OBJECTIVE: To study the effect of early intervention with lipoxin A4 (LXA4) on septic mice. METHODS: Healthy male Balb/c mice aged 6-8 weeks were randomly divided into sham-operation group, sepsis group, 1-hour intervention group (intervention at 1 hour after sepsis), and 6-hour intervention group (intervention at 6 hours after sepsis) (n=8 each). A sepsis model was prepared by cecal ligation and puncture. The intervention groups received LXA4 at 0.01 μg/g body weight 1 or 6 hours after the model was established. Blood was taken from eyeballs at 24 hours after operation. Peritoneal lavage fluid and liver and lung tissue samples were collected. The bacterial colonies of whole blood and peritoneal lavage fluid were counted by dilution plating. The serum levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and monocyte chemoattractant protein-1 (MCP-1) were determined by cytometric bead array. The serum level of high mobility group box-1 (HGMB1) was determined using ELISA. The percentages of macrophages and neutrophils in peritoneal lavage fluid were determined by flow cytometry. Paraffin sectioning and hematoxylin-eosin staining were performed for the liver and lung tissue samples to observe pathological damage. RESULTS: Compared with the sham-operation group, the sepsis group had a significantly decreased percentage of macrophages and a significantly increased percentage of neutrophils in peritoneal lavage fluid (P<0.05), as well as significantly increased serum levels of IL-6, TNF-α, MCP-1, and HMGB1 (P<0.05); in addition, the sepsis group showed more vacuolar degeneration, hepatocyte swelling, and inflammatory cell infiltration in liver tissue, and more capillary congestion, pulmonary septal thickening, inflammatory cell infiltration, and partial tissue destruction in lung tissue. Compared with the sepsis group, the 1-hour and 6-hour intervention groups had a significantly increased percentage of macrophages in peritoneal lavage fluid (P<0.05) and significantly reduced bacterial load in whole blood (P<0.05), serum levels of IL-6, TNF-α, MCP-1, and HMGB1 (P<0.05), and degree of liver and lung tissue damage and inflammatory cell infiltration, but there was no significant difference in the percentage of neutrophils and bacterial load in peritoneal lavage fluid (P>0.05). Compared with the 6-hour intervention group, the 1-hour intervention group had a significantly decreased serum level of HMGB1 (P<0.05), but there was no significant difference in other indicators between the two groups (P>0.05). CONCLUSIONS Early intervention with LXA4 may attenuate liver and lung injuries in septic mice, which may be explained by the decrease in serum levels of IL-6, TNF-α, MCP-1, and HMGB1, and it also may reduce the bacterial dissemination in the whole blood of septic mice, which may be explained by the increase in the percentage of peritoneal macrophages.
Animals ; Early Intervention (Education) ; Interleukin-6 ; Lipoxins ; Male ; Mice ; Sepsis ; Tumor Necrosis Factor-alpha

Nonsteroidal anti-inflammatory drug (NSAID)-exacerbated respiratory disease (NERD) has attracted a great deal of attention because of its association with severe asthma. However, it remains widely underdiagnosed in asthmatics as well as the general population. Upon pharmacological inhibition of cyclooxygenase 1 by NSAIDs, production of anti-inflammatory prostaglandin E2 and lipoxins ceases, while release of proinflammatory cysteinyl leukotrienes increases. To determine the underlying mechanisms, many studies have attempted to elucidate the genetic variants, such as single nucleotide polymorphisms, responsible for alterations of prostaglandins and leukotrienes, but the results of these genetic studies could not explain the whole genetic pathogenesis of NERD. Accordingly, the field of epigenetics has been introduced as an additional contributor to genomic alteration underlying the development of NERD. Recently, changes in CpG methylation, as one of the epigenetic components, have been identified in target tissues of NERD. This review discusses in silico analyses of both genetic and epigenetic components to gain a better understanding of their complementary roles in the development of NERD. Although the molecular mechanisms underlying NERD pathogenesis remain poorly understood, genetic and epigenetic variations play significant roles. Our results enhance the understanding of the genetic and epigenetic mechanisms involved in the development of NERD and suggest new approaches toward better diagnosis and management.
Anti-Inflammatory Agents, Non-Steroidal ; Asthma ; Computer Simulation ; Cyclooxygenase 1 ; Diagnosis ; Dinoprostone ; Epigenomics ; Genetics ; Leukotrienes ; Lipoxins ; Methylation ; Polymorphism, Single Nucleotide ; Prostaglandins

PURPOSE: An unresolved inflammatory state contributes to the pathogenesis of periodontal disease and metabolic syndrome (MetS). Therefore, the purpose of this study was to evaluate the role of lipoxin A4 (LXA4), a proresolving lipid mediator, in the association between periodontal disease and MetS. METHODS: Sixty-seven patients with MetS and 65 patients without MetS were included in the study. Sociodemographic information was obtained via a questionnaire, and detailed medical diagnoses were made. Periodontal parameters (plaque index [PI], gingival index [GI], probing pocket depth [PD], and clinical attachment level [CAL]) and metabolic parameters were measured, and serum LXA4 levels were determined. The associations among MetS, periodontal parameters, and serum LX levels were evaluated by adjusted multivariate linear regression analyses. RESULTS: Patients with MetS were older and had a higher body mass index than patients without MetS. Periodontal parameters (PI, GI, PD, and CAL) were higher in patients with MetS than in those without MetS. Serum LXA4 levels were higher in patients without MetS. Multivariate linear regression analysis indicated a positive association between MetS and periodontal parameters (PD and CAL). Negative associations were established between MetS and LXA4 levels, and between LXA4 and periodontal parameters (PI, PD, and CAL). CONCLUSIONS: The presence of higher values of periodontal parameters in patients with MetS and the negative relationship of LXA4 with MetS and periodontal disease may support the protective role of proresolving lipid mediators in the association between periodontal disease and MetS.
Body Mass Index ; Diagnosis ; Humans ; Inflammation ; Linear Models ; Lipoxins ; Metabolic Syndrome X ; Periodontal Diseases ; Periodontal Index

OBJECTIVETo study the protective effect of lipoxin A4 (LXA4) against sepsis induced by lipopolysaccharide (LPS) in rats with obesity and its effect on the expression of Toll-like receptor 4 (TLR4) and TNF receptor-associated factor 6 (TRAF6) in the liver.

METHODSA total of 60 male Sprague-Dawley rats aged three weeks were randomly divided into a normal group and an obesity group, with 30 rats in each group. A rat model of obesity was established by high-fat diet. Each of the two groups was further randomly divided into control group, sepsis group, and LXA4 group, and 8 rats were selected from each group. The rats in the control, sepsis, and LXA4 groups were treated with intraperitoneal injection of normal saline, LPS, and LXA4+LPS respectively. Twelve hours later, blood samples were collected from the heart and liver tissue samples were also collected. ELISA was used to measure the serum levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). Western blot was used to measure the protein expression of TLR4 and TRAF6 in liver tissue. Quantitative real-time PCR was used to measure the mRNA expression of TLR4 and TRAF6.

RESULTSAfter being fed with high-fat diet for 6 weeks, the obesity group had significantly higher average weight and Lee's index than the normal group (P<0.05). Compared with the normal group, the obesity group had significant increases in the serum levels of IL-6 and TNF-α (P<0.05). In the normal group or the obesity group, the sepsis subgroup had significant increases in the serum levels of IL-6 and TNF-α compared with the control subgroup (P<0.05), while the LXA4 subgroup had significant reductions in the two indices compared with the sepsis subgroup (P<0.05). Compared with the normal group, the obesity group had significant increases in the protein and mRNA expression of TLR4 and TRAF6 (P<0.05). In the normal group or the obesity group, the sepsis subgroup had significant increases in the protein and mRNA expression of TLR4 and TRAF6 compared with the control subgroup (P<0.05). Compared with the sepsis subgroup, the LXA4 subgroup had significant reductions in the protein and mRNA expression of TLR4 and TRAF6 (P<0.05).

CONCLUSIONSLXA4 can reduce the serum levels of IL-6 and TNF-α and alleviate inflammatory response. LXA4 can inhibit the expression of TLR4 and TRAF6 in the liver of septic rats, possibly by inhibiting the TLR4 signaling pathway.

Animals ; Humans ; Interleukin-6 ; genetics ; metabolism ; Lipoxins ; administration & dosage ; Liver ; drug effects ; metabolism ; Male ; Obesity ; complications ; drug therapy ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Sepsis ; complications ; drug therapy ; genetics ; metabolism ; Signal Transduction ; drug effects ; TNF Receptor-Associated Factor 6 ; genetics ; metabolism ; Toll-Like Receptor 4 ; genetics ; metabolism ; Tumor Necrosis Factor-alpha ; genetics ; metabolism

BackgroundLipoxin A4 (LXA4) can alleviate lipopolysaccharide (LPS)-induced acute lung injury (ALI) and acute respiratory distress syndrome through promoting epithelial sodium channel (ENaC) expression in lung epithelial cells. However, how LXA4 promote ENaC expression is still largely elusive. The present study aimed to explore genes and signaling pathway involved in regulating ENaC expression induced by LXA4.

MethodsA549 cells were incubated with LPS and LXA4, or in combination, and analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) of ENaC-α/γ. Candidate genes affected by LXA4 were explored by transcriptome sequencing of A549 cells. The critical candidate gene was validated by qRT-PCR and Western blot analysis of A549 cells treated with LPS and LXA4 at different concentrations and time intervals. LXA4 receptor (ALX) inhibitor BOC-2 was used to test induction of candidate gene by LXA4. Candidate gene siRNA was adopted to analyze its influence on A549 viability and ENaC-α expression. Phosphoinositide 3-kinase (PI3K) inhibitor LY294002 was utilized to probe whether the PI3K signaling pathway was involved in LXA4 induction of candidate gene expression.

ResultsThe A549 cell models of ALI were constructed and subjected to transcriptome sequencing. Among candidate genes, N-myc downstream-regulated gene-1 (NDRG1) was validated by real-time-PCR and Western blot. NDRG1 mRNA was elevated in a dose-dependent manner of LXA4, whereas BOC-2 antagonized NDRG1 expression induced by LXA4. NDRG1 siRNA suppressed viability of LPS-treated A549 cells (treatment vs. control, 0.605 ± 0.063 vs. 0.878 ± 0.083, P = 0.040) and ENaC-α expression (treatment vs. control, 0.458 ± 0.038 vs. 0.711 ± 0.035, P = 0.008). LY294002 inhibited NDRG1 (treatment vs. control, 0.459 ± 0.023 vs. 0.726 ± 0.020, P = 0.001) and ENaC-α (treatment vs. control, 0.236 ± 0.021 vs. 0.814 ± 0.025, P < 0.001) expressions and serum- and glucocorticoid-inducible kinase 1 phosphorylation (treatment vs. control, 0.442 ± 0.024 vs. 1.046 ± 0.082, P = 0.002), indicating the PI3K signaling pathway was involved in regulating NDRG1 expression induced by LXA4.

ConclusionOur research uncovered a critical role of NDRG1 in LXA4 alleviation of LPS-induced A549 cell injury through mediating PI3K signaling to restore ENaC expression.

A549 Cells ; Acute Lung Injury ; metabolism ; Cell Cycle Proteins ; metabolism ; Cell Line ; Epithelial Sodium Channels ; metabolism ; Humans ; Intracellular Signaling Peptides and Proteins ; metabolism ; Lipopolysaccharides ; pharmacology ; Lipoxins ; pharmacology ; Signal Transduction ; drug effects

Previous studies have shown that oxidative stress and corporal fibrosis in penile tissues of rats were key pathological factors of erectile dysfunction induced by diabetic mellitus (DMED). Lipoxin A4 (LXA4) was reported to inhibit oxidative stress and fibrosis diseases, while whether it could exert a protective role on erectile function was not clear. Type I diabetic mellitus (DM) was induced in thirty male 10-week-old Sprague-Dawley rats using streptozotocin. Ten weeks later, twenty-two rats with DMED confirmed by an apomorphine test were divided into two groups: the DMED group (n = 11) and the DMED + LXA4 group (n = 11; LXA4 injection daily for 4 weeks). In addition, another ten age-matched rats formed the Control group. We found that erectile function was significantly impaired in the DMED group compared with the Control group, but was improved in the DMED + LXA4 group. Similarly, the over-activated oxidative stress and impaired endothelial function in the DMED group were both improved in the DMED + LXA4 group. Moreover, the DMED group showed serious corporal fibrosis, which was also inhibited by the treatment of LXA4 in the DMED + LXA4 group. Taken together, LXA4 could exert an inhibition role on oxidative stress and fibrosis to improve DMED effectively.
Actins/metabolism* ; Animals ; Anti-Inflammatory Agents, Non-Steroidal/pharmacology* ; Diabetes Mellitus, Experimental/physiopathology* ; Diabetes Mellitus, Type 1/physiopathology* ; Erectile Dysfunction/physiopathology* ; Fibrosis ; Lipoxins/pharmacology* ; Male ; Nitric Oxide/metabolism* ; Nitric Oxide Synthase Type III/metabolism* ; Oxidative Stress/drug effects* ; Penile Erection/drug effects* ; Penis/pathology* ; Rats ; Rats, Sprague-Dawley

Liopxin A4 (LXA4) is considered to be a crucial modulator in the inflammatory responses. In the present study, we aimed to study the effect of LXA4 on the inflammatory cytokines production induced by lipopolysaccharide (LPS) and the possible mechanism in normal human epidermal keratinocytes (NHEKs). NHEKs were isolated and cultured. The expression of toll-like receptor 4 (TLR4), LXA4 receptor (ALXR) and aryl hydrocarbon receptor (AhR) in NHEKs was detected by reverse transcription polymerase chain reaction (RT-PCR). The mRNA and protein levels of tumor necrosis factor-alpha (TNF-α) and interleukin-1β (IL-1β) were determined in NHEKs stimulated by LPS (10 μg/mL) with or without preincubation with LXA4 (100 nmol/L) for 30 min by real-time quantitative PCR (real-time qPCR) and enzyme-linked immunosorbent assay (ELISA), respectively. The expression levels of tumor necrosis factor receptor-associated factor 6 (TRAF6) and suppressors of cytokine signaling 2 (SOCS2) mRNAs and proteins, and nuclear translocation of NF-kB-p65 were measured by real-time qPCR and Western blotting, respectively. The results showed that NHEKs expressed TLR4, ALXR and AhR. LXA4 significantly inhibited the mRNA and protein expression levels of TNF-α, IL-1β and TRAF6 induced by LPS in NHEKs, and LXA4 obviously increased the expression of SOCS2 at mRNA and protein levels. The nuclear NF-kB-p65 protein expression induced by LPS was inhibited after preincubation with LXA4 in NHEKs. It was concluded that LXA4 inhibits the LPS-induced production of TNF-α and IL-1β in NHEKs by up-regulating SOCS2 and down-regulating TRAF6.
Anti-Inflammatory Agents, Non-Steroidal ; pharmacology ; Cells, Cultured ; Gene Expression Regulation ; drug effects ; Humans ; Keratinocytes ; Lipopolysaccharides ; pharmacology ; Lipoxins ; pharmacology ; NF-kappa B ; genetics ; metabolism ; Suppressor of Cytokine Signaling Proteins ; genetics ; metabolism ; TNF Receptor-Associated Factor 6 ; genetics ; metabolism ; Toll-Like Receptor 4 ; genetics ; metabolism ; Tumor Necrosis Factor-alpha ; genetics ; metabolism

OBJECTIVE: To investigate the change of serum myeloperoxidase (MPO) and lipoxin A4 (LXA4) in coronary heart disease (CHD) patients with anxiety and depression and its clinical significance. METHODS: From December 2010 to February 2011, 143 CHD patients and 44 non-CHD patients (the control group) hospitalized in the Department of Cardiology at the Second Xiangya Hospital were enrolled. The hospital anxiety and depression scale (HADS) was used to evaluate the psychological state of all patients and the CHD patients were assigned to an anxiety and depression group (n=57) or a non-depression and anxiety group (n=86). The serum levels of high sensitive C-reactive protein (Hs-CRP), MPO, and LXA4 were examined, and the ratio of MPO and LXA4 (M/L) was calculated. RESULTS: The levels of Hs-CRP, MPO, and LXA4 as well as M/L ratios in both CHD groups were significantly higher than those in the control group (all P<0.01). Compared with the non-anxiety and depression group, the levels of MPO and LXA4, and M/L ratios in the anxiety and depression group increased (all P<0.05). Correlation analysis showed that MPO was positively correlated with the score of HADS-total (HADS-t), HADS-anxiety (HADS-a), or HADS-depression (HADS-d), while LXA4 was negatively correlated with HADS-t or HADS-d. Multiple ordinal logistic regression analysis revealed that higher HADS-t score, stable angina, unstable angina, and acute myocardial infarction were the independent impact factors for the elevation of M/L ratio. CONCLUSION Anxiety and depression may aggravate the inflammatory response in CHD patients. The imbalance between inflammation and anti-inflammation may be part of the mechanism.
Aged ; Anxiety ; complications ; Case-Control Studies ; Coronary Disease ; blood ; complications ; Depression ; complications ; Female ; Humans ; Inflammation ; Lipoxins ; blood ; Male ; Middle Aged ; Peroxidase ; blood

This study examined in vitro effect of lipoxin A(4) (LXA(4)) on interleukin-1β (IL-1β) production of monocytes and its possible mechanism in severe preeclampsia (PE). Peripheral venous blood was drawn from 15 patients with severe preeclampsia (PE group) and 20 normal pregnant women (control group) to prepare monocytes which were then treated with LXA(4) at different concentrations of 0, 10, 100 nmol/L respectively. IL-1β level in the supernatant of monocytes was detected by enzyme linked immunoassay. The [Ca(2+)](i) of monocytes was measured by laser scanning confocal microscopy. The results showed that the IL-1β level and the [Ca(2+)](i) of monocytes in the PE group were significantly higher than those in the control group. LXA(4) significantly decreased the generation of IL-1β in a dose-dependent manner in the PE group. After treatment with 100-nmol/L LXA(4), in the PE group, the [Ca(2+)](i) concentration of monocytes was significantly reduced. It was concluded that LXA(4) may inhibit the IL-1β production of monocytes from severe preeclampsia women by inhibiting extracellular calcium influx.
Adult ; Anti-Inflammatory Agents, Non-Steroidal ; pharmacology ; Calcium ; metabolism ; Cells, Cultured ; Female ; Humans ; Interleukin-1beta ; biosynthesis ; Lipoxins ; pharmacology ; Monocytes ; cytology ; metabolism ; Pre-Eclampsia ; blood ; physiopathology ; Pregnancy

BACKGROUNDLipoxins (LXs), endogenous anti-inflammatory and pro-resolving eicosanoids generated during various inflammatory conditions, have novel immunomodulatory properties. Because dendritic cells (DCs) play crucial roles in the initiation and maintenance of immune response, we determined whether LXs could modulate the maturation process of DCs and investigated the effects of lipoxin A(4) (LXA(4)) on lipopolysaccharide (LPS)-induced differentiation of RAW264.7 cells into dendritic-like cells.

METHODSRAW264.7 cells were cultured in vitro with 1 microg/ml LPS in the absence or presence of LXA(4) for 24 hours, and cellular surface markers (MHC-II, CD80 (B7-1), CD86 (B7-2)) were measured by flow cytometry (FCM). Mixed lymphocyte reaction was performed to evaluate the allostimulatory activity. Cytoplastic IkappaB degradation and nuclear factor kappa B (NF-kappaB) translocation were detected by Western blotting. Luciferase reporter plasmid was transiently transfected into RAW264.7 cells, and luciferase activity was determined to measure the transcriptional activity of NF-kappaB.

RESULTSLXA(4) reduced the ratio of LPS-treated RAW264.7 cells to DCs with morphological characteristics and inhibited the expression of MHC II. LPS-induced up-regulation of CD86 was moderately suppressed by LXA(4) but no obvious change of CD80 was observed. Moreover, LXA(4) weakened the allostimulatory activity of LPS-treated RAW264.7 cells. These alterations of LPS+LXA(4)-treated cells were associated with a marked inhibition of IkappaB degradation, NF-kappaB translocation and then the transcriptional activity of NF-kappaB.

CONCLUSIONSLXA(4) negatively regulates LPS-induced differentiation of RAW264.7 cells into dendritic-like cells. This activity reveals an undescribed mechanism of LXA(4) to prevent excessive and sustained immune reaction by regulating maturation of DCs.

Animals ; Biological Transport ; drug effects ; Cell Differentiation ; drug effects ; Cells, Cultured ; Dendritic Cells ; cytology ; I-kappa B Kinase ; metabolism ; Lipopolysaccharides ; pharmacology ; Lipoxins ; pharmacology ; Macrophages ; cytology ; drug effects ; Mice ; NF-kappa B ; metabolism ; Phenotype ; Transcription, Genetic ; drug effects