The Effect of Oleoylethanolamide (OEA) Add-On Treatment on Inflammatory, Oxidative Stress, Lipid, and Biochemical Parameters in the Acute Ischemic Stroke Patients: Randomized Double-Blind Placebo-Controlled Study

Abstract

Methods:
Sixty patients with a mean age of 68.60 ± 2.10 comprising 29 females (48.33%), who were admitted to an academic tertiary care facility within the first 12 hours poststroke symptoms onset or last known well (LKW), in case symptom onset time is not clear, were included in this study. AIS was confirmed based on a noncontrast head CT scan and also neurological symptoms. Patients were randomly and blindly assigned to OEA of 300 mg/day (n = 20) or 600 mg/day (n = 20) or placebo (n = 20) in addition to the standard AIS treatment for three days. A blood sample was drawn at 12 hours from symptoms onset or LKW as the baseline followed by the second blood sample at 72 hours post symptoms onset or LKW. Blood samples were assessed for inflammatory and biochemical parameters, oxidative stress (OS) biomarkers, and lipid profile.

Results:
Compared to the baseline, there is a significant reduction in the urea, creatinine, triglyceride, high-density lipoprotein, cholesterol, alanine transaminase, total antioxidant capacity, malondialdehyde (MDA), total thiol groups (TTG), interleukin-6 (IL-6), and C-reactive protein levels on the follow-up blood testing in the OEA (300 mg/day) group. In patients receiving OEA (600 mg/day) treatment, there was only a significant reduction in the MDA level comparing baseline with follow-up blood testing. Also, the between-group analysis revealed a statistically significant difference between patients receiving OEA (300 mg/day) and placebo in terms of IL-6 and TTG level reduction when comparing them between baseline and follow-up blood testing.

Conclusion:
OEA in moderate dosage, 300 mg/day, add-on to the standard stroke treatment improves short-term inflammatory, OS, lipid, and biochemical parameters in patients with AIS. This effect might lead to a better long-term neurological prognosis.

Full text

Research Article
The Effect of Oleoylethanolamide (OEA) Add-On Treatment on
Inflammatory, Oxidative Stress, Lipid, and Biochemical
Parameters in the Acute Ischemic Stroke Patients: Randomized
Double-Blind Placebo-Controlled Study
Mohammadmahdi Sabahi ,
1,2
Sara Ami Ahmadi,
1
Azin Kazemi,
1
Maryam Mehrpooya ,
3
Mojtaba Khazaei ,
4
Akram Ranjbar ,
5,6
and Ashkan Mowla
7
1
Neurosurgery Research Group (NRG), Student Research Committee, Hamadan University of Medical Sciences, Hamadan, Iran
2
Behavioral Disorders and Substances Abuse Research Center, Hamadan University of Medical Sciences, Hamadan, Iran
3
Department of Clinical Pharmacy, School of Pharmacy, Hamadan University of Medical Sciences, Hamadan, Iran
4
Department of Neurology, School of Medicine, Hamadan University of Medical Sciences, Hamadan, Iran
5
Department of Pharmacology and Toxicology, School of Pharmacy, Hamadan University of Medical Sciences, Hamadan, Iran
6
Nutrition Health Research Center, Hamadan University of Medical Sciences, Hamadan, Iran
7
Department of Neurological Surgery, Keck School of Medicine, University of Southern California (USC), Los Angeles, CA, USA
Correspondence should be addressed to Mojtaba Khazaei; khazaeimojtaba@yahoo.com
and Akram Ranjbar; akranjbar2015@gmail.com
Mohammadmahdi Sabahi, Sara Ami Ahmadi, and Azin Kazemi contributed equally to this work.
Received 25 January 2022; Revised 1 April 2022; Accepted 5 June 2022; Published 8 September 2022
Academic Editor: Anna M. Giudetti
Copyright © 2022 Mohammadmahdi Sabahi et al. This is an open access article distributed under the Creative Commons
Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work
is properly cited.
Background and Objective. There is a growing body of evidence for the efficacy of oleoylethanolamide (OEA) in patients with
inflammatory disorders. The present randomized double-blind placebo-controlled study is aimed at evaluating the efficacy of
OEA add-on treatment in patients with acute ischemic stroke (AIS). Methods. Sixty patients with a mean age of 68:60 ± 2:10
comprising 29 females (48.33%), who were admitted to an academic tertiary care facility within the first 12 hours poststroke
symptoms onset or last known well (LKW), in case symptom onset time is not clear, were included in this study. AIS was
confirmed based on a noncontrast head CT scan and also neurological symptoms. Patients were randomly and blindly
assigned to OEA of 300 mg/day (n=20) or 600 mg/day (n=20) or placebo (n=20) in addition to the standard AIS treatment
for three days. A blood sample was drawn at 12 hours from symptoms onset or LKW as the baseline followed by the second
blood sample at 72 hours post symptoms onset or LKW. Blood samples were assessed for inflammatory and biochemical
parameters, oxidative stress (OS) biomarkers, and lipid profile. Results. Compared to the baseline, there is a significant
reduction in the urea, creatinine, triglyceride, high-density lipoprotein, cholesterol, alanine transaminase, total antioxidant
capacity, malondialdehyde (MDA), total thiol groups (TTG), interleukin-6 (IL-6), and C-reactive protein levels on the follow-
up blood testing in the OEA (300 mg/day) group. In patients receiving OEA (600 mg/day) treatment, there was only a
significant reduction in the MDA level comparing baseline with follow-up blood testing. Also, the between-group analysis
revealed a statistically significant difference between patients receiving OEA (300 mg/day) and placebo in terms of IL-6 and
TTG level reduction when comparing them between baseline and follow-up blood testing. Conclusion. OEA in moderate
dosage, 300 mg/day, add-on to the standard stroke treatment improves short-term inflammatory, OS, lipid, and biochemical
parameters in patients with AIS. This effect might lead to a better long-term neurological prognosis.
Hindawi
Oxidative Medicine and Cellular Longevity
Volume 2022, Article ID 5721167, 11 pages
https://doi.org/10.1155/2022/5721167

1. Introduction
The prevalence and mortality of noncommunicable diseases
have surpassed infectious diseases globally. Neurological dis-
orders such as stroke, Alzheimer’s, and Parkinson’s are asso-
ciated with high mortality and morbidity, and thus far, there
is no cure for them. These disorders have revealed common
pathological features such as inflammation, oxidative stress
(OS) production, abnormal protein accumulation, disrup-
tion of normal calcium homeostasis, and apoptosis. Among
these, stroke is one of the leading causes of death worldwide
and a major cause of disability in adults [1–3].
Stroke is a neurological disorder that is divided into
ischemic and hemorrhagic types [4, 5]. In the ischemic type,
the hypoxia of the brain tissue occurs with the cessation of
blood flow to the brain tissue and leads to the destruction
of neurons and glial cells [2, 3, 6, 7]. The sequence of the
events responding to ischemia is known as the ischemic cas-
cade, including glutamate release, calcium influx, OS,
inflammation, and ultimately, apoptosis, which leads to irre-
versible neuronal death [8, 9].
Oleoylethanolamide (OEA) is a member of the N-
acylethanolamine (NAE) family, which are endogenously bio-
active lipids and are formed from amidation of membrane
phospholipids. This fatty acid is mostly found in sources such
as olives and sesame [10, 11]. Pure olive oil counteracts cell
death by reducing lipid peroxidation (LPO), brain prostaglan-
din E2, and nitric oxide production, while increasing glutathi-
one concentrations. The family of NAEs is involved in a wide
rangeofbodyprocessessuchasinflammation, nerve protec-
tion,acutestress,painrelief,anxiety,hypotension,sleep,and
energy balance [11]. Based on previous findings, using olive
oil fatty acid supplements reduces damage and protects the
brain tissue during an ischemic stroke [3]. Peroxisome
proliferator-activated receptor alpha (PPAR-α) and cannabi-
noid receptors (CBR) have critical roles in regulating inflam-
mations [12]. The activation of the CBR1 can protect neural
tissues against ischemic injury or tissue reperfusion injury by
reducing OS, releasing lactate dehydrogenase, and activating
caspase-3 in vitro [13]. The members of the NAE family have
been identified as the natural ligands of the CBR. In addition
to the CBR, OEA can bind to PPAR-α, and consequently, exert
its anti-inflammatory effects [12], since PPAR-αacts as a neg-
ative regulator of the inflammatory response through direct
binding to the p65–nuclearfactor(NF)-κB, which subsequently
antagonizes NF-κB transcription factor pathways [14, 15].
This study sought to determine the effect of OEA supple-
mentation on the biomarkers of OS, inflammatory parame-
ters, lipid profile, and renal and hepatic parameters in
patients with acute ischemic stroke.
2. Materials and Methods
2.1. Trial Setting and Design. This single-center three-day
randomized, placebo-controlled, double-blind, parallel-
group trial was conducted in Farshchian (Sina) Hospital,
an affiliate of the Hamadan University of Medical Sciences,
Hamadan, Iran. All patients were screened and enrolled
between April 21
st
, 2020 and July 22
nd
, 2020.
2.2. Standard Protocol Approvals, Registrations, and Patient
Consents. The trial was registered in the Iranian Registry of
Clinical Trials (Identifier: IRCT20130501013194N4) and
approved by the Ethics Committee of the Hamadan Univer-
sity of Medical Sciences with the ethical code of IR.UM-
SHA.REC.1398.720-722. Before participation in the study,
all participants and/or their next of kin signed a written
informed consent form in compliance with the Declaration
of Helsinki.
2.3. Patients. In general, 82 patients were assessed for their
eligibility; among whom, 12 and 10 patients did not meet
the inclusion criteria or declined to participate, respectively.
As such, 60 patients aged 30-93 years who met the inclusion
and exclusion criteria were consented to participate in the
study (Figure 1). No patients lost to follow-up since they were
all admitted in the hospital for at least three days after the
onset of their neurological symptoms or last known well
(LKW), in case stroke onset time was unclear. The National
Institute of the Health Stroke Scale (NIHSS) score and the
modified Rankin Scale (mRS) were used to measure the neuro-
logical deficits and functional outcome poststroke, respec-
tively. The NIHSS is a scoring system which offers a
quantitative assessment neurological deficit caused by stroke.
This 15-item scale measures the deficits caused by acute stroke
on several domains including awareness, language, eye move-
ments, motor force, ataxia, and sensorium. Each item is
graded on a three- to five-point scale leading to scores between
0 and 42, where 0 indicates no deficit and 42 translates to
death [16]. Furthermore, the mRS is a functional outcome
measure, which ranges from 0 to6, where 0 defines as no neu-
rological symptoms and 6 indicates death [17].
The patients were included if they were reaching the
emergency department within 12 hours from the neurologi-
cal symptom onset or LKW in case the onset time was
unclear, had no evidence of intracranial hemorrhage on their
initial noncontrast head CT, and had NIHSS score ≥2 and
<20 [18]. Exclusion criteria is as follows: patients aged <18,
had a temperate ≥38.0
°
C and/or a white blood cell count
≥16,000 cells/mm
3
, given we did this study during the
COVID pandemic, history or suspicion of active malig-
nancy, inflammatory vasculopathy, systemic inflammatory
disease, connective tissue disease, hypercoagulable state,
and chronic renal failure, as well as patients who had a glo-
merular filtration rate <30, hepatic failure and/or cirrhosis,
gastrointestinal bleeding, uncontrolled diabetes mellitus,
chronic respiratory diseases, hematologic illnesses, and sei-
zure at presentation. Also, use of medications/supplements,
having either anti-inflammatory or anti-OS effect, prior sen-
sitivity to compounds, alcohol and drug abuse, and a vegeta-
tive state were the other exclusion criteria.
These 60 patients underwent quick neurological assess-
ment upon admission, as per standard practice [19, 20].
Patients meeting both inclusion and exclusion criteria were
divided into two categories. Patients who were eligible for
acute reperfusion therapy including intravenous and/or
intra-arterial thrombolytic therapy and those who were not
eligible for acute treatment. In both categories, in addition
to OEA or placebo, patients’underlying diseases such as
2 Oxidative Medicine and Cellular Longevity

diabetes mellitus, hypertension, and dyslipidemia were
addressed as per standard of care. They also receive anti-
platelets or anticoagulants when appropriate as per standard
of care. Patients were not allowed to receive any additional
treatment in the course of the trial.
The baseline blood sample was drawn at 12 hours after
LKW, testing OS, and lipid, along with renal and hepatic
parameters during the acute phase. The follow-up sample
was drawn at 72 hours after LKW.
Patients were randomly divided into three groups using
Minitab software, patients who received OEA 300 mg/day,
600 mg/day, or placebo capsules with respect to concealment,
respectively. All three groups received the treatment for three
consecutive days. Each individual received a computer code,
and the code was disclosed during the treatment period. The
allocation was hidden behind a series of sequentially num-
bered, opaque, and sealed envelopes. A study coordinator
opened the envelope after the recruitment of each patient.
The patients were randomly assigned and assessed by two
physicians. The statistician, the patients, the referring physi-
cian, and the physician evaluating the patients and providing
the trial drugs, were all blinded to the allocation. The treat-
ment codes were revealed at the end of the study and after
closing the database. All three groups were adjusted for age
and gender, extent and severity of ischemic stroke using the
NIHSS score, baseline mRS, acute infarct volumes on noncon-
trast head CT, door to needle time in case intravenous throm-
bolytic therapy was administered, whether acute reperfusion
therapy was performed, body mass index, history of smoking,
and history of hypertension. Furthermore, groups were
adjusted for history of hyperlipidemia, coronary artery disease,
diabetes mellitus, deep vein thrombosis, prior stroke, systolic
and diastolic blood pressure, and home medication prior to
admission (Table 1). Frequency matching methods were used
for this purpose.
2.4. Chemicals. The applied chemicals in this investigation
included 5,5’-Dithiobis-(2-nitrobenzoic acid) (DTNB),
2,4,6-tripyridyl-S-triazine (TPTZ), Thiobarbituric acid
(TBA), Nitro blue tetrazolium (NBT), and sodium phos-
phate buffer that were obtained from the Sigma-Aldrich
(St. Louis, MO, USA). OEA supplements were provided by
Karen Pharma and Food Supplement Co.
2.5. Biochemical Analysis. Phlebotomy was employed to col-
lect ten milliliters of venous blood into chilled ethylenedi-
aminetetraacetic acid-containing tubes, which were then
centrifuged for plasma separation, aliquoted in 1.5 ml vials,
Allocation
Randomized (n=60)
Assessed for eligibility (n=82)
Excluded (n=22)
󳴴 Not meeting inclusion criteria (n=12)
󳴴 Declined to participate (n=10)
Enrollment
Intervention groups, OEA 300 mg/day
(n=20) and OEA 600mg/day (n=20)
Placebo group
(n=20)
Lost to follow-up (n=0)
Non-adherence to treatment (n=0)
Adverse eects (n=0)
Lost to follow-up (n=0)
Non-adherence to treatment (n=0)
Adverse eects (n=0)
Analysed (n=40)
󳴴 Excluded from analysis (n=0)
Analysed (n=20)
󳴴 Excluded from analysis (n=0)
Analysis
Figure 1: The flow diagram of the study.
3Oxidative Medicine and Cellular Longevity

snap-frozen, and kept at −80
°
C until further analyses. After the
treatment period, interleukin- (IL-) 6 levels were measured in
the blood samples of the subjects by enzyme-linked immuno-
sorbent assay, and C-reactive protein (CRP) levels were mea-
sured by turbidometry. Portion of the blood sample was sent
to the laboratory to measure the lipid profile including triglyc-
erides (TG), total cholesterol, and high-density lipoprotein
(HDL) in addition to serum hematological parameters com-
prising urea, creatinine, aspartate transaminase (AST), and ala-
nine transaminase (ALT). On the other portion of the blood
sample, the following parameters were measured: LPO level
by assessing malondialdehyde (MDA), capacity of the serum
antioxidants which is called total antioxidant capacity (TAC),
the level of total thiol groups (TTG), and the activity of the
superoxide dismutase (SOD) enzyme by a spectrophotometer
using the TBA reagent at 532 nm, the TPTZ reagent at
593 nm, the DTNB reagent at 412 nm, and the NBT reagent
at 560 nm wavelength, respectively.
Table 1: Demographic and clinical characteristics of the study participants.
Number (%) or mean ± SD
All (n=60)Placebo
(n=20)
OEA 300 mg/day
(n=20)
OEA 600 mg/day
(n=20)Pvalue
Male/female 31/29 11/9 10/10 10/10 0.935
Age (years) 68:60 ± 2:10 65:4±3:21 67:2±2:65 69:7±3:09 0.648
BMI (kg/m
2
)26:9±4:12 27:2±3:71 26:4±4:32 27:1±3:45 0.762
Risk factors
Smoking 34 (56.6%) 11 (55%) 9 (45%) 14 (70%) 0.275
Hypertension 51 (85%) 16 (80%) 17 (85%) 18 (90%) 0.676
Hyperlipidemia 47 (78.3%) 14 (70%) 16 (80%) 15 (75%) 0.766
CAD 35 (58.3%) 17 (85%) 12 (60%) 16 (80%) 0.155
Diabetes mellitus 16 (26.6%) 5 (25%) 4 (20%) 7 (35%) 0.551
Prior stroke 17 (28.3%) 7 (35%) 4 (20%) 6 (30%) 0.563
DVT 6 (10%) 1 (5%) 2 (10%) 2 (10%) 0.804
SBP, mmHg 149 ± 8:29 152 ± 11:01 149 ± 10:73 148 ± 12:14 0.742
DBP, mmHg 87 ± 10:02 87 ± 8:73 89 ± 10:20 88 ± 12:65 0.933
Acute ischemic
stroke event
characteristics
NIHSS 5:2±1:26 5:12 ± 1:72 5:18 ± 1:50 5:26 ± 1:37 0.790
Infarct volume (cm
3
)1:2±0:87 1:1±1:02 1:2±0:98 1:4±1:25 0.720
mRS 2:1±0:34 2:03 ± 0:53 2:12 ± 0:36 1:96 ± 0:27 0.502
DTN, hours 8:3±4:57 7:2±5:19 7:9±4:21 8:3±4:71 0.191
Thrombolytic therapy 27 (45%) 9 (45%) 8 (40%) 10 (50%) 0.817
Nonthrombolytic therapy 33 (55%) 11 (55%) 12 (60%) 10 (50%) 0.817
Medications prior
to admission
Antiplatelet therapy 18 (30%) 8 (40%) 4 (20%) 6 (30%) 0.386
Anticoagulation therapy 18 (30%) 8 (40%) 4 (20%) 6 (30%) 0.386
Statin therapy 47 (78.3%) 14 (70%) 16 (80%) 15 (75%) 0.766
ACEI therapy 50 (83.3) 16 (80%) 17 (85%) 17 (85%) 0.877
ARB therapy 4 (6.6%) 1 (5%) 2 (10%) 1 (5%) 0.765
BB therapy 29 (48.3%) 11 (55%) 8 (40%) 10 (50%) 0.627
CCB therapy 36 (60%) 12 (60%) 14 (70%) 10 (50%) 0.435
Diuretics therapy 9 (15%) 3 (15%) 3 (15%) 3 (15%) 1.00
Insulin with oral
antidiabetic therapy 10 (16.6%) 3 (15%) 2 (10%) 5 (25%) 0.432
Oral anti diabetic therapy 6 (10%) 2 (10%) 2 (10%) 2 (10%) 1.00
Underlying mechanism
for stroke
Atherothrombotic 33 (55%) 12 (60%) 10 (50%) 11 (55%) 0.817
Cardioembolic 9 (15%) 3 (15%) 2 (10%) 4 (20%) 0.676
Lacunar infract 9 (15%) 3 (15%) 3 (15%) 3 (15%) 1.00
Other causes 6 (10%) 2 (10%) 2 (10%) 2 (10%) 1.00
Undetermined 3 (5%) 1 (5%) 1 (5%) 1 (5%) 1.00
Abbreviations: BMI, body mass index; CAD, coronary artery disease; DVT, deep vein thrombosis; SBP, systolic blood pressure; DBP, diastolic blood pressure;
NIHSS, National Institutes of Health Stroke Scale; mRS, Modified Rankin scale; DTN, door-to-needle time; ACEI, angiotensin-converting-enzyme inhibitors;
ARB, Angiotensin II receptor blockers; BB, beta blocker; CCB, calcium channel blocker.
4 Oxidative Medicine and Cellular Longevity

2.6. Statistical Analysis. Descriptive analysis and comparison
of differences between demographics in each group were
performed by SPSS 16 software. The mean, standard devia-
tion, frequency, and percentage were used to represent the
data. The Kolmogorov-Smirnov test was applied to deter-
mine if the distribution was normal. The Chi-squared test
and Fisher’s exact test were employed for comparing qualita-
tive variables between the groups. In addition, the indepen-
dent t-test and Mann–Whitney Utest were used to
compare quantitative variables. Furthermore, intragroup dif-
ferences between baseline and follow-up parameters were
examined using the Wilcoxon signed-rank test. Finally, the
between-group difference analysis was conducted through
the Kruskal-Wallis test, followed by a post hoc test, and P
values less than 0.05 were considered statistically significant.
2.7. Data Availability Policy. Any competent investigator can
request anonymized data from the corresponding author for
replicating techniques and findings.
3. Results
Thirty-one men (51.66%) and 29 women (48.33%) partici-
pated in this study, and their mean age was 68:60 ± 2:10
(range 30-93). Demographic and clinical characteristics of
the study participants including the cardiovascular risk fac-
tors, acute ischemic stroke event characteristics, medications
prior to admission, and the underlying mechanism for
stroke are summarized in Table 1. No significant differences
were observed between the baseline characteristics of these
three groups. Our primary outcome was to evaluate the
effect of OEA on inflammatory and OS parameters. The sec-
ondary outcome was to investigate the effect of OEA on the
lipid profile, as well as renal and hepatic parameters.
3.1. Renal Parameters. As shown in Table 2, although the
between-group analysis revealed no statistically significant
difference in the blood urea and creatinine levels, there was
a statistically significant decline in the level of urea in
patients receiving 300 mg/day OEA. Overall, OEA 300 mg/
day reduced both urea and creatinine levels and might act
as a renoprotective factor in acute ischemic stroke patients.
3.2. Lipid Profile. As demonstrated in Table 2, a statistically
significant reduction in total cholesterol and TG and a rise
in HDL levels were seen in patients receiving OEA 300 mg/
day, while changes in other groups were not statistically sig-
nificant in either between-group or within-group analysis.
3.3. Hepatic Parameters. As shown in Table 2, although
between-group analysis revealed no statistically significant
difference regarding ALT and AST levels, there was a statis-
tically significant decline in the level of ALT in patients
receiving OEA 300 mg/day.
3.4. OS Parameters. The levels of OS parameters are shown
in Table 2. Although SOD levels have no significant differ-
ence between the groups, within-group analysis indicated
that consumption of OEA could significantly alter TAC,
MDA only in the 300 mg/day group, and TTG in both
300 mg/day and 600 mg/day groups. Also, between-group
analysis indicated statistically significant differences in
TTG, and the results of post hoc test revealed that this differ-
ence was noticeable between patients receiving OEA 300 mg/
day and a placebo (P=0:047).
3.5. Inflammatory Profile. The levels of inflammatory param-
eters are displayed in Table 2. In within-group analysis,
patients who received moderate dose of OEA, 300mg/day,
showed statistically significant decrease in IL-6 and CRP
levels. Between-group analysis demonstrated statistically sig-
nificant differences in IL-6, and based on the results of post
hoc test, this difference was observed between patients receiv-
ing OEA, 300mg/day, and placebo (P=0:037).
3.6. Complications. Table 3 presents the occurrence of
adverse effects. Nausea, vomiting, dyspepsia, and headache
were the most frequently reported adverse effects among
the participants regardless of their treatment group. There
were no statistically significant differences among different
groups regarding the reported side effects.
4. Discussion
OS and inflammation are considered as an important path-
ophysiological mechanism in acute ischemic stroke. OS is a
consequence of the failure in the equilibrium between the
endogenous reactive oxygen species (ROS) production and
their cleansing by endogenous antioxidant defense systems
[21]. The rapid increase in ROS production immediately
after acute ischemic stroke quickly overwhelms the antioxi-
dant capacity of the brain tissue and causes further tissue
damage. ROS can damage cell macromolecules, resulting in
autophagy, apoptosis, and necrosis. Furthermore, the rapid
restoration of the blood flow after reperfusion increases the
level of oxygen delivery to the tissue, leading to a new wave
of ROS production and thus further tissue damage [22].
Inflammation is also one of the main pathological mecha-
nisms of acute ischemic stroke and may cause OS [23]. Vari-
ous cytokines such as tumor necrosis factor (TNF), IL-1, and
IL-6 are known to regulate the tissue damage in stroke models,
and therefore, play a crucial role in poststroke therapies. The
effect of these cytokines on the development and progression
of infarction in human and animal modelsdepends on various
factors, including their availability in the penumbra of the
stroke area at the onset of early symptoms [24].
Diet and lifestyle are known to play important roles in
preventing noncommunicable diseases. High-fat diets may
cause metabolic changes, and obesity can induce chronic
inflammation. Diet-induced obesity and related metabolic
disorders such as hyperlipidemia are also considered risk
factors for cardiovascular diseases and stroke [25].
A Mediterranean diet, which has widely known to be
beneficial on health, is a diet that is characterized by the
presence of useful bioactive compounds such as monounsat-
urated fatty acids (MUFAs) and polyunsaturated fatty acids
or polyphenols [26].
OEA is derived from the unsaturated fatty acid oleic
acid, which is part of MUFA. This fatty acid is mostly found
5Oxidative Medicine and Cellular Longevity

Table 2: Laboratory characteristics of the study participants.
Lab parameter Before or after
receiving the capsule
Mean ±SD Pvalue
Placebo (n=20) OEA 300 mg/day (n=20) OEA 600 mg/day (n=20)
Ur
Before 43:16 ± 10:98 52:25 ± 12:65 41:8±12:63
0.796
After 38:5±12:34 42:25 ± 8:22 36:6±14:04
Pvalue 0.56 0.046 0.168
Cr
Before 1:12 ± 0:32 1:34 ± 0:15 1:06 ± 0:17
0.466
After 1:05 ± 0:23 1:17 ± 0:24 0:98 ± 0:13
Pvalue 0.353 0.030 0.210
TG
Before 109:5±37:22 116:12 ± 44:87 108 ± 60:81
0.964
After 77:75 ± 7:22 88:37 ± 27:72 74:5±20:5
Pvalue 0.126 0.044 0.449
Chl
Before 149:5±41:58 163:4±37:62 156:5±30:55
0.659
After 135:25 ± 47:61 136:8±28:60 140:9±30:96
Pvalue 0.217 0.027 0.138
HDL
Before 47:85 ± 28:32 33:25 ± 6:38 35 ± 3:69
0.317
After 47:42 ± 30:70 37:83 ± 5:89 39 ± 7:89
Pvalue 0.834 0.035 0.288
ALT
Before 16:71 ± 6:87 19:33 ± 4:50 15 ± 3:31
0.170
After 18:57 ± 7:67 18:22 ±4:65 14:2±1:92
Pvalue 0.374 0.007 0.374
AST
Before 13:5±2:51 19:66 ± 2:65 19:57 ± 7:16
0.424
After 16 ± 8:28 19 ± 2:75 19:14 ± 6:98
Pvalue 0.591 0.235 0.078
IL-6
Before 2:42 ± 1:32 3:24 ± 1:81 3:20 ± 1:57
0.035
After 3:18 ± 1:54 2:13 ± 0:59 3:26 ± 1:19
Pvalue 0.216 0.040 0.888
CRP
Before 4:25 ± 3:77 4:98 ± 3:16 4:17 ± 2:20
0.235
After 4:59 ± 4:12 3:04 ± 1:54 4:44 ± 1:28
Pvalue 0.831 0.049 0.743
SOD
Before 0:24 ± 0:011 0:251 ± 0:013 0:25 ± 0:01
0.369
After 0:25 ± 0:018 0:253 ± 0:023 0:24 ± 0:02
Pvalue 0.187 0.792 0.435
TAC
Before 0:59 ± 0:08 0:63 ± 0:082 0:70 ± 0:08
0.464
After 0:61 ± 0:12 0:71 ± 0:099 0:73 ± 0:14
Pvalue 0.637 0.020 0.478
MDA
Before 0:089 ± 0:011 0:106 ± 0:021 0:097 ± 0:026
0.054
After 0:090 ± 0:017 0:091 ± 0:018 0:080 ± 0:016
Pvalue 0.820 0.026 0.007
TTG
Before 0:140 ± 0:017 0:151 ± 0:016 0:155 ± 0:017
0.022
After 0:134 ± 0:025 0:167 ± 0:016 0:151 ± 0:017
Pvalue 0.494 0.016 0.266
Abbreviations: Cr, Creatinine; Ur, Urea; Chl, Cholesterol; TG, Triglyceride; HDL, high-density lipoprotein; AST, aspartate transaminase; ALT, alanine
transaminase; CRP, C-reactive protein; IL-6, interlukin-6; TAC, total antioxidant capacity; TTG, total thiol groups; MDA, malondialdehyde; SOD,
superoxide dismutase.
6 Oxidative Medicine and Cellular Longevity

in olives and sesame [10, 11]. In an experimental study on
the effect of high MUFA diet on cerebral ischemia, an
improvement was observed in the neurological and motor
function in acute ischemic stroke mice models receiving
olive oil fatty acid supplements compared to placebo [3].
Targeting CBR2 has several effects on ROS-induced neu-
roinflammation, as it can reduce ROS/reactive nitrogen spe-
cies (RNS) production in active glial cells, reduce vascular
inflammation, improve blood-brain barrier (BBB) function,
and inhibit leukocyte cell uptake and thus reduce nerve cell
death [13].
The results of the present study confirmed the effect of
OEA supplementations on the biomarkers of OS, inflamma-
tory parameters (IL-6, CRP, and lipid profile), and renal and
hepatic parameters in patients with acute ischemic stroke.
Our investigation indicated that OEA has remarkable
effects on OS parameters, which can reduce OS and increase
anti-OS marker in the patients with acute ischemic stroke. In
a neuron-like SH-SY5Y cell line, Giusti et al. observed that
10μM oleocanthal, as a phenolic component of extra virgin
olive oil (EVOO), neutralizes OS which was induced by
H
2
O
2
, and leads to increased cell viability, decreased produc-
tion of ROS, and an increased intracellular glutathione
(GSH) level [27]. Furthermore, Tasset et al. demonstrated that
EVOO, which represents 10% of calorie intake in the total
standard daily diet of rats, reduces oxidative damage in Hun-
tington’s disease-like rat model induced by 3-nitropropionic
acid (3NP) [28]. They further found that in all studied sam-
ples, 3NP increased lipid peroxides but decreased GSH levels
[28]. However, their results revealed that EVOO reduced the
level of LPO and blocked the GSH deficiency caused by 3NP
in the striatum and other parts of the brain of Wistar rats
[28]. In our study,TAC, TTG, and MDA levels had significant
changes, confirming the antioxidant effects of OEA.
Previous studies have shown that mitochondrial CBR1
expression plays a unique role in cannabinoid-driven neuro-
protection and might directly regulate mitochondrial ROS
formation under this pathological condition [13]. Moreover,
targeting CBR2 has several effects on ROS-induced neuroin-
flammation, as it can reduce ROS/RNS production in active
glial cells, reduce vascular inflammation, improve blood-
brain barrier function, inhibit leukocyte cell uptake, and
consequently, decrease nerve cell death [13]. Therefore, the
antioxidative effect of OEA in our study might be due to
the activation of both CBR1 and CBR2.
Although the neuroprotective effects of OEA after acute
cerebral ischemic injury in the experimental model have
been reported, to the best of our knowledge, there are no
clinical trials on humans to prove this concept [29]. It has
been indicated that OEA (40 mg/kg, intraperitoneally (ip))
attenuates apoptosis by inhibiting the Toll-like receptor
(TLR4)/NF-κB and ERK1/2 signaling pathways in mice
model of acute ischemic stroke [29]. N15, an analogue of
OEA, has the ability to protect the brain against ischemic
injury. Li et al. assessed both neuropreventive (50, 100, or
200 mg/kg, ip) and neurotherapeutic effects of N15
(200 mg/kg, ip) in mice model of acute ischemic stroke and
also lipopolysaccharide- (LPS-) stimulated BV-2 microglial
cells [30]. Furthermore, the anti-inflammatory properties
of N15 may contribute to its neuroprotective effects on cere-
bral ischemia, at least in part, by boosting PPAR/dual signal-
ing and suppressing the activation of the NF-κB, STAT3,
and ERK1/2 signaling pathways [30]. These data imply that
OEA might be a promising therapy option for ischemic
stroke prevention and treatment.
The evaluation of inflammatory parameters revealed that
in the OEA 300 mg/day group, the IL-6 and CRP levels were
significantly different before and after OEA administration.
Contrarily, these levels were not significantly different in
other groups before and after OEA administration.
Between-group analysis showed that IL-6 in patients receiv-
ing OEA 300 mg/day was statistically lower than its level in
patients who received placebo. A controlled clinical trial
evaluated the anti-inflammatory and antioxidative effects
of OEA (250 mg/day) on obesity, as well as assessing LPO,
TAC, CRP, IL-6, and TNF-αlevels. Based on the results,
IL-6 and TNF-αconcentrations were significantly reduced
in the intervention group, but other changes were not signif-
icantly different [31]. Sayd et al. demonstrated that systemic
administration of OEA (10 mg/kg, ip) could decrease levels
of IL-1βand IL-6 as proinflammatory cytokines and also
markers of nitrosative/OS nitrites and MDA which was
induced by LPS (0.5 mg/kg, ip) in rats [32]. In another study,
Xu et al. investigated the anti-inflammatory effect of differ-
ent doses of OEA (10 μM, 20 μM, 50 μM, and 100 μM) and
concluded that OEA reduces inflammatory cytokines (IL-6
and IL-8) and adhesion molecules on TNF-α(20 ng/ml)-
induced inflammation in human umbilical vein endothelial
cells through the activation of the CBR2 and PPAR-α[12].
Zhou et al. assessed both neuropreventive (10, 20,or 40 mg/
kg, intragavage (ig)) and neurotherapeutic effects of OEA
(40 mg/kg, ig) in a mice model of ischemic stroke and con-
cluded that orally administered OEA protects mice from
focal cerebral ischemic injury particularly BBB disruption
by activating PPAR-α[33]. This impact is tremendously
important since poststroke BBB disruption can exacerbate
ischemic damage by raising edema and inducing bleeding
[34]. During an acute ischemic stroke, cerebral edema is
the most prevalent cause of neurological impairment and
death [35]. The BBB’s integrity can help avoid brain swelling
and subsequent tissue damage. Since OEA is rapidly
depleted in vivo due to hydrolysis, its therapeutic potential
is limited. As a result, encapsulating OEA in a nanoparticu-
late structure like cubosomes, which may be utilized to target
Table 3: Frequency of drug-related adverse effects among patients
in each groups.
Number (%)
All
(n=60)
Placebo
(n=20)
OEA
300 mg/day
(n=20)
OEA
600 mg/day
(n=20)
P
value
Nausea 16 (26.6%) 4 (20%) 6 (30%) 6 (30%) 0.711
Vomiting 11 (18.3%) 3 (15%) 4 (20%) 4 (20%) 0.895
Dyspepsia 24 (40%) 6 (30%) 8 (40%) 10 (50%) 0.435
Headache 26 (43.3%) 7 (35%) 10 (50%) 9 (45%) 0.622
Dizziness 15 (25%) 5 (25%) 5 (25%) 5 (25%) 1.000
7Oxidative Medicine and Cellular Longevity

the BBB, protects it from hydrolysis and allows therapeutic
amounts to reach the brain [36]. Another study by Wu
et al. showed that the survival rate, behavioral score, cerebral
infarct volume, edema degree, spatial learning, and memory
capacity of stroke-model rats could all be improved greatly
using endogenous OEA crystals loaded lipid nanoparticles
[37]. Further studies should be done in order to find the
most effective format for OEA administration.
Likewise, Luo et al. in both in vitro and in vivo experi-
ments found that OEA (10-50 μM) inhibits glial activation
via modulating PPAR-αand promotes motor function
recovery after brain ischemia [38]. Also, SUL (3 and
10 mg/kg) treatment, a stable OEA-modeled compound, in
addition to PPAR-αantagonist, GW6471 (1 mg/kg),
improves the brain damage and accompanying motor and
cognitive impairments caused by hypoxia-ischemia in mice,
most likely through regulating alterations in neuroinflam-
mation/immune system mediators [39].
A meta-analysis demonstrated that acute kidney injury is
a common complication following acute ischemic stroke and
is associated with increased mortality following acute ische-
mic stroke [40]. Additionally, it was shown that impaired
kidney function is associated with the presence of cerebral
microbleeds in acute ischemic stroke [41], and renal dys-
function increases the risk of recurrent stroke in those
patients [42]. Thus, kidney function preservation is crucially
important in acute ischemic stroke patients. In our study,
within-group analysis revealed that blood urea and creati-
nine, as the markers of renal function, had significantly
changed in patients receiving OEA 300 mg/day, indicating
that OEA might also be used as a renal protective factor in
acute ischemic patients. Thus, OEA not only has no renal
toxicity character, but also can prevent deleterious effects
on kidneys.
In terms of the lipid profile, within-group analysis indi-
cated a statistically significant decrease in the TG and cho-
lesterol levels, while a rise in the HDL level in patients
receiving OEA 300 mg/day. In terms of hepatic function, a
statistically significant reduction was found in ALT in
patients receiving OEA 300 mg/day. It is concluded that
OEA has no hepatotoxic character, but can improve liver
function at moderate doses (300 mg/day). In an experimen-
tal study on KDS-5104, a nonhydrolyzable lipid OEA ana-
log, Thabuis et al. showed that the most significant
bioindicator of OEA activity is adipose tissue fatty-acid
translocase (FAT)/CD36 expression, which appears to be a
determining player in the OEA fat-lowering response [43].
In addition, Fu et al. demonstrated PPAR-αand other
PPAR-target genes, such as FAT/CD36, liver fatty-acid bind-
ing protein (L-FABP), and uncoupling protein-2 (UCP-2),
are activated by subchronic OEA administration (5 mg/kg/
day, ip, for two weeks) in Zucker rats [44]. Furthermore,
OEA lowers hepatocyte neutral lipid content as well as blood
cholesterol and TG levels. The findings imply that OEA
might modulate lipid metabolism [44].
Similarly, Li et al. evaluated the effect of 17 weeks of
OEA administration (5 mg/kg/day, ip) on nonalcoholic fatty
liver disease (NAFLD) in Sprague Dawley rats and found
that the treatment with OEA delayed the progression of
NAFLD by regulating plasma TG and cholesterol levels
and reducing ALT, AST, and inflammatory liver cytokines
compared with controls [45]. On the other hand, the study
of the liver and plasma tissue gene expression in these ani-
mal models showed that OEA increases lipid oxidation
through PPAR-αactivation [45]. The results of this study
also represented that treatment with OEA inhibits the
expression of genes involved in fat synthesis [45].
Higher levels of TG than HDL-C are associated with pre-
mature neuronal degradation, while lower ratios are linked
with early clinical improvements. Several studies reported
that TG/HDL-C can predict mortality and worsen clinical
outcomes after acute ischemic stroke, and thus it is a simple
and inexpensive indicator for predicting disease prognosis.
The TG/HDL-C ratio is independently associated with mor-
tality and poor prognosis in acute ischemic stroke patients
[46, 47]. As such and considering the importance of lower-
ing lipid profile, OEA might be considered in patients with
acute ischemic stroke.
In terms of preclinical use of OEA in acute ischemic
stroke treatment, an experimental study on Sprague Dawley
rats demonstrated that chronic OEA therapy (30 mg/kg/day
for 28 days) can promote neurogenesis in the hippocampus
through increasing the expression of brain-derived neuro-
trophic factor (BDNF) and PPAR-αresulting in functional
recovery of cognitive deficits and neuroprotective benefits
against cerebral ischemic insult, indicating that OEA might
be used therapeutically for cerebral ischemia [48].
In addition, by increasing collagen content and decreas-
ing necrotic core size in plaques and also by modulating
macrophage polarization both via the AMPK- PPAR-α
pathway, OEA increased atherosclerotic plaque stability in
both in vivo and in vitro experiments [49]. These data imply
preventive role of OEA in ischemic events.
In general, a wide range of studies have pointed out the
role of OS, inflammatory parameters, and lipid profile in
the pathogenesis and prognosis of acute ischemic stroke.
Accordingly, it is recommended that OEA, as a member of
the NAE family, might be used to reduce the complications
of acute ischemic stroke.
4.1. Limitations and Strengths. Despite the uniqueness of our
findings, several limitations warn against extrapolating the
findings too far including a relatively small number of
patients enrolled in this study. More studies with larger sam-
ple sizes are warranted. The second drawback of this study is
that each patient only had two samples obtained at baseline
and 72 hours posttreatment. As a result, long-term under-
standing of the biomarker changes postacute ischemic stroke
is not feasible. Third, the patients in our trial were given
OEA supplements orally. As such, no conclusion can be
withdrawn on intravenous (IV) OEA treatment effects.
Fourth, testing the effects of OEA supplementation in a
short length of time and lack of long-term follow-up is a dis-
advantage. Fifth, the effects of OEA on clinical outcome of
these patients were not assessed. Sixth, the window for treat-
ment with OEA in our trial was within 12 hours following
the onset of stroke symptoms; earlier treatment may have
different efficacy. Finally, IV thrombolytics are the only
8 Oxidative Medicine and Cellular Longevity

FDA-approved pharmacological treatment for acute ische-
mic stroke [50–60] and roughly half of our patients received
this treatment given they were eligible. Thus, this treatment
might have influenced our results.
Our study has the following advantages. It benefited
from randomization, double-blinding, and the presence of
a control group as a pilot clinical trial. Furthermore, the
changes in inflammatory markers after treatment with
OEA might be an indication of reduction in brain tissue
inflammation happening after acute ischemic stroke.
5. Conclusion
Our results indicate that OEA add-on to standard acute
ischemic stroke treatment improves the short-term inflam-
matory, OS status, and lipid and biochemical parameters in
those patients, particularly in moderate dosage, 300 mg/
day, which might lead to the better functional outcome.
Our findings need to be confirmed in larger-scale studies
with larger sample sizes and longer intervention duration
and follow-up.
Data Availability
The authors confirm that the data supporting the findings of
this study are available within the article.
Ethical Approval
All procedures performed in this study involving human
participants were in accordance with the ethical standards
and approved by the Hamadan University of Medical Sci-
ences Ethical Committee (IR.UMSHA.REC.1398.720-722)
and the 1964 Helsinki Declaration and its later amendments
or comparable ethical standards. The trial was registered in
the Iranian Registry of Clinical Trials (Identifier: IRCT2013
0501013194N4).
Consent
Informed consent was obtained from all individual partici-
pants and/or their proxies included in the study. Informed
consent to participate in this study was obtained from par-
ticipants included in the study. All authors give their consent
to the submission of this article to the Oxidative Medicine
and Cellular Longevity.
Conflicts of Interest
All authors certify that they have no affiliations with or
involvement in any organization or entity with any financial
interest (such as honoraria; educational grants; participation
in speakers’bureaus; membership, employment, consultan-
cies, stock ownership, or other equity interest; and expert
testimony or patent-licensing arrangements) or nonfinancial
interest (such as personal or professional relationships, affil-
iations, knowledge or beliefs) in the subject matter or mate-
rials discussed in this manuscript.
Authors’Contributions
Mohammadmahdi Sabahi, Sara Ami Ahmadi, and Azin
Kazemi contributed equally to this work.
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11Oxidative Medicine and Cellular Longevity