|Year : 2015 | Volume
| Issue : 2 | Page : 55-61
Urinary lipoxin A4 as a biomarker for systemic lupus erythematosus
Manal M Sedky Abdou1, Dina A Effat1, Lamiaa A Mansour2, Noha M Abd El Baky MSc 1, Mona M Abdul Salam2
1 Department of Rheumatology & Rehabilitation, Faculty of Medicine, Cairo University, Giza, Egypt
2 Department of Clinical & Chemical Pathology, Faculty of Medicine, Cairo University, Giza, Egypt
|Date of Submission||05-Mar-2015|
|Date of Acceptance||07-Mar-2015|
|Date of Web Publication||29-May-2015|
Noha M Abd El Baky
Department of Rheumatology and Rehabilitation, Faculty of Medicine, Cairo University, Giza
Source of Support: None, Conflict of Interest: None
Systemic lupus erythematosus (SLE) is an autoimmune disorder that has multiorgan involvement. The continuation of inflammation in lupus could be attributed to failure of the resolution process because of deficiency of potent endogenous proresolution-inducing molecules such as lipoxin A4 (LXA4), leading to progression and flares of lupus and lupus nephritis.
The aim of this study was to assess the levels of urinary LXA4 in SLE patients and in healthy controls, and to correlate them with various clinical and laboratory data as well as renal biopsy and disease activity indices (DAIs).
Patients and methods
A total of 40 adult female SLE patients were included in this study. Forty healthy women age-matched with SLE patients served as the control group. All patients were subjected to a full assessment of history, clinical examination, assessment of DAIs (SLEDAI and renal SLEDAI), and laboratory investigations including the urinary LXA4/creatinine ratio assessed by enzyme linked immunosorbent assay.
Urinary LXA4/creatinine ratio levels were found to be significantly higher in all SLE patients compared with the healthy controls (P = 0.037). The median level of the urinary LXA4/creatinine ratio was lower in SLE patients with nephritis than in patients without nephritis (0.1 and 0.3 ng/ml, respectively), but with no statistical significance (P = 0.11). The urinary LXA4/creatinine ratio levels were found to be significantly lower in SLE patients with cardiovascular manifestations as well as those with neuropsychiatric manifestations (P = 0.009, 0.04 respectively).
This was a novel study that was carried out to assess the levels of urinary LXA4 in SLE patients. It showed that the urinary LXA4/creatinine ratio levels were significantly lower in SLE patients with cardiovascular and neuropsychiatric manifestations and nonsignificantly lower in patients with nephritis, suggesting that insufficiency of LXA4 in the human body may be responsible for major organ involvement in SLE patients. Accordingly, LXA4 is suggested to be an inflammatory biomarker not only for lupus nephritis but also for other systemic manifestations in SLE.
Keywords: activity scores, lupus nephritis, systemic lupus erythematosus, urinary lipoxin A4
|How to cite this article:|
Sedky Abdou MM, Effat DA, Mansour LA, Abd El Baky NM, Abdul Salam MM. Urinary lipoxin A4 as a biomarker for systemic lupus erythematosus. Egypt Rheumatol Rehabil 2015;42:55-61
|How to cite this URL:|
Sedky Abdou MM, Effat DA, Mansour LA, Abd El Baky NM, Abdul Salam MM. Urinary lipoxin A4 as a biomarker for systemic lupus erythematosus. Egypt Rheumatol Rehabil [serial online] 2015 [cited 2019 Jun 26];42:55-61. Available from: http://www.err.eg.net/text.asp?2015/42/2/55/157861
| Introduction|| |
Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by diverse multisystem involvement and the production of an array of autoantibodies. Clinical features in individual patients can be quite variable, ranging from mild joint and skin involvement to severe life-threatening internal organ disease  . The pathogenesis of SLE, which involves the various facets of the immune system, is complex and perplexing  .
Renal involvement is common in SLE and is a significant cause of morbidity and mortality. It is estimated that up to 90% of SLE patients have pathologic evidence of nephritis on biopsy, but clinically significant nephritis develops in only 50% of patients with SLE. Up to 25% of these patients still develop end-stage renal disease 10 years after the onset of renal compromise. In terms of outcome, the 5- and 10-year renal survival rates of lupus nephritis in the 1990s ranged between 83-93 and 74-84%, respectively  .
A number of biochemical markers are currently used to clinically assess SLE renal disease activity, such as anti-dsDNA antibodies and complement component levels. Nevertheless, the correlation between these markers and lupus nephritis is imperfect, and their usefulness in reflecting disease activity remains controversial  . Thus, novel biomarkers that can discriminate lupus renal activity and its severity, predict renal flares, and monitor treatment response and disease progress are clearly necessary  .
Essential ω3 fatty acids, in particular eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are precursors to a new genus of potent lipid mediators (LM) that are both proresolving and anti-inflammatory (specialized proresolving mediators) and play a physiologic role defining programmed resolution  . Specialized proresolving mediators are endogenously biosynthesized chemical mediators identified in exudates and consist of four distinct new chemical families: lipoxins (LXs), resolvins, protectins, and maresins  .
LXA4 and LXB4 were the first anti-inflammatory LMs recognized to have proresolving actions. LXs are lipoxygenase interaction products derived from the enzymatic conversion of arachidonic acid by transcellular biosynthesis during cell-cell interactions occurring during inflammation. They act as 'braking signals' of further polymorphonuclear leukocyte infiltration and as potent stimuli for the nonphlogistic recruitment of monocytes and macrophage efferocytosis  .
Proresolution mediators such as LXs may also have therapeutic potential in settings in which sustained inflammation and impaired resolution are components of disease pathophysiology, thus providing new avenues for the development of treatment strategies and the development of resolution-based pharmacology and lipidomics-based therapeutics  .
Failure of resolution of inflammation could be the cause of continued inflammatory events observed in lupus, leading to disturbance of the balance between inflammation and resolution more in favor of proinflammatory events and/or failure of resolution. Thus, the healing/repair process is delayed and tissue/organ damage continues  .
On the basis of this hypothesis, it is suggested that progression and flares of lupus are because of the increased production of proinflammatory molecules interleukin-6 (IL-6), tumor necrosis factor-α, macrophage migration inhibitory factor, high-mobility group box 1, free radicals, and LMs such as prostaglandins, leukotrienes (LTs), and/or decreased formation, and the release of anti-inflammatory molecules: IL-4, IL-10, transforming growth factor-β, and LXs, resolvins, protectins, maresins, and nitrolipids  .
| Objective|| |
The aim of this study was to assess the levels of urinary LXA4 in SLE patients and in healthy controls and to correlate them with various clinical and laboratory data as well as renal biopsy and disease activity indices (DAIs).
| Patients and methods|| |
Forty adult female patients with SLE, diagnosed according to the American College of Rheumatology (ACR) revised criteria for SLE  , were included in this study as well as 40 healthy age-matched women who served as the control group. SLE patients were conveniently recruited over a period of 1 month on the basis of the following exclusion criteria.
- Patients with a diagnosis of overlap syndrome (coexistence of lupus with other connective tissue diseases such as rheumatoid arthritis or scleroderma).
- Conditions that could affect the level of LXA4 such as diabetes mellitus, inflammatory lung diseases (e.g. bronchial asthma), and coronary heart disease.
The study was approved by the local ethics committee of Cairo University scientific review board and informed consent was obtained from all participants according to the Declaration of Helsinki; General Assembly, October 2008.
All patients were subjected to the following: full assessment of history, clinical examination, laboratory investigations including erythrocyte sedimentation rate (ESR), complete blood count, liver function tests, kidney function tests, complete urine analysis, 24 h urinary proteins, antinuclear antibodies, anti-dsDNA, C3, and C4 as well as assessment of urinary LXA4, and assessment of disease activity using SLEDAI  and the renal SLEDAI  .
Renal biopsy was performed in patients with established lupus nephritis and the renal pathology was classified according to the revised ISN/RPS system  .
Assessment of urinary lipoxin A4
Freshly voided urine samples were obtained from all patients and controls and stored at −20°C for later analysis of LXA4 levels using the enzyme linked immunosorbent assay. Measurement of LXA4 in urine samples was performed using an enzyme linked immunosorbent assay kit with product No: EA45, purchased from Oxford Biomedical Research Inc., USA. LXA4 was extracted from urine samples before analysis using the C18 Sep-Pak® Light Column (Waters® Corporation, #23501; USA) according to the manufacturer's instructions. Urinary creatinine was measured with enzymatic reaction. The results were expressed as the LXA4/creatinine ratio.
Statistical analysis was carried out using the Statistical package for social science software, version 15.0, 2006; Echosoft Corporation, Roswell, Georgia, USA. Quantitative parametric data were summarized as mean and SD, whereas nonparametric data were summarized as median and percentiles. Frequency and percentages were used for qualitative variables. Comparison between groups was carried out using the χ2 -test for qualitative variables. One-way analysis of variance and the Kruskal-Wallis test for parametric and nonparametric data, respectively, were used to assess the differences between groups. If significant, post-hoc multiple comparison and Mann-Whitney tests were used to identify exactly where the differences were. The Pearson ranked correlation test and the Spearman ranked correlation test were used for correlation analysis between variables. P value was considered significant if less than 0.05.
| Results|| |
The 40 adult SLE patients were all women, ranging in age from 18 to 50 years, mean age 29.55 ± 7.7 years, and disease duration ranging from 1 to 20 years, mean 6.99 ± 4.72 years. Forty healthy age-matched women served as the control group; their ages ranged from 18 to 50 years, with a mean of 32.9 ± 10.35 years. The SLE patients were divided into two groups.
Group I included 20 SLE patients without nephritis.
Group II included 20 SLE patients with nephritis, defined as those patients with a renal SLEDAI of 8 or more (at least two abnormal results for renal parameters on at least two occasions).
The pathology of renal biopsy of the SLE patients with nephritis was as follows:
- Four (20%) patients were lupus nephritis class II.
- Six (30%) patients were class III.
- Seven (35%) patients were class IV.
- Three (15%) patients were class V.
The results were as follows
There was a significant statistical difference between all SLE patients and the control group in the urinary LXA4/creatinine ratio (P = 0.037) ([Table 1] and [Table 2]).
|Table 2 Comparison between the 40 SLE patients and the control group in the urinary LXA4/creatinine ratio|
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The median level of the urinary LXA4/creatinine ratio was lower in SLE patients with nephritis than patients without nephritis (0.1, 0.3 ng/ml, respectively) and the lowest in the control group (0.058 ng/ml), but with no statistical significance (P = 0.113) ([Table 3]).
|Table 3 Comparison between the median levels of the urinary LXA4/creatinine ratio in groups I and II and the control group|
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Comparison between SLE patients with and without various clinical manifestations in the urinary LXA4/creatinine ratio showed that its level was significantly lower in SLE patients with cardiovascular manifestations (0.07 ng/ml, P = 0.009) as well as those with neuropsychiatric manifestations (0.1 ng/ml, P = 0.04) ([Table 4]).
|Table 4 Comparison between SLE patients with and without various clinical manifestations in the urinary LXA4/creatinine ratio|
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No statistically significant correlation was found between the urinary LXA4/creatinine ratio and the age (r = −0.200, P = 0.182) or disease duration (r = −0.151, P = 0.381) of the SLE patients.
There was a positive significant correlation between the urinary LXA4/creatinine ratio and ESR (r = 0.432, P = 0.008), but not with other laboratory parameters.
In terms of the level of the urinary LXA4/creatinine ratio, no statistically significant difference was found between SLE patients with positive or negative anti-dsDNA (P = 0.469) and between SLE patients with normal or consumed C3 and C4 (0.87 and 0.58, respectively).
There was no significant correlation between the level of the urinary LXA4/creatinine ratio and SLEDAI (r = 0.299, P = 0.076), or renal SLEDAI (r = 0.076, P = 0.658) of all SLE patients, as well as the activity scores of the SLE patients when divided into two groups ([Table 5]).
|Table 5 Correlation of the urinary LXA4/creatinine ratio with activity scores in groups I and II SLE patients|
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No statistically significant difference was found between WHO classes of lupus nephritis in SLE patients with nephritis in the urinary LXA4/creatinine ratio (P = 0.99) ([Table 6]).
|Table 6 Comparison of urinary LXA4/creatinine ratio levels in WHO classes of lupus nephritis in group II SLE patients|
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| Discussion|| |
Das  proposed that progression and flares of lupus and lupus nephritis are because of the decreased production of LXA4 and enhanced production of LTs by the renal tissue and/or infiltrating leukocytes and macrophages.
In our study, in which we assessed urinary LXA4 in SLE patients and controls, we found that SLE patients had a higher median level of the urinary LXA4/creatinine ratio compared with the control group (0.12 vs. 0.058 ng/ml), with a statistically significant difference (P = 0.037). The differential expression of biomarkers in serum and urine of SLE patients may reflect the pathophysiological status of disease development and may therefore be used as biomarkers for early diagnosis and prognosis. Generally, urinary substances are likely to reflect kidney damage better than serum components. Urine is a source of biofluid that is easy to harvest and the biomarkers in urine usually reflect the renal function directly in various kinds of nephritic diseases  .
It is noteworthy that Wu et al.  , who studied the temporal changes in blood and urinary LXA4, LTB4, and urinary LTE4 in 49 children with Henoch-Schönlein purpura, showed that blood and urinary LXA4 in the active phase (on the day before treatment) were also higher than those of the controls and further increased in early resolution, and this was in contrast to the blood LTB4 and urinary LTB4 and LTE4, which showed an early peak in the active phase and a subsequent decrease during early resolution. This temporal changes between gradually enhanced LXA4 production and gradually suppressed LTB4 and LTE4 generation suggested eicosanoid class switching during acute inflammation and resolution.
Our results showed that the median level of the urinary LXA4/creatinine ratio was lower in SLE patients with nephritis than in patients without nephritis (0.1, 0.3 ng/ml, respectively), but with no statistical significance (P = 0.113). This is in agreement with the hypothesis of Das  , who reported that a deficiency of LXA4 and excess of LTs may be responsible for lupus/lupus nephritis and he proposed that the urinary levels of LXA4 and LTs may be used to predict prognosis and response to treatment. He postulated that if the urinary LXA4 levels revert to normal or are slowly increasing with and without a decrease in urinary levels of LTs, it can be considered an indication that the patient is responding to treatment and that both systemic and renal lesions of lupus are ameliorating.
The urinary LXA4/creatinine ratio levels in our study were found to be lower in SLE patients with cardiovascular and neuropsychiatric manifestations.
The significant relation with cardiovascular manifestations could be compared with the study carried out by Merched et al.  that tested the hypothesis that atherosclerosis results from a failure in the resolution of local inflammation. Results indicated that 12/15-lipoxygenase expression protects mice against atherosclerosis through its role in the local biosynthesis of LMs, including LXA4, resolvin D1, and protectin D1. These mediators exert potent agonist actions on macrophages and vascular endothelial cells that can control the magnitude of the local inflammatory response. Taken together, these findings suggest that a failure of local endogenous resolution mechanisms may underlie the unremitting inflammation that fuels atherosclerosis.
Indeed, LXs regulate key pathways of vascular hemostasis. Early studies showed LX stimulation of prostacyclin secretion by endothelial cells and vasorelaxant effects, indicating that LXs may regulate the vascular tone. The regulatory effect of aspirin-triggered lipoxin A (ATLa) on nitric oxide release was also reported, although no evidence of a direct impact of LX on nitric oxide biosynthesis has been reported as yet. Consistent with a vasoprotective profile, ATLa inhibited the generation of reactive oxygen species by endothelial cells  .
Also, Das  reported that supplementation of EPA and DHA, respectively suppresses arrhythmias. It is likely that leukocyte and/or myocardial deficiency of EPA and DHA and the consequent reduced formation of LXs, resolvins, protectins, and maresins enhance inflammation and MPO activity, which leads to myocardial damage and fibrosis and initiation and progression of cardiac arrhythmias. On the basis of these evidences, he proposed that LXs, resolvins, and protectins function as endogenous antiarrhythmic molecules and their stable synthetic analogs could be useful in the management of cardiac arrhythmias.
The significant relation with neuropsychiatric manifestations could be explained by the study carried out by Ye et al.  that showed that the LXA4 analog protects the brain and reduces inflammation in a rat model of focal cerebral ischemia reperfusion. Transient focal cerebral ischemia was induced by middle cerebral artery occlusion for 2 h. Intracerebroventricular administration of the LXA4 analog immediately after the onset of ischemia ameliorated neurological dysfunctions, reduced infarction volume, and attenuated neuronal apoptosis. Moreover, treatment with the LXA4 analog suppressed neutrophils infiltration and lipid peroxidation levels; inhibited the activation of microglia and astrocytes; reduced the expression of proinflammatory cytokines tumor necrosis factor-α and IL-1β; and upregulated the expression of anti-inflammatory cytokines IL-10 and transforming growth factor-β1 in the ischemic brain. These results indicate that treatment of LXA4 analog exerts a strong neuroprotective effect against cerebral ischemia-reperfusion injury, and that these effects might be associated with its anti-inflammatory property.
Yao et al.  examined the anti-inflammatory effects of ATL in the central nervous system using rat astrocyte cultures stimulated with lipopolysaccharide (LPS). They found that pretreatment with ATL exerted potent anti-inflammatory effects by inhibiting LPS-induced production of nitric oxide and prostaglandin E2 and reduced the expression of cyclooxygenase 2 and inducible nitric oxide synthase mRNA and protein. Furthermore, ATL suppressed the LPS-induced translocation of the NF-κB p65 subunit to the nucleus. These findings suggest that ATL attenuates neuroinflammation by inhibiting the NF-κB signal transducer pathway in cultured cortical astrocytes.
Our results showed that there was a positive significant correlation between the urinary LXA4/creatinine ratio and ESR (r = 0.43, P = 0.008), but not with other laboratory parameters including anti-dsDNA, C3, and C4.
However, in their study of the changes in LXA4 and LTs in children with Henoch-Schönlein purpura, Wu et al.  showed that with the gradually increased proteinuria, the levels of urinary LXA4 decreased gradually and the levels of urinary LTE4 and LTB4 increased gradually.
Also, Wu et al.  studied the expressions of 15-lipoxygenase and LXA4 in 22 children with acute poststreptococcal glomerulonephritis, and they reported that the temporal changes in urinary LXA4 and glomerular filtration rate of the patients showed a positive correlation, and the correlation coefficient was 0.528 (P<0.05). However, no significant correlations were found between the temporal changes of urinary LXA4 and the degree of proteinuria, and hematuria of the patients with acute poststreptococcal glomerulonephritis.
Our results showed that there was no significant correlation between the level of the urinary LXA4/creatinine ratio and the activity scores (SLEDAI and renal SLEDAI) of all SLE patients, as well as the activity scores of the two SLE groups. There was no statistical significance in the urinary LXA4/creatinine ratio levels within different WHO classes of lupus nephritis in SLE patients with nephritis.
However, Wu et al.  , who studied the changes in LXA4 and LTs in 22 children with Henoch-Schönlein purpura with nephritis, found that concordant with the gradually increased grade of mesangial proliferation, the levels of urinary LXA4 decreased gradually and urinary LTE4 and LTB4 increased gradually. Correlation analysis between urinary LXA4 and the grade of mesangial proliferation showed a negative correlation, and the correlation coefficient was −0.657 (P < 0.05) in their study.
| Conclusion|| |
This was a novel study to assess changes in the levels of urinary LXA4 in SLE patients and to correlate them with various clinical and laboratory data. It showed that the urinary LXA4/creatinine ratio levels were lower in SLE patients with cardiovascular and neuropsychiatric manifestations as well as those with lupus nephritis, suggesting that insufficiency of LXA4 in the human body may be responsible for major organ involvement, making the disease more severe and progressive. Accordingly, LXA4 is suggested to be an inflammatory biomarker not only for lupus nephritis but also for other systemic manifestations in SLE.
We, therefore, recommend that longitudinal studies be carried out with larger number of patients to follow changes in serum and urinary LXA4 levels, and compare their levels with other proinflammatory molecules such as LTs. Furthermore, researchers should evaluate the efficacy of administration of aspirin in SLE patients to induce ATL, which has potent anti-inflammatory properties, and use LXA4 as a therapeutic target in the management of lupus/lupus nephritis.
| Acknowledgements|| |
Conflicts of interest
| References|| |
Dall′Era M, Wofsy D. Clinical features of systemic lupus erythematosus. In: eds Firestein GS, Budd RC, Gabriel SE, McInnes IB, O′Dell JR. Kelley′s Textbook of Rheumatology. 9th ed. Philadelphia, PA: Saunders, an imprint of Elsevier Inc.; 2013: 1283-1303.
Yap DYH, Lai KN. The role of cytokines in the pathogenesis of systemic lupus erythematosus - from bench to bedside. Nephrology 2013; 18
Mok CC. Biomarkers for lupus nephritis: a critical appraisal. J Biomed Biotechnol 2010; 2010
El-Shehaby A, Darweesh H, El-Khatib M, Momtaz M, Marzouk S, El-Shaarawy N, Emad Y. Correlations of urinary biomarkers, TNF-like weak inducer of apoptosis (TWEAK), osteoprotegerin (OPG), monocyte chemoattractant protein-1 (MCP-1), and IL-8 with lupus nephritis. J Clin Immunol 2011; 31
Serhan CN, Krishnamoorthy S, Recchiuti A, Chiang N. Novel anti-inflammatory - pro-resolving mediators and their receptors. Curr Top Med Chem 2011; 11
Schwab JM, Chiang N, Arita M, Serhan CN. Resolvin E1 and protectin D1 activate inflammation-resolution programmes. Nature 2007; 447
Recchiuti A, Serhan CN. Proresolving lipid mediators (SPMs) and their actions in regulating miRNA in novel resolution circuits in inflammation. Front Immunol 2012; 3
Serhan CN, Chiang N, van Dyke TE. Resolving inflammation: dual anti-inflammatory and pro-resolution lipid mediators. Nat Rev Immunol 2008; 8
Das UN. Lipoxins as biomarkers of lupus and other inflammatory conditions. Lipids Health Dis 2011; 10
Hochberg MC. Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum 1997; 40
Bombardier C, Gladman DD, Urowitz MB, Caron D, Chang CH. Derivation of the SLEDAI. A disease activity index for lupus patients. The Committee on Prognosis Studies in SLE. Arthritis Rheum 1992; 35
Pitashny M, Schwartz N, Qing X, Hojaili B, Aranow C, Mackay M, Putterman C Urinary lipocalin-2 is associated with renal disease activity in human lupus nephritis. Arthritis Rheum 2007; 56
Weening JJ, D′Agati VD, Schwartz MM, Seshan SV, Alpers CE, Appel GB, et al.
International Society of Nephrology Working Group on the Classification of Lupus Nephritis; Renal Pathology Society Working Group on the Classification of Lupus Nephritis The classification of glomerulonephritis in systemic lupus erythematosus revisited. Kidney Int 2004; 65
Li Y, Fang X, Li QZ. Biomarker profiling for lupus nephritis. Genomics Proteomics Bioinformatics 2013; 11
Wu SH, Liao PY, Yin PL, Zhang YM, Dong L. Inverse temporal changes of lipoxin A4 and leukotrienes in children with Henoch-Schönlein purpura. Prostaglandins Leukot Essent Fatty Acids 2009; 80
Merched AJ, Ko K, Gotlinger KH, Serhan CN, Chan L Atherosclerosis: evidence for impairment of resolution of vascular inflammation governed by specific lipid mediators. FASEB J 2008; 22
Nascimento-Silva V, Arruda MA, Barja-Fidalgo C, Fierro IM. Aspirin-triggered lipoxin A4 blocks reactive oxygen species generation in endothelial cells: a novel antioxidative mechanism. Thromb Haemost 2007; 97
Das UN. Lipoxin A4 may function as an endogenous anti-arrhythmic molecule. Med Hypotheses; 2011; 76
Ye XH, Wu Y, Guo PP, Wang J, Yuan SY, Shang Y, Yao SL Lipoxin A4 analogue protects brain and reduces inflammation in a rat model of focal cerebral ischemia reperfusion. Brain Res 2010; 1323
Yao C, Yang D, Wan Z, Wang Z, Liu R, Wu Y, et al.
Aspirin-triggered lipoxin A4 attenuates lipopolysaccharide induced inflammatory response in primary astrocytes. Int Immunopharmacol 2014; 18
Wu SH, Liao PY, Yin PL, Zhang YM, Dong L. Elevated expressions of 15-lipoxygenase and lipoxin A4 in children with acute poststreptococcal glomerulonephritis. Am J Pathol 2009; 174
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]