf2 expression. shNrf2 and ZnPP prohibited LXA4-induced increase of BBB scores, and PWT and PWL, decrease of lesion volume of spinal cord, reduction of Bax expression and increase of Bcl-2 expression. The results indicate that LXA4 protects against SCI through Akt/Nrf2/HO-1 signaling. The data provide novel insights into the mechanisms of LXA4-mediated neuprotective effects against SCI and suggest that LXA4 may be a potential therapeutic agent for SCI and its associated complications.

Signal transduction involved in lipoxin A4‑induced protection of tubular epithelial cells against hypoxia/reoxygenation injury.

Previous studies have reported that lipoxin A4 (LXA4) may exert a renoprotective effect on ischemia/reperfusion injury in various animal models. The underlying mechanism of LXA4-induced renoprotection during ischemia/reperfusion injury remains to be elucidated. The present study investigated LXA4-induced protection on renal tubular cells subjected to hypoxia/reoxygenation (H/R) injury, and determined the effects of peroxisome proliferator-activated receptor-γ (PPARγ) and heme oxygenase-1 (HO-1) on LXA4 treatment. HK-2 human tubular epithelial cells exposed to H/R injury were pretreated with LXA4, signal molecule inhibitors or the HO-1 inhibitor zinc protoporphyrin-IX, or were transfected with PPARγ small interfering RNA (siRNA) or nuclear factor E2-related factor 2 (Nrf2) siRNA. The protein and mRNA expression levels of PPARγ and HO-1 were analyzed using western blotting and reverse transcription-quantitative polymerase chain reaction. Binding activity of Nrf2 to the HO-1 E1 enhancer was determined using chromatin immunoprecipitation. Nrf2 binding to the HO-1 antioxidant responsive element (ARE) was assessed using electrophoretic mobility shift assay. Preincubation of cells with LXA4 exposed to H/R injury led to a decreased production of inducible nitrogen oxide synthase, malondialdehyde, γ-glutamyl transpeptidase, leucine aminopeptidase and N-acetyl-β-glucosaminidase. In addition, LXA4 pretreatment increased cell viability, protein and mRNA expression levels of PPARγ and HO-1 and PPARγ and HO-1 promoter activity. SB20358 is a p38 mitogen-activated protein kinase (p38 MAPK) pathway inhibitor, which reduced LXA4-induced PPARγ expression levels. LXA4 treatment upregulated p38 MAPK activation, Nrf2 nuclear translocation and increased binding activity of Nrf2 to HO-1 ARE and E1 enhancer in cells exposed to H/R injury. Transfection of the cells with PPARγ siRNA reduced the LXA4-induced Nrf2 translocation. Transfection of the cells with PPARγ siRNA or Nrf2 siRNA also reduced the LXA4-induced increase in HO-1 expression. In conclusion, LXA4-induced protection of renal tubular cells against H/R injury was associated with the induction of PPARγ and HO-1, via activation of the p38 MAPK pathway, as well as Nrf2 nuclear translocation and binding to HO-1 ARE and E1 enhancer. Therefore, LXA4-induced renoprotection is associated with activation of the p38 MAPK/PPARγ/Nrf2-ARE/HO-1 pathway.