Lipoxins, RevD1 and 9, 13 HODE as the most important derivatives after an early incident of ischemic stroke

There is limited information available regarding the association of plasma free fatty acids (FFA) and inflammation mediators with ischemic stroke. At the same time, new treatment strategies are being pursued. The aim of this study was to carry out a thorough analysis of inflammation with multiple FFA-derivative mediators after and ischemic stroke and standard treatment. HPLC separations of 17 eicosanoids were performed using an Agilent Technologies 1,260 liquid chromatograph. The profiles of the esters of fatty acids were labelled by means of gas chromatography. FFA, and eicosanoid profiles in the group of patients after ischemic stroke significantly differed from the profile of the control group. Studies confirmed the involvement of derivative synthesis pathways responsible for the inflammation, especially palmitic acid (9 and 13 HODE), arachidonic acid, EPA and DHA. Arachidonic acid derivatives were synthesised on 5LOX, 15 LOX and COX pathways with the participation of prostaglandins while omega 3 derivatives strengthened the synthesis of resolvins, RevD1 in particular. The ability to accelerate the quenching of inflammation after ischemic stroke seems to be a promising strategy of stroke treatment in its early stage. In this context, our study points to lipoxins, RevD1, and 9, 13 HODE as the most important derivatives.

Scientific RepoRtS | (2020) 10:12849 | https://doi.org/10.1038/s41598-020-69831-0 www.nature.com/scientificreports/ associated with severe functional disability and high mortality 16 . The limitation of the functional consequences of a stroke may help patients recover faster and limit the costs of long-term treatment and rehabilitation. So far, a number of publications have focused on the connection between the risk of cardiovascular illnesses/mortality and FFA, focusing on docosahexaenoic acid (DHA), linoleic acid, arachidonic acid and palmitic acid, but the results are unclear [17][18][19][20][21] . A study by Harris et al. did not show any connection between the level of linoleic acid and mortality, whereas the research conducted by De Goede et al. revealed no connection with stroke 18,21 . However, higher levels of linoleic acid were associated with a decreased risk of stroke in general in the population of Swedish men 22 , and of ischemic stroke in the ARIC study 23 . Similar connections referring to linoleic acid were observed in Asian populations 24,25 . When evaluating the influence of palmitic acid, it was determined that the circulating palmitic acid is associated with the increased risk of ischemic stroke in women after menopause 26 and with the increased risk of a stroke in general in men 22 . In ARIC, although the levels of saturated fatty acids (SFA) in general were associated with the 64% increased risk of stroke, the results were not significant for palmitic acid itself 23 . Other studies did not show a connection between palmitic acid and ischemic heart disease 17 . The differences in the interpretation of the connection between FFA (palmitic acid in particular) and stroke could have resulted from a number of factors, including the timing of the study after the stroke and not accounting for the fact that individual acids are the precursors of mediators of inflammation whose synthesis could have been amplified and whose use could have been elevated in the inflammatory response. Therefore, higher use of FFA for the processes of synthesis of derivative compounds could have obscured the influence of individual acids on the occurrence of stroke. This is why we decided to study FFA and their derivatives. Elaidic acid (18: 1n9) and arachidonic acid (20: 4n6) were positively correlated, whereas lignoceric acid (24: 0) had a negative correlation with cardioembolic (CE) stroke in the Korean population 27 . No significant difference was observed in relation to EPA/AA and DHA/AA between three subgroups of strokes (hemorrhagic, ischemic strokes and lacunar) but the DGLA/AA relation was much higher in patients with lacunar infarction (LI) than in CE patients 28 . The high ratio of EPA/AA was associated with a good prognosis in ischemic stroke, suggesting that nutritional habits before the stroke influence the exacerbation and course of ischemic stroke in patients 28 . Moreover, it has been established that the proportions of EPA/AA and DHA/AA may be specific markers for younger patients after a stroke. The EPA/AA ratio may be associated with the calcification of the aortic arch in older stroke patients, with numerous infarctions and with the disease of white matter in middle-aged stroke patients 29 . For a better understanding of the cascade of FFA transformation into inflammatory mediators, a diagram is shown in Fig. 1. Acetylsalicylic acid (aspirin) and statins are the basic drugs for the secondary prevention of stroke 30 . They also have an influence on the eicosanoids. Aspirin is the permanent inhibitor of COX-1. Moreover, both aspirin and statins can modify products of COX-2 activity, such as 15-HETE, HEPE and 17(H)DHA, which are precursors of specialized pro-resolving mediators 31 Antiplatelet therapy is the basis for the secondary prevention of strokes 32 . No response to antiplatelet treatment is associated with worse clinical results 33 . The average annual risk of a future ischemic stroke after an initial ischemic stroke/TIA (transient ischemic attack) is from 3 to 4% 34 . Some scientists interpret such individual differences in relation to antiplatelet drugs as "aspirin/clopidogrel resistance" [35][36][37] . The aim of this study was to thoroughly analyze inflammation with multiple FFA derivative mediators after ischemic stroke and standard treatment.  The study group consisted of 75 patients of Caucasian origin with ischemic stroke (IS) and 35 potentially healthy patients constituting the control group (CG). Patients with ischemic stroke were hospitalized at the Department of Neurology of the District Hospital in western Poland, whereas the control group consisted of participants of University of the Third Age in western Poland. The inclusion criteria included a diagnosis of ischemic stroke basing on symptoms, computer tomography (TK) of the brain and/or brain nuclear magnetic resonance (NMR), and a conscious consent to participate in the study. Computer tomography was performed by means of Somatom Emotion 16 slices (Siemens 2015, Munich, Germany), and for brain NMR a Magnetom Essenza 1,5 T (Siemens 2017, Munich, Germany) was used. The exclusion criteria included speech and/or consciousness disorders that would make it impossible to receive conscious consent to participate in the study, and the presence of an active infection or a neoplastic disease. Fasting blood was collected from study group patients on the seventh day after the incident. Blood samples for chromatographic determinations were collected from patients into 10 ml polypropylene tubes containing EDTA. Then, the blood samples were centrifuged at 3,000 rpm for 10 min using a refrigerated centrifuge. The separated plasma was collected into Eppendorf tubes and stored at − 80 °C until analysis. Standard treatment was conducted in accordance with the up-to-date recommendations for the management of acute stroke, based on the guidelines of the Expert Group of the Section of Cerebrovascular Diseases of the Polish Neurological Association 38 and guidelines for the prevention of stroke in patients with stroke and transient ischemic attack from the American Heart Association/American Stroke Association 39 . Only five patients with IS used sporadic fish oil or omega-3 capsules. The description of both groups and the participation of sexes is presented in Table 1.
Gas chromatography (GC) analysis. GC was performed using an Agilent Technologies 7890A GC System equipped with a SUPELCOWAX 10 Capillary GC Column (L × I.D. 15 m × 0.10 mm, df 0.10 μm; Supelco, cat no. 24343). The temperature was held at 40 °C for 0.5 min, and was then increased by 25 °C/min up to 195 °C for 0 min, then by 3 °C/min to 205 °C for 0 min, and by 8 °C/min to 250 °C for 0.5 min. The total analysis time was 16.158 min and the gas flow rate was 1 ml/min, with nitrogen as the carrier gas.
FAs were identified by comparing their retention times with those of Food Industry FAME Mix (cat. no 35077) (Restek), supplemented with the following single standards: C18:4 (stearidonic acid methyl ester, cat. no 10005000,), C22:4n6 (docosatetraenoic acid methyl ester, cat. no 10006866) and C22:5n3 (docosapentaenoic acid methyl ester, cat. no 21124, Cayman Chemical) by using ChemStation Software (Agilent Technologies, Cheadle, UK). To control the fatty acid retention times, C21:0 (heneicosanoic acid, cat. no H5149, Merck KGaA) was used as the internal standard. Results are presented as the percentage of the individual fatty acids in the total mass of fatty acids from the examined samples (Fig. 2).  For the extraction of eicosanoids, 0.5 ml plasma was added to 1 ml acetonitrile for protein precipitation and 50 µl of internal standard (1 µg/ml). After 15 min of incubation at − 20 °C, samples were centrifuged at 10,000 rpm for 10 min using a chilled centrifuge (Eppendorf, Centrifuge 5804R). Supernatants were transferred to new collection tubes and 4.5 ml of 1 mM HCl was added. The pH of each sample was adjusted to 3 by adding 30-50 µl of 1 M HCl. The columns were activated with consecutive washes of 3 ml 100% acetonitrile and 3 ml 20% acetonitrile in water. The samples were loaded and double washed with 3 ml 20% acetonitrile in water. Eicosanoids were then eluted with 1.5 ml of a mixture of methanol and ethyl acetate (1/1 v/v), dried under a vacuum and dissolved in 100 µl of 60% methanol in water with 0.1% acetic acids. Samples were immediately analyzed using HPLC.
HPLC operating parameters. The HPLC separations were performed using an Agilent Technologies The content of buffer B in the mobile phase was 30% at 0 to 2 min of separation, increased linearly to 80% at 33 min, was 98% between 33.1 and 37.5 min, and was 30% between 40.
When analyzing the level of individual mediators that are FFA derivatives, significant differences between the studied groups (IS and CG) were observed, as shown in Table 3, Figs. 4 and 5.

Discussion
Cerebral circulation is strictly regulated by vasoactive substances. There is a delicate balance between vasoconstrictor and vasodilating factors. During ischemia stroke, the autoregulation of cerebral circulation is handicapped, causing hyperaemia or hypoperfusion that disrupts the flow of blood. This situation contributes to the death of neuron cells due to incorrect blood flow. Traditional vasoactive mediators, such as nitric oxide and the peptide bound to calcitonin gene, were documented in order to ensure vasodilatation and for neuroprotection in the ischemic brain. The recurring domain is the identification of FFA and their derivatives which, as extracellular  www.nature.com/scientificreports/ signalling particles, may lead to vasodilatation, probably also causing a neuroprotective effect 45,46 . Neuroprotection as an approach to brain therapy has been the subject of numerous discussions, fuelled by the necessity to improve the care of patients with acute stroke and by the identification of new medicines. When analyzing the FFA profile in the plasma of ischemic stroke patients, it was observed that medium chain acids, such as C13:0 Tridecanoic acid, elongate into C16: 0 Palmitic acid. Additionally, the supply with the diet may also increase the amount of this acid in the plasma, resulting in a substrate in the course of further cascade 46,47 . Subsequently, C16:0 palmitic acid is desaturated into palmitoleic acid (C16:1) and elongated into stearic acid (C18:0), which is desaturated into its metabolites 48 . The pathway of this synthesis with the participation of SREBP-1c is stimulated by the high concentration of insulin and cholesterol. This is why the influence of the diet seems to be one of the key elements modulating the course of the reaction 49 .
One of the key metabolites is oleic acid, as presented in Fig. 6. Higher concentrations of oleic acid (C18:1w9) may be the cause of the growth and accumulation of lipids, supporting the occurrence of atherosclerosis and cardiovascular issues 50 . LA is the most widely present fatty acid in atherosclerotic plaques. These findings should not be extrapolated to dietary oleic acid intake because numerous epidemiological and intervention studies confirmed this observation, pointing to a strong relationship between the Mediterranean diet and circulatory system diseases. In this context, it seems that extra virgin olive oil (EVOO), the most representative element of this diet and a source of oleic acid, is important for the reduction of the frequency of occurrence of cardiovascular events, including myocardial infarction and stroke 51 . Diets providing increased amounts of LA can increase the synthesis of 13-HODE in the vascular endothelium, decreasing the adhesion of platelets and thrombogenicity. Another synthesized acid is C18:2n6 linoleic acid, which is the precursor of the synthesis of proinflammatory mediators 9 and 13 HODE, while gamma-linolenic acid (C18:3n-6) is the precursor of subsequent chains from the ɷ-6 family-eicosatrienoic acid (C20: 3n6) and arachidonic acid (C20:4n6). In this study, we observed an increase of all of these derivatives, except C18:2n6 linoleic acid which, while becoming a substrate for the synthesis of proinflammatory mediators 9 and 13 HODE, was partially used up. The lower level of these mediators in the IS group than in the control group means that the synthesis of both mediators is insufficient in the inflammatory reaction associated with ischemic stroke. Hydroxyoctadecadienoic acids (HODEs) are stable oxidation products, the generation of which is increased when oxidative stress is increased, such as in diabetes 52 . The influence of these derivatives has not been studied yet with reference to ischemic stroke. However, it was observed that in prior atherosclerosis, 13-HODE is created in macrophages through lipoxygenase-1, which increases protective mechanisms and leads to the increased removal of lipids from cells loaded with lipids from the arterial wall. Therefore, the activation of peroxisome proliferator-activated receptor (PPAR) is initiated and in later atherosclerosis both 9-HODE and 13-HODE are formed non-enzymatically 53 . Fatty acids and eicosanoids are activated PPAR nuclear transcription factors. PPAR-γ2 is the predominant form in adipose tissue. 13-HODE, created through the activity of 15-LOX-1, can also increase the reverse transportation of cholesterol through a mechanism encompassing PPAR-α 54 . Although the increased expression of 15-LOX-1 is a quality of early atherosclerosis, the activity of 13-HODE may in fact have a protective effect at this stage of the disease. However, at a later stage, both 9 and 13 HODE increase the susceptibility of vascular cells to apoptosis, which may contribute to the formation of atherosclerotic plaque 55 . Moreover, vascular smooth muscle cells (VSMC) may be stimulated by HODE generated by neighbouring cells, including platelets and macrophages. Limor et al. observed that vascular smooth muscle cells express 12/15-LOX, and hence are capable of synthesizing 12-HETE, 15-HETE, and 13-HODE 56 .
In the ischemic stroke environment, in which the flow of cerebral blood is obstructed, brain tissue that lacks oxygen initiates the separation of arachidonic acid (AA) from the double-layer phospholipid membrane. After release, AA undergoes both oxidative metabolism independent of enzymes and mediated by enzymes such as www.nature.com/scientificreports/ lipoxygenase and cyclooxygenase. As a result of the amplified synthesis reactions, numerous biologically active metabolites occur, which contribute to the prognosis of the pathological stroke 47 . When analyzing further elongation and increases in the synthesis of arachidonic acid and the cascade of its transformations, which enable the synthesis of prostaglandins (PG), thromboxanes (TX) series 2, leukotrienes (LT) series 4, and 5, 12, 15 HETE, we observed a significant amplification of inflammation in the levels of selected mediators. HETE derivatives are strongly pro-inflammatory and stimulate clotting, atherosclerotic changes, inflammatory and allergic reactions, the proliferation of cells and malignant tissue growth 57 . In our study, we observed the activation of three synthesis pathways from the cascade of arachidonic acid: -COX-1,2-leading to the synthesis of PGE2, -LOX-15-leading to the synthesis of 15 HETE, -LOX-5-leading to the synthesis of leukotrienes.
Here, the LOX-5 pathway appears to have been activated and its metabolites were partially used up. However, we cannot be absolutely sure because we did not study the entire leukotrienes family (LTC4, LTD4, LTE4). The outline of this part of our discussion is presented in Fig. 7.
The pathways of lipoxin synthesis, which are specialised pro-resolving mediators (SPMs), were not activated. Therefore, particles separating inflammation could be used in the early treatment stages of ischemic stroke. Analogues like BML-111 have extended biological activity and can, therefore, be a better therapeutic option (synthetic LXA4 analogue also called heptanoate 5 (S), 6 (R)-7-trihydroxymethyl) 58,59 . Due to the fact that 12/15-lipoxygenase (12/15-LOX-the key enzymes of arachidonic acid cascade) contribute to both neural cell death and damage, the inhibition of 12/15-LOX can ensure the multifactor protection against ischemic issues, as shown by our study and the studies of other authors 60 .
When performing the analysis of long chain fatty acids of the omega-3 family, it was observed that they are involved at this stage of ischemic stroke, especially DHA-derivative resolvins. However, the synthesis through cyclooxygenase proceeds to a lesser, yet still significant, extent. In the case of EPA and DHA derivatives, we did not observe the involvement of all pathways responsible for the quenching of inflammation. This relationship was not observed with reference to the synthesis of maresins through 12LOX, as presented in Fig. 8. However, www.nature.com/scientificreports/ maresins may also serve as therapeutic alternatives during the later stages of treatment, particularly due to the fact that the 7S-MaR1 analogue accelerates and improves the clearance of neutrophils and reduces the accumulation of macrophages in the damaged spinal cord 61 . Further studies of this subject are necessary. A limitation in our study is the fact that we did not account for the APOE-ε4 of hosts who, due to increased insulin resistance markers, are more susceptible to both the positive and negative biological effects of fatty acids  www.nature.com/scientificreports/ in response to dietary changes [62][63][64] . Another limitation is the unequal gender distribution in the control group relative to stroke patients, although no differences were found in the level of inflammatory mediators by gender.

Conclusions
Ischemic stroke is associated with the presence of a strong inflammatory reaction, in which arachidonic acid plays a key role. Derivatives are synthesized using three pathways-5LOX, 15LOX and COX1,2-with the participation of prostaglandins; it seems that 12LOX is not involved in the process. The pathways leading to the synthesis of EPA derivatives, particularly DHA, are also intensified, leading to the increased consumption of resolvins, which are DHA derivatives. Furthermore, the pathway of palmitic acid derivatives (9 and 13 HODE) is also activated, though it seems to play a lesser role. The potential to affect and accelerate the stage of inflammation quenching after stroke seems to be a promising strategy in the treatment of stroke in its early stage. Our study points to lipoxins, RevD1 and HODE as the most important derivatives for this task.