The International Journal of Biochemistry & Cell Biology 75 (2016) 1122

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The International Journal of Biochemistry

& Cell Biology
journal homepage: www.elsevier.com/locate/biocel

Inhibitory effects of omega-3 fatty acids on early brain injury after

subarachnoid hemorrhage in rats: Possible involvement of G
protein-coupled receptor 120/-arrestin2/TGF- activated kinase-1
binding protein-1 signaling pathway
Jia Yin a,b,1 , Haiying Li a,1 , Chengjie Meng a,c,1 , Dongdong Chen a , Zhouqing Chen a ,
Yibin Wang a , Zhong Wang a, , Gang Chen a,
a
Department of Neurosurgery & Brain and Nerve Research Laboratory, The First Afliated Hospital of Soochow University, 188 Shizi Street, Suzhou 215006,
China
b
Department of Neurosurgery, Taixing Peoples Hospital, 1 Chang Zheng Road, Taixing 225400, China
c
Department of Neurosurgery, Yancheng First Peoples Hospital, 16 Yue He Road, Yancheng 224000, China

a r t i c l e

i n f o

Article history:
Received 16 September 2015
Received in revised form 25 February 2016
Accepted 17 March 2016
Available online 18 March 2016
Keywords:
Apoptosis
Early brain injury
G protein-coupled receptor 120
Omega-3 fatty acids
Subarachnoid hemorrhage

a b s t r a c t
Omega-3 fatty acids have been reported to improve neuron functions during aging and in patients affected
by mild cognitive impairment, and mediate potent anti-inammatory via G protein-coupled receptor 120
(GPR120) signal pathway. Neuron dysfunction and inammatory response also contributed to the progression of subarachnoid hemorrhage (SAH)-induced early brain injury (EBI). This study was to examine
the effects of omega-3 fatty acids on SAH-induced EBI. Two weeks before SAH, 30% Omega-3 fatty acids
was administered by oral gavage at 1 g/kg body weight once every 24 h. Specic siRNA for GPR120 was
exploited. Terminal deoxynucleotidyl transferase dUTP nick end labeling, uoro-Jade B staining, and
neurobehavioral scores and brain water content test showed that omega-3 fatty acids effectively suppressed SAH-induced brain cell apoptosis and neuronal degradation, behavioral impairment, and brain
edema. Western blot, immunoprecipitation, and electrophoretic mobility shift assays results showed
that omega-3 fatty acids effectively suppressed SAH-induced elevation of inammatory factors, including
cyclooxygenase-2, monocyte chemoattractant protein-1, and inducible nitric oxide synthase. In addition,
omega-3 fatty acids could inhibit phosphorylation of transforming growth factor activated kinase-1
(TAK1), MEK4, c-Jun N-terminal kinase, and IkappaB kinase as well as activation of nuclear factor kappa B
through regulating GPR120/-arrestin2/TAK1 binding protein-1 pathway. Furthermore, siRNA-induced
GPR120 silencing blocked the protective effects of omega-3 fatty acids. Here, we show that stimulation
of GPR120 with omega-3 fatty acids pretreatment causes anti-apoptosis and anti-inammatory effects
via -arrestin2/TAK1 binding protein-1/TAK1 pathway in the brains of SAH rats. Fish omega-3 fatty acids
as part of a daily diet may reduce EBI in an experimental rat model of SAH.
2016 Elsevier Ltd. All rights reserved.

1. Introduction

Abbreviations: COX-2, cyclooxygenase-2; DAPI, 4,6-diamino-2-phenyl indole;

DHA, docosahexaenoic acid; EBI, early brain injury; EMSA, electrophoretic mobility
shift assays; EPA, eicosapentaenoic acid; FJB, Fluoro-Jade B; GAPDH, glyceraldehyde3-phosphate dehydrogenase; GPR120, G protein-coupled receptor 120; IKK, IkappaB
kinase; iNOS, inducible nitric oxide synthase; JNK, c-Jun N-terminal kinase; MCP-1,
monocyte chemoattractant protein-1; NF-B, nuclear factor kappa B; SAH, subarachnoid hemorrhage; SD, Sprague Dawley; TAK1, transforming growth factor
activated kinase-1; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end
labeling; -3, omega-3 fatty acids.
Corresponding authors.
E-mail address: nju neurosurgery@163.com (Z. Wang).
1
These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.biocel.2016.03.008
1357-2725/ 2016 Elsevier Ltd. All rights reserved.

Intracranial aneurysm rupture is the most common cause of

subarachnoid hemorrhage (SAH) (Chen et al., 2014; Fujii et al.,
2013). Although an increasing number of clinicians could diagnose
and treat aneurysms in a timely manner using surgical clipping,
endovascular embolization, and effective drug treatment, disability and mortality rates after aneurysm rupture are still high, and
the underlying mechanisms remain elusive (Sehba et al., 2012;
Tso and Macdonald, 2014). Early brain injury (EBI) occurs within
2448 h following aneurysm rupture (Sabri et al., 2013), and is
considered as one of the most import EMSA ant factors affecting the prognosis of patients with SAH (Kikkawa et al., 2015;

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J. Yin et al. / The International Journal of Biochemistry & Cell Biology 75 (2016) 1122

Song et al., 2015). Inammation responses and neuronal death are

involved in the pathological processes of acute brain injury caused
by stroke and other diseases (An et al., 2014; Ayer and Zhang, 2010;
Marbacher et al., 2014; Suzuki et al., 2010). Thus, pro-apoptosis and
pro-inammatory factors may serve as potential targets for SAH
treatment.
Omega-3 fatty acids (also titled -3 FAs), eicosapentaenoic acid
(EPA) and docosahexaenoic acid (DHA), are rich in sh oil, and
are known to exert anti-inammatory effects (Oh et al., 2010).
As the primary source of Omega-3 fatty acids, sh oil capsules
have been widely used for many years in developed countries
(Delgado-Lista et al., 2012). The omega-3 fatty acids in sh oil have
been studied in a wide variety of human disease, such as hypertriglyceridemia, heart disease, and macular degeneration (Calder,
2015). These ndings suggest the health benets of sh oil and
omega-3 fatty acids. In Japan, Mishina et al., 2013 reported that
sh oil supplements as part of a daily diet may improve the
prognosis of patients with ischemic stroke. In addition, it was
reported that oral EPA could reduce cerebral infarction and the frequency of symptomatic vasospasm and cerebral infarction caused
by aneurysmal SAH (Yoneda et al., 2014), and also could improve
clinical prognosis (Yoneda et al., 2008), yet mechanisms underlie these conditions have not been veried. Previous research
has indicated that the primary mechanism underlying the antiinammation effects of omega-3 fatty acids involves regulation of
the 2-arrestin/transforming growth factor activated kinase-1
(TAK1) binding protein-1/TAK1 pathway via its receptor, G proteincoupled receptor 120 (GPR120) (Oh et al., 2010). It has been shown
that endogenous TAK1 is constitutively associated with TAB1, and
this interaction is required for phosphorylation-dependent TAK1
activation (Blonska et al., 2005). And, GPR120 has been conrmed
as the target of sh oil (Im, 2015). However, until now, no study
reported the effects of omega-3 fatty acids in sh oil on SAHinduced EBI.
The aim of this study was to investigate the effects of omega-3
fatty acids in sh oil on SAH-induced EBI in a rat SAH model as well
as the underlying mechanisms.

2. Materials and methods

2.1. Experimental animals
All procedures were approved by the Institutional Animal Care
Committee of the Soochow University and were performed in
accordance with the guidelines of the National Institutes of Health
on the care and use of animals. Adult male Sprague Dawley (SD)
rats weighing 250300 g were provided by the Shanghai Experimental Animal Center of Chinese Academy of Sciences. All the rats
were fed with a regular laboratory rodent diet (Tianshi, Q/320500,
SHS02-2006). The diet contains 6.1% fat by weight, and 5.46% of the
total lipid content was provided by fatty acids. Specic fatty acid
contents in the diet were shown in Supplemental Table S1. Food
intake in rats was monitored throughout the study. Diet restriction was not required since the rats in all the groups were found to
have similar mean food intakes throughout the experiment. Every
effort was made to minimize the number of animals used and their
suffering.

2.2. Omega-3 fatty acids

Fish Oil (cholesterol free) containing 30% omega-3 fatty acids
was purchased from Natures Bounty Inc. Per 1000 mg the Fish
Oil contains 300 mg omega-3 fatty acids, including180 mg EPA and
120 mg DHA.

2.3. Establishing a rat model of SAH

As described in a previous study (Wang et al., 2015), we established a SAH model by injecting blood into the prechiasmatic cistern
of rats. Briey, 0.3 mL of non-heparinized autologous arterial blood
was collected and injected within 20 s into the prechiasmatic cistern via stereotactic injection under sterile conditions. After the
procedure, 5 mL of saline was injected subcutaneously into the back
to prevent excessive postoperative loss of body uids. Rats in the
sham group received only craniotomies. All rats were kept warm
until awake, provided ad libitum access to water and food. It was
observed in SAH group that inferior basal temporal lobe was always
stained by blood. The severity of SAH was evaluated by an observer
who was blind to the experimental groups, as previously described
(Fujii et al., 2014). Briey, after removing the brain from the skull,
a photograph of the base of the brain was taken and divided into
six parts. According to the presence of blood in the subarachnoid
space, each part was subscored (03), and a total score was calculated as the sum of all subscores. Operated rats received a total
score ranging from 0 (no SAH) to 18 (most severe SAH), and SAH
rats with a score of 7 or less were excluded from this study. General
physiological parameters, including blood pressure and heart rate,
were measured via the cannulated right femoral artery. The brain
tissue adjacent to or under the blood clots was taken for the analysis in this study. The areas taken for assay were shown in Fig. 1A
and B.
2.4. Animal grouping and treatment
Experiments were performed in a rat model of SAH. A total of
70 rats were used.
Experiment 1 was to evaluate the enrichment of EPA and DHA in
rat brain induced by omega-3 fatty acids pretreatment. 16 rats were
randomly divided into 4 groups (4 rats per group): control group,
placebo group, 2-week omega-3 fatty acids pretreatment group,
and 3-week omega-3 fatty acids pretreatment group. Omega-3
fatty acids pretreatment groups received oral gavage of 30% omega3 fatty acids at 1 g/kg body weight once every 24 h for indicated
time. Placebo group received oral gavage of an equal volume of
corn oil using the same procedure for 3 weeks. After the indicated
treatments, absolute omega-3 fatty acids content in rat brain for
each group was analyzed (Fig. 1C).
Experiment 2 was to evaluate the effects of omega-3 fatty acids
on SAH induced-EBI and the possible mechanisms (Fig. 1C). 60 rats
were randomly divided into 4 groups: sham group (12/12 survived), SAH group (12/16 survived), SAH + placebo group (12/18
survived), and SAH + omega-3 fatty acids group (12/14 survived). 2
weeks before the SAH, rats in the SAH + omega-3 fatty acids group
and SAH + placebo group received treatment using the same procedure shown in Experiment 1. 24 h after the SAH model established,
behavioral activity was examined in all groups. And then all the
rats were killed and samples were collected. The brain cortex of
six rats were extracted for terminal deoxynucleotidyl transferasemediated dUTP nick end labeling (TUNEL) staining, uoro-jade B
(FJB) staining to detect brain cell apoptosis and neuronal degradation, and for western blot analysis, electrophoretic mobility
shift assay (EMSA), and immunoprecipitation analysis to examine the possible mechanisms of the action of omega-3 fatty acids.
The remaining six rats were subjected to brain edema evaluation
(Fig. 1D).
Experiment 3 was to further examine the mechanism of the
action of omega-3 fatty acids and characterize the role of GPR120 in
this action (Fig. 1E). 24 rats were randomly divided into 3 groups:
SAH + omega-3 fatty acids group (6/7 survived), SAH + omega-3
fatty acids + control siRNA group (6/8 survived), and SAH + omega-3
fatty acids + siGPR120 group (6/9 survived). Similar to Experiment

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13

Fig. 1. Experimental Design. (AB) Schematic representation of the areas taken for assay. (A) Sham group. (B) Subarachnoid hemorrhage (SAH) group. (C) Experimental design
for evaluating the enrichment of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in rat brain induced by omega-3 fatty acids pretreatment. (D) Experimental
design for studying the effects of omega-3 fatty acids on SAH induced-EBI and the possible mechanisms. (E) Experimental design for studying the mechanism of the action
of omega-3 fatty acids and the role of GPR120 in this action. -3: omega-3 fatty acids.

2, rats received omega-3 fatty acids by oral gavage beginning at 2

weeks before SAH. Transfection of si RNA in rat brain was performed
48 h before SAH. After the indicated treatments, rats were killed and
underwent EBI assessment at 24 h after SAH, as mentioned above.

2.5. Lipid extraction and gas chromatography analysis

After the indicated treatments, lipid extraction from brains was
performed as described previously (Wang et al., 2014). Briey, fatty
acid compositions were detected by capillary gas chromatography

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Fig. 2. The effects of omega-3 fatty acids pretreatment on fatty acid composition, brain cell death, neuronal degradation, brain edema, neurological impairment, and the
levels of inammatory factors in brain tissues after SAH. The contents of EPA (A) and DHA (B) in the rat brain of control and omega-3 fatty acids pretreatment group. Data
are presented as mean SEM, n = 4. *p < 0.05 vs. placebo group. N.S. = No signicant differences. EPA: F (3, 12) = 9.265; DHA: F (3, 12) = 4.560. (C) Terminal deoxynucleotidyl
transferase dUTP nick end labeling (TUNEL) staining. Sections were labeled by TUNEL (green) to detect apoptotic brain cells and counterstained with DAPI (blue) to detect
nuclei. Arrows point to TUNEL-positive brain cells. Asterisks mark the outside of the brain. Scale Bar = 64 m. Percentage of TUNEL-positive brain cells was calculated. Data
are expressed as mean SEM, n = 6. **p < 0.001 vs. sham group; # p = 0.021 vs. SAH + placebo group, F (3, 20) = 124.5. (D) Fluoro-jade B (FJB) staining. Asterisks mark the
outside of the brain. Scale bar = 64 m. The number of FJB-positive brain cells was calculated. Data are expressed as mean SEM, n = 6. **p <0.001 vs. sham group; # p = 0.042
vs. SAH + placebo group, F (3, 20) = 63.32. Brain water content (E) and clinical behavior scores (F) of each group. Data are means SEM. **p < 0.01 vs sham group; # p < 0.05
vs. SAH + placebo group. Brain water content: F (3, 20) = 7.252; Clinical behavior scores: F (3, 44) = 15.99. (G) The protein levels of cyclooxygenase-2 (COX-2), monocyte
chemoattractant protein-1 (MCP-1), and inducible nitric oxide synthase (iNOS) in brain tissues determined using western blots. Statistical analysis of the protein levels was
shown. Protein levels were normalized to that of the loading control. Data are expressed as mean SEM, n = 6. *p < 0.05, **p < 0.01 vs. sham group; # p < 0.05, ## p < 0.01 vs.
SAH + placebo group. F value and degrees of freedom were as follows: COX-2, F (3, 20) = 96.82; MCP-1, F (3, 20) = 89.70; iNOS, F (3, 20) = 9.249. One-way ANOVA followed by
StudentNewmanKeuls post hoc tests were used. -3: omega-3 fatty acids. (For interpretation of the references to color in this gure legend, the reader is referred to the
web version of this article.)

using a Clarus 500 Gas Chomatograph (PerkinElmer, USA). Components were determined by comparing the peak retention times to
a 30-component methyl ester standard (Sigma-Aldrich, USA). Fatty
acid concentrations were expressed as pmol/mg of total lipid.
2.6. Neurobehavioral scores
At 24 h after SAH, all the rats in the Experiment 2 were examined
for behavioral impairment using a previously published scoring system and monitored for appetite, activity, and neurological
defects (Table 1) (Cui et al., 2015).

Table 1
Behavior and activity scores.
Category

Behavior

Score

Appetite

Finished meal
Left meal unnished
Scarcely ate
Walk and reach at least three corners of the cage
Walk with some stimulations
Almost always lying down
No decits
Unstable walk
Impossible to walk

0
1
2
0
1
2
0
1
2

Activity

Decits

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Fig. 3. The effects of omega-3 fatty acids pretreatment on GPR120 expression and GPR120/-arrestin2, -arrestin2/TAB1, and TAB1/TAK1 interactions after SAH. (A) Western blot analysis and quantication of GPR120 protein levels in brain tissues of different groups. Data are expressed as mean SEM, n = 6. (BD) GPR120/-arrestin2,
-arrestin2/TAB1, and TAB1/TAK1 interactions in brain tissues were determined using immunoprecipitation (IP). Quantitative histogram analysis was performed. Data are
expressed as mean SEM, n = 6. ANOVA followed by StudentNewmanKeuls post hoc tests were used. A, **p = 0.002 vs. sham group; ## p = 0.004 vs. SAH + placebo group. F
(3, 20) = 19.88. B, *p = 0.012 vs. SAH + placebo group. C, **p = 0.003 vs. SAH + placebo group. D, **p = 0.002 vs. SAH + placebo group. -3: omega-3 fatty acids; WB: western blot.

2.7. Brain edema

The index of brain edema was evaluated using the wet/dry
method as described previously (Cui et al., 2015). Briey, after
brain tissue was removed and collected, the samples were weighed
immediately (wet weight), dried in 100 C for 72 h, and then
weighed (dry weight). The percentage of water content was calculated as [(wet weigh t dry weight)/wet weight] 100%.

2.8. Transfection of si RNA in rat brain

Specic siRNAs against GPR120 were obtained from GenScript.
The knockdown efciency of all the three siRNAs was separately
tested by in vivo transfection and western blot (data not shown).

To improve the knockdown efciency, siRNA used in this study is a

pool of 3 different siRNA duplexes.
GPR120 siRNA sequences:
(I) Sense: 5 GCUUGGUGCUCAACCUCUU dTdT 3
Antisense: 3 dTdT CGAACCACGAGUUGGAGAA 5
(II) Sense: 5 UCUUCUACGUGAUGACCAU dTdT 3
Antisense: 3 dTdT AGAAGAUGCACUACUGGUA 5
(III) Sense: 5 GCGCUGCUGGCUUUCAUAU dTdT 3
Antisense: 3 dTdT CGCGACGACCGAAAGUAUA 5
According to the manufacturers instructions for Entranster-in
vivo RNA transfection reagent (Engreen), the transfection complex of siRNA was prepared as follows. Briey, 500 pmol GPR120
siRNA and 500 pmol control siRNA were respectively dissolved
in 5 L RNase-free water. Then, 10 L Entranster-in vivo RNA
transfection reagent was added to 5 L siRNA or 5 L control

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J. Yin et al. / The International Journal of Biochemistry & Cell Biology 75 (2016) 1122

Fig. 4. The effects of omega-3 fatty acids pretreatment on TAK1, MEK4, JNK, and IKK phosphorylation and NF-B activity after SAH. (A) TAK1, MEK4, JNK, and IKK phosphorylation levels in brain tissues of different groups were revealed by western blot analysis. (B) Data are expressed as mean SEM, n = 6. (C) NF-B DNA-binding activity was
assessed by electrophoretic mobility shift assay (EMSA) analysis. (D) Levels of NF-B DNA binding activity was quantied. Data are expressed as mean SEM, n = 6. ANOVA
followed by StudentNewmanKeuls post hoc tests were used. B, *p < 0.05 vs. sham group; # p < 0.05 vs. SAH + placebo group. F value and degrees of freedom were as follows:
p-TAK1, F (3, 20) = 183.9; p-MEK4, F (3, 20) = 56.53; p-JNK, F (3, 20) = 45.63; p-IKK: F (3, 20) = 28.01. D, **p = 0.009 vs. sham group; # p = 0.024 vs. SAH + placebo group. F (3,
20) = 13.99. -3: omega-3 fatty acids.

siRNA immediately. The solution was mixed for 15 min at room

temperature. Finally, 15 L Entranster-in vivosiRNA mixture was
injected intracerebroventricularly 48 h before SAH. Briey, after
anesthetized, rats were placed in a stereotactic frame. And then,
a burr hole was drilled into the skull 1.0 mm lateral to and 1.5 mm
posterior to bregma over the left hemisphere. The needle of 100 L
Hamilton syringe was slowly inserted through the burr hole into
the left lateral ventricle 4.0 mm below the dural surface. Reagent
was infused into the left lateral ventricle at a rate of 0.5 L/min.

low-uorescence, styrene-based mounting medium (DPX, SigmaAldrich). Microscopy of the stained tissue sections was performed
by an experienced observer blind to the experimental condition
groups.
2.11. Immunouorescence staining

Parafn sections (46 m) were dried for 1 h at 70 C, and

deparafnized in xylene and graded ethanol solutions (100%, 95%,
90%, 80%, and 70%). Then, TUNEL staining was performed according to the manufacturers instructions (Roche). The TUNEL-positive
cells were counted in the basal temporal cortex by an observer
who was blind to the experimental condition. The microscopic ten
areas were recorded and the TUNEL-positive cells were calculated
by Image-Pro Plus 6.0.

Immunouorescence staining was performed for GPR120 to

detect its protein level in brains. The sections were incubated
with primary antibody for GPR120 (Santa Cruz, 1:100 dilution)
overnight at 4 C. Secondary antibody, Alexa Fluor 488 donkey
anti-rabbit IgG antibody (Life Technologies, 1:300 dilution), was
added for 1 h at 37 C and then the samples were washed for
three times. After nal washing, sections were protected with
cover slips with anti-fading mounting medium containing 4,6diamino-2-phenyl indole (DAPI, Southern Biotech). Normal rabbit
IgG served as the negative control for immunouorescence assay
(data not shown). Sections were observed using a uorescence
microscope (OLYMPUS BX50/BX-FLA/DP70, Olympus). The relative
uorescence intensity was analyzed using ImageJ program.

2.10. FJB staining

2.12. Western blot analysis

FJB (Histo-Chem) staining served as a marker of neuronal degeneration. Brain sections were deparafnized and rehydrated as
described in TUNEL staining section. Then, the slides were incubated in 0.06% K permanganate solution (Sigma-Aldrich) for 15 min.
Slides were then rinsed in deionized water and immersed in
0.001% uoro-jade working solution for 30 min. Then they were
washed and dried in an incubator (5060 C) for 10 min. Sections
were cleared in xylene and coverslipped with a non-aqueous,

Western blot analysis was performed as described previously

(Li et al., 2014). Briey, The brain samples were mechanically
lysed in RIPA lysis buffer (Beyotime) for western blot. The protein concentrations were measured by the bicinchoninic acid (BCA)
method using enhanced BCA protein assay kit (Beyotime). Protein
samples (50 g/lane) were loaded on a 12% SDS-polyacrylamide
gel, separated, and electrophoretically transferred to a polyvinylidene diuoride membrane (Millipore), which was blocked with

2.9. TUNEL staining

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17

Fig. 5. Efciency of GPR120 siRNA transfection. (A) Immunouorescence analysis was performed with antibody for GPR120 (green). Nuclei were uorescently labeled with
DAPI (blue). Asterisks mark the outside of the brain. Scale bar = 64 m. (B) Western blot analysis and quantication of GPR120 protein levels in brain tissues of different
groups. Data are expressed as mean SEM, n = 6. ANOVA followed by StudentNewmanKeuls post hoc tests were used. B, **p < 0.001 vs. SAH + -3 + ctrsiRNA group. F (2,
15) = 27.55. -3: omega-3 fatty acids; ctr: control. (For interpretation of the references to color in this gure legend, the reader is referred to the web version of this article.)

5% bovine serum albumin (BIOSHARP) for 1 h at room temperature. The membrane was then incubated overnight at 4 C with
primary antibodies against GPR120 (Santa Cruz, 1:1000 dilution),
cyclooxygenase-2 (COX-2, Cell Signaling Technology, 1:2000 dilution), monocyte chemoattractant protein-1 (MCP-1, Abcam, 1:2000
dilution), inducible nitric oxide synthase (iNOS, Abcam, 1:2000
dilution), Phos-TAK1 (Cell Signaling Technology, 1:1500 dilution), Phos-MEK4 (Abcam, 1:1000 dilution), Phos-c-Jun N-terminal
kinase (Phos-JNK, Cell Signaling Technology, 1:1000 dilution), and
Phos-IkappaB kinase (Phos- IKK, Abcam, 1:1000 dilution). The
primary antibody against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was diluted 1:5000 and served as the loading
control. The membrane was then incubated with HRP-conjugated
secondary antibodies (Santa Cruz, 1:5000 dilution) for 2 h at room
temperature. The band signal was developed using SuperSignal
West Pico chemiluminescent substrate (Thermo Fisher Scientic).
The relative quantity of proteins was analyzed using Image J and
normalized to that of the loading control. Phosphorylation levels
were evaluated by the ratio of phosphoprotein to loading control.
2.13. Immunoprecipitation analysis
For immunoprecipitation, the brain tissue lysate was incubated
with specic antibodies against -arrestin2 (Santa Cruz, 2 g per
500 g of total protein in 1 mL lysate), TAK1 (Santa Cruz, 2 g per
500 g of total protein in 1 mL lysate) or normal mouse IgG (negative control) overnight at 4 C with rotary agitation. Protein A + G
Sepharose beads (Beyotime) were then added to each immune
complex and the lysate-bead mixture was incubated for 4 h at 4 C
with rotary agitation. The sample was then centrifuged at 1000g
for 5 min at 4 C and the supernatant was discarded. The beads
were rinsed 3 times with lysate, and 4 SDS sample buffer was
added. Finally, the samples were boiled at 100 C for 5 min and then
centrifuged at 1000g for 5 min at 4 C. The supernatant was collected, and SDS-PAGE and immunoblotting were then performed
for further protein separation and detection.
2.14. EMSA
Nuclear extracts were prepared from the cortical tissues.
EMSA was performed by a commercial kit (Gel Shift Assay
System; Promega). The nuclear factor kappa B (NF-B) oligonucleotide probe (5 -AGTTGAGGGGACTTTCCCAGGC-3 ), which was

end-labeled with -32P ATP, was provided by the Beijing Free

Biotech Corp.
2.15. Data analysis
All data are expressed as mean SEM. One-way ANOVA for
multiple comparisons and StudentNewmanKeuls post hoc test
were performed for TUNEL staining, immunouorescence staining, immunoprecipitation analysis, western blot, and EMSA assay.
p < 0.05 were considered statistically signicant.
3. Results
3.1. General observation
No signicant difference in mortality rate, body weight, mean
arterial blood pressure, heart rate, temperature, or blood gas data
was detected in any of the experimental groups. And there was no
signicant difference in SAH grade between SAH, SAH + placebo,
and SAH + omega-3 fatty acids pretreatment group (data not
shown).
3.2. Omega-3 fatty acids suppresses brain cell death, neurological
impairment and brain edema after SAH
Firstly, lipid extraction and gas chromatography analysis of fatty
acid composition showed that both 2-week and 3-week fatty acids
pretreatments resulted in signicant increases in the concentrations of EPA and DHA in rat brains (Fig. 2A and B). In addition,
it also showed that 3-week omega-3 fatty acids pretreatment did
not induced higher content of EPA and DHA in the rat brain, compared with the 2-week pretreatment group. Thus, we chose 2-week
omega-3 fatty acids pretreatment in the following study.
Secondly, to examine the effects of Omega-3 fatty acids on brain
cell apoptosis and neuronal degeneration, we performed TUNEL
and FJB staining. 24 h after SAH, the number of TUNEL and FJB positive cells in the brain tissues increased signicantly compared with
the sham group. However, compared with SAH + placebo group, the
number of TUNEL- and FJB-positive cells was signicantly reduced
in the omega-3 fatty acids pretreatment group (Fig. 2C and D).
This suggests that omega-3 fatty acids pretreatment could inhibit
SAH-induced brain cell apoptosis and neuronal degeneration.

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Fig. 6. The effect of GPR120 siRNA on GPR120/-arrestin2, -arrestin2/TAB1, and TAB1/TAK1 interactions in brain tissues of SAH rats with omega-3 fatty acids pretreatment.
A and C, -arrestin2/TAB1, and TAB1/TAK1 interactions in brain tissues were determined using immunoprecipitation (IP). B and D, Quantitative analysis. Data are expressed
as mean SEM, n = 6. ANOVA followed by StudentNewmanKeuls post hoc tests were used. B, **p = 0.003 vs. SAH + -3 + ctrsiRNA group. F (2, 12) = 60.81. D, *p = 0.033 vs.
SAH + -3 + ctrsiRNA group. F (2, 15) = 8.459. -3: omega-3 fatty acids; ctr: control.

Fig. 7. The effect of GPR120 siRNA on TAK1, MEK4, JNK, and IKK phosphorylation and NF-B activity in brain tissues of SAH rats with omega-3 fatty acids pretreatment. (A)
The effects of GPR120 siRNA on TAK1, MEK4, JNK, and IKK phosphorylation were revealed by western blot. (B) Quantitative analysis. Data are expressed as mean SEM, n = 6.
(C) NF-B activity in brain tissues of each group was revealed by electrophoretic mobility shift assays (EMSA). (D) Quantitative analysis. Data are expressed as mean SEM,
n = 6. ANOVA followed by StudentNewmanKeuls post hoc tests were used. B, **p < 0.01, *p < 0.05 vs. SAH + -3 + ctrsiRNA group. F value and degrees of freedom were as
follows: p-TAK1, F (2, 15) = 43.55; p-MEK4, F (2, 15) = 12.04; p-JNK, F (2, 15) = 5.138; p-IKK: F (2, 15) = 15.60. D, *p = 0.015 vs. SAH + -3 + ctr siRNA group. F (2, 15) = 9.746.
-3: omega-3 fatty acids; ctr: control.

Finally, brain water content was found to be signicantly higher

in brain samples of SAH group than that in rats subjected to
the sham group, while the mean brain water content was lower
in rats with SAH + omega-3 fatty acids pretreatment than in the
SAH + placebo group (Fig. 2E). And the rats after induction of SAH
showed severe neurological behavior impairment relative to the
sham group. When given omega-3 fatty acids pretreatment, the
impairment of neurological behavior was signicantly less pronounced (Fig. 2F).

3.3. Omega-3 fatty acids suppresses SAH-mediated inammatory

responses in the brain
We used western blot analysis to determine the protein levels
of COX-2, chemokine MCP-1, and cytokine iNOS in brain tissues.
The results showed that, compared with the sham group, the levels
of these proteins were all signicantly elevated in the SAH group.
However, compared with SAH + placebo group, omega-3 fatty acids
pretreatment signicantly downregulated the levels of these proteins (Fig. 2G). These results suggest that after SAH, signicant

J. Yin et al. / The International Journal of Biochemistry & Cell Biology 75 (2016) 1122

19

Fig. 8. The effect of GPR120 siRNA on brain cell death, neuronal degradation, and the levels of inammatory factors in brain tissues of SAH rats with omega-3 fatty acids
pretreatment. (A) Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining results. Sections were labeled by TUNEL (green) to detect apoptotic
brain cells and counterstained with DAPI (blue) to detect nuclei. Arrows point to TUNEL-positive brain cells. Asterisks mark the outside of the brain. Scale Bar = 64 m. (B)
Percentage of TUNEL-positive brain cells was calculated. Data are expressed as mean SEM, n = 6. (C) Fluoro-jade B (FJB) staining. Asterisks mark the outside of the brain. Scale
bar = 64 m. (D) The number of FJB-positive brain cells was calculated. Data are expressed as mean SEM, n = 6. (E) The protein levels of cyclooxygenase-2 (COX-2), monocyte
chemoattractant protein-1 (MCP-1), and inducible nitric oxide synthase (iNOS) in brain tissues determined using western blots. Statistical analysis of the protein levels was
shown. Protein levels were normalized to that of the loading control. Data are expressed as mean SEM, n = 6. One-way ANOVA followed by StudentNewmanKeuls post
hoc tests were used. B, *p = 0.017 vs. SAH + -3 + ctrsiRNA group. F (2, 15) = 39.83. D, * p = 0.041 vs. SAH + -3 + ctrsiRNA group. F (2, 15) = 35.52. E, * p < 0.05, ** p < 0.01 vs.
SAH + -3 + ctrsiRNA group. F value and degrees of freedom were as follows: COX-2, F (2, 15) = 5.707; MCP-1, F (2, 15) = 39.58; iNOS, F (2, 15) = 26.81. -3: omega-3 fatty
acids; ctr: control. (For interpretation of the references to color in this gure legend, the reader is referred to the web version of this article.)

inammatory responses occur in the brain tissue and omega-3 fatty

acids pretreatment suppresses this action.

inammatory effects via GPR120/-arrestin2/TAB1/TAK1 signaling

pathways in the brains of SAH rats.

3.4. Effects of omega-3 fatty acids on

GPR120/-arrestin2/TAB1/TAK1 signaling pathway

3.5. Omega-3 fatty acids acts in a GPR120-dependent manner

To explore further the mechanism underlying the effects of

omega-3 fatty acids, we used western blot analysis to measure the
protein level of GPR120 in brain tissues of each group. We found
that, compared with the sham group, the GPR120 protein level was
signicantly reduced in the SAH group. Furthermore, omega-3 fatty
acids signicantly inhibited the reduction in SAH-induced GPR120
protein levels (Fig. 3A). In addition, immunoprecipitation analysis
showed that compared to the SAH + placebo group, omega-3 fatty
acids pretreatment signicantly enhanced the binding between
GPR120 and -arrestin2, and between -arrestin2 and TAK1, and
signicantly inhibited binding between TAK1 and TAB1 (Fig. 3BD).
Western blot analysis also revealed that after SAH, phosphorylation levels of TAK1, MEK4, JNK, and IKK in brain tissues were
signicantly enhanced, and omega-3 fatty acids pretreatment signicantly inhibited phosphorylation of these proteins (Fig. 4A and
B). More importantly, EMSA results showed that Omega-3 fatty
acids pretreatment signicantly suppressed activation of NF-B,
a key factor in inammatory signaling pathway (Fig. 4C and D). In
summary, Omega-3 fatty acids may exert anti-apoptosis and anti-

Western blot and immunouorescence staining results showed

that after SAH + omega-3 fatty acids pretreatment, GPR120 siRNA
signicantly reduced GPR120 protein levels (Fig. 5). In addition,
GPR120 siRNA blocked omega-3 fatty acids regulation of the arrestin2/TAB1/TAK1 pathway (Fig. 6). Omega-3 fatty acids could
no longer suppress the SAH-induced phosphorylation of TAK1,
MEK4, JNK, and IKK and activation of NF-B (Fig. 7). Finally,
GPR120 silencing inhibited the ability of omega-3 fatty acids to suppress SAH-induced brain cell apoptosis, neuronal degeneration, and
increases in levels of inammatory factors including COX-2, MCP1, and iNOS (Fig. 8). These results indicate that following SAH, the
protective effects of Omega-3 fatty acids was mediated by GPR120
via -arrestin2/TAB1/TAK1 signaling pathway.
4. Discussion
The results of this and previous studies (Yoneda et al., 2008,
2014) demonstrate that omega-3 fatty acids exert protective effects
during SAH. To date, the possible mechanism of the action of
omega-3 fatty acids is not clear. Several studies conrmed that

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J. Yin et al. / The International Journal of Biochemistry & Cell Biology 75 (2016) 1122

Fig. 9. Mode pattern illustrating the possible mechanisms underlying omega-3 fatty acids-mediated protective effects on EBI after SAH. Blue colored letters and arrows indicate
the omega-3-mediated anti-apoptosis and anti-inammation effects, and black colored letters and arrows indicate the SAH-induced pro-apoptosis and pro-inammation
pathway. SAH activates TAK1 by causing the association of TAB1 with TAK1, and subsequently activates TAK1-NF-B axis. Omega-3 fatty acids pretreatment leads to the
associations of GPR120/-arrestin 2 and -arrestin 2/TAB1, which largely blocks TAK1/TAB1 association. When there is insufcient TAB1 to activate TAK1, downstream IKK
and the key transcription factor NF-B are in the resting state. In addition, omega-3 fatty acids-induced protective effects were signicantly abolished by GPR120 silencing.
Red colored letters and arrows indicate the effects of GPR120 silencing. (For interpretation of the references to color in this gure legend, the reader is referred to the web
version of this article.)

the mechanisms underlying the effects of omega-3 fatty acids

include: 1) Biochemical competition for COX-2 and Prostaglandin
E2, known mediators for inammation, 2) Activation of peroxisome
proliferator-activated receptor , which is an anti-inammatory
molecule, 3) Inhibition of Toll-like receptor 2/3/4, and 4) Synthesis
of pro-resolving metabolites of DHA and EPA (Im, 2015). However,
more recent studies have conrmed GPR120 as the target of omega3 fatty acids (Im, 2015). In this study, SAH activates TAK1 by causing
the association of TAB1 with TAK1, and subsequently activates
TAK1-NF-B axis, which has been considered as a critical regulator
of inammation (Blonska et al., 2005; Sakurai, 2012). Omega-3 fatty
acids pretreatment leads to the associations of GPR120/-arrestin
2 and -arrestin 2/TAB1, which largely blocks TAK1/TAB1 association. When there is insufcient TAB1 to activate TAK1, downstream
IKK and the key transcription factor NF-B are in the resting state.
In addition, in this study, gene silencing study using siRNA demonstrated that omega-3 fatty acids-induced protective effects were
signicantly abolished by knock-down of endogenous GPR120. On
the other hand GPR120 silencing also signicantly impairs the ability of omega-3 fatty acids to suppress TAK1-NF-B activation in

response to SAH condition. The mechanisms map is shown in Fig. 9.

We reasoned that sh oil supplements as part of a daily diet may
contribute to the benecial outcome of SAH patients. GPR120 and
TAK1-NF-B axis may serve as new targets for prevention and treatment of post-SAH EBI. To the best of our knowledge, this is the rst
study demonstrating such involvement.
Studies have shown that the components that play antiinammatory roles by activating GPR120 are omega-3 fatty acids
themselves, such as 14-DHA, resolvins, and proteins, rather than
their metabolites (Oh et al., 2010). However, DHA cannot be synthesized by the human body (Oh et al., 2010), and its content in
brain tissues, 0.33% of the wet weight of an adult brain, is higher
than in other tissues (Igarashi et al., 2010; Martinez, 1992). Omega3 fatty acids could effectively cross the bloodbrain barrier via its
receptors to enter brain tissue (Nguyen et al., 2014). Brain tissues
of other primates (Diau et al., 2005), rats (DeMar et al., 2006), and
mice (Orr et al., 2010) are also rich in DHA. In contrast, EPA content
in brain tissue is very low (Orr et al., 2010). Thus, the component
of sh oil that plays an anti-inammatory role in the brain is more
likely to be DHA.

J. Yin et al. / The International Journal of Biochemistry & Cell Biology 75 (2016) 1122

In addition, DHA can reduce COX-2 expression and

prostaglandin E2 synthesis in a concentration- and time-dependent
manner, and GPR120 knockdown blocks these effects (Li et al.,
2013). Through coupling of GPR120 and -arrestin-2, DHA inhibits
Akt/JNK phosphorylation, which subsequently inhibits TLR4 signaling and nuclear translocation of p65. This suggests that DHA
causes LPS-induced COX-2 reduction via GPR120. In the present
study using an in vivo model of SAH, we found that GPR120 was
expressed in brain cells, and its expression level was signicantly
reduced after SAH. Omega-3 fatty acids inhibited the reduction in
SAH-induced GPR120 protein levels and this signicantly affected
phosphorylation of downstream pro-inammatory cytokines
TAK1, MEK4, JNK, and IKK, as well as NF-B activation, thereby
exhibiting anti-inammatory effects. In rat brain tissues, GPR120
siRNA signicantly suppressed the anti-inammatory and antiapoptosis effects of omega-3 fatty acids. These results show that
omega-3 fatty acids plays anti-inammatory and anti-apoptosis
roles by inhibiting two pathways, IKK/NF-B and MEK4/JNK, and
that the anti-inammatory effects depend on its receptor, GPR120.
The anti-inammatory effects of omega-3 fatty acids are timeand dose-dependent (Li et al., 2013). In the current study, we utilized the pretreatment time and dose that are typically used for
enteral administration (Ji et al., 2012). The present study had the following limitations. Firstly, we used healthy adult rats, which did not
maximally mimic human high-risk populations, such as the elderly,
women, and patients with cardiovascular diseases (Lanzino et al.,
1999). And, the oral route should be the most appropriate means of
administration for preventive treatment. However, omega-3 fatty
acids are metabolized quickly in the gastrointestinal tract. Thus,
methods of administration that promote transport of omega-3 fatty
acids to brain tissues should be investigated further.
In summary, we report that, through the GPR120/arrestin2/TAB1/TAK1 anti-inammatory signaling pathway,
omega-3 fatty acids pretreatment could inhibit the downstream
IKK-/NF-B and MEK4/JNK pathways, thereby inhibiting SAHmediated apoptosis and inammatory responses. In addition,
GPR120 siRNA suppresses the anti-inammatory effects of omega3 fatty acids, which demonstrates that GPR120 is a key factor in
the anti-apoptosis and anti-inammatory mechanisms after SAH.
For people with a high risk of aneurysm, sh oil supplements as
part of a daily diet may contribute to an improvement clinical
outcomes, in case the aneurysm rupture.
Acknowledgements
This work was supported by Suzhou Key Medical Center
(Szzx201501), grants from the National Natural Science Foundation of China (No. 81571115, 81422013, and 81471196), Scientic
Department of Jiangsu Province (No. BL2014045), Suzhou Government (No. LCZX201301, SZS201413, and SYS201332), and A Project
Funded by the Priority Academic Program Development of Jiangsu
Higher Education Institutions.
Appendix A. Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.biocel.2016.03.
008.
References
An, C., Shi, Y., Li, P., Hu, X., Gan, Y., Stetler, R.A., et al., 2014. Molecular dialogs
between the ischemic brain and the peripheral immune system: dualistic roles
in injury and repair. Prog. Neurobiol. 115, 624.
Ayer, R., Zhang, J., 2010. Connecting the early brain injury of aneurysmal
subarachnoid hemorrhage to clinical practice. Turk. Neurosurg. 20, 159166.

21

Blonska, M., Shambharkar, P.B., Kobayashi, M., Zhang, D., Sakurai, H., Su, B., et al.,
2005. TAK1 is recruited to the tumor necrosis factor-alpha (TNF-alpha)
receptor 1 complex in a receptor-interacting protein (RIP)-dependent manner
and cooperates with MEKK3 leading to NF-kappaB activation. J. Biol. Chem.
280, 4305643063.
Calder, P.C., 2015. Functional roles of fatty acids and their effects on human health.
JPEN J. Parenteral Enter. Nutr. (in press).
Chen, S., Feng, H., Sherchan, P., Klebe, D., Zhao, G., Sun, X., et al., 2014.
Controversies and evolving new mechanisms in subarachnoid hemorrhage.
Prog. Neurobiol. 115, 6491.
Cui, Y., Duan, X., Li, H., Dang, B., Yin, J., Wang, Y., et al., 2015. Hydrogen sulde
ameliorates early brain injury following subarachnoid hemorrhage in rats.
Mol. Neurobiol. (in press).
DeMar Jr., J.C., Lee, H.J., Ma, K., Chang, L., Bell, J.M., Rapoport, S.I., et al., 2006. Brain
elongation of linoleic acid is a negligible source of the arachidonate in brain
phospholipids of adult rats. Biochim. Biophys. Acta 1761, 10501059.
Delgado-Lista, J., Perez-Martinez, P., Lopez-Miranda, J., Perez-Jimenez, F., 2012.
Long chain omega-3 fatty acids and cardiovascular disease: a systematic
review. British J. Nutr. 107 (Suppl. 2), S201S203.
Diau, G.Y., Hsieh, A.T., Sarkadi-Nagy, E.A., Wijendran, V., Nathanielsz, P.W., Brenna,
J.T., 2005. The inuence of long chain polyunsaturate supplementation on
docosahexaenoic acid and arachidonic acid in baboon neonate central nervous
system. BMC Med. 3, 11.
Fujii, M., Yan, J., Rolland, W.B., Soejima, Y., Caner, B., Zhang, J.H., 2013. Early brain
injury, an evolving frontier in subarachnoid hemorrhage research. Transl.
Stroke Res. 4, 432446.
Fujii, M., Sherchan, P., Krafft, P.R., Rolland, W.B., Soejima, Y., Zhang, J.H., 2014.
Cannabinoid type 2 receptor stimulation attenuates brain edema by reducing
cerebral leukocyte inltration following subarachnoid hemorrhage in rats. J.
Neurol. Sci. 342, 101106.
Igarashi, M., Ma, K., Gao, F., Kim, H.W., Greenstein, D., Rapoport, S.I., et al., 2010.
Brain lipid concentrations in bipolar disorder. J. Psychiatr. Res. 44, 177182.
Im, D.S., 2015. Functions of omega-3 fatty acids and FFA4 (GPR120) in
macrophages. Eur. J. Pharmacol.
Ji, A., Diao, H., Wang, X., Yang, R., Zhang, J., Luo, W., et al., 2012. n-3
polyunsaturated fatty acids inhibit lipopolysaccharide-induced microglial
activation and dopaminergic injury in rats. Neurotoxicology 33, 780788.
Kikkawa, Y., Kurogi, R., Sasaki, T., 2015. The single and double blood injection
rabbit subarachnoid hemorrhage model. Transl. Stroke Res. 6, 8897.
Lanzino, G., Kassell, N.F., Dorsch, N.W., Pasqualin, A., Brandt, L., Schmiedek, P., et al.,
1999. Double-blind, randomized, vehicle-controlled study of high-dose
tirilazad mesylate in women with aneurysmal subarachnoid hemorrhage Part
I. A cooperative study in Europe, Australia, New Zealand, and South Africa. J.
Neurosurg. 90, 10111017.
Li, X., Yu, Y., Funk, C.D., 2013. Cyclooxygenase-2 induction in macrophages is
modulated by docosahexaenoic acid via interactions with free fatty acid
receptor 4 (FFA4). FASEB J. 27, 49874997.
Li, H., Gao, A., Feng, D., Wang, Y., Zhang, L., Cui, Y., et al., 2014. Evaluation of the
protective potential of brain microvascular endothelial cell autophagy on
blood-brain barrier integrity during experimental cerebral
ischemia-reperfusion injury. Transl. Stroke Res. 5, 618626.
Marbacher, S., Nevzati, E., Croci, D., Erhardt, S., Muroi, C., Jakob, S.M., et al., 2014.
The rabbit shunt model of subarachnoid haemorrhage. Transl. Stroke Res. 5,
669680.
Martinez, M., 1992. Abnormal proles of polyunsaturated fatty acids in the brain,
liver, kidney and retina of patients with peroxisomal disorders. Brain Res. 583,
171182.
Mishina, M., Kim, K., Kominami, S., Mizunari, T., Kobayashi, S., Katayama, Y., 2013.
Impact of polyunsaturated fatty acid consumption prior to ischemic stroke.
Acta Neurol. Scand. 127, 181185.
Nguyen, L.N., Ma, D., Shui, G., Wong, P., Cazenave-Gassiot, A., Zhang, X., et al., 2014.
Mfsd2a is a transporter for the essential omega-3 fatty acid docosahexaenoic
acid. Nature 509, 503506.
Oh, D.Y., Talukdar, S., Bae, E.J., Imamura, T., Morinaga, H., Fan, W., et al., 2010.
GPR120 is an omega-3 fatty acid receptor mediating potent anti-inammatory
and insulin-sensitizing effects. Cell 142, 687698.
Orr, S.K., Tong, J.Y., Kang, J.X., Ma, D.W., Bazinet, R.P., 2010. The fat-1 mouse has
brain docosahexaenoic acid levels achievable through sh oil feeding.
Neurochem. Res. 35, 811819.
Sabri, M., Lass, E., Macdonald, R.L., 2013. Early brain injury: a common mechanism
in subarachnoid hemorrhage and global cerebral ischemia. Stroke Res. Treat.
2013, 394036.
Sakurai, H., 2012. Targeting of TAK1 in inammatory disorders and cancer. Trends
Pharmacol. Sci. 33, 522530.
Sehba, F.A., Hou, J., Pluta, R.M., Zhang, J.H., 2012. The importance of early brain
injury after subarachnoid hemorrhage. Prog. Neurobiol. 97, 1437.
Song, J., Li, P., Chaudhary, N., Gemmete, J.J., Thompson, B.G., Xi, G., et al., 2015.
Correlating cerebral (18)FDG PET-CT patterns with histological analysis during
early brain injury in a rat subarachnoid hemorrhage model. Transl. Stroke Res.
6, 290295.
Suzuki, H., Ayer, R., Sugawara, T., Chen, W., Sozen, T., Hasegawa, Y., et al., 2010.
Protective effects of recombinant osteopontin on early brain injury after
subarachnoid hemorrhage in rats. Crit. Care Med. 38, 612618.
Tso, M.K., Macdonald, R.L., 2014. Subarachnoid hemorrhage: a review of
experimental studies on the microcirculation and the neurovascular unit.
Transl. Stroke Res. 5, 174189.

22

J. Yin et al. / The International Journal of Biochemistry & Cell Biology 75 (2016) 1122

Wang, J., Shi, Y., Zhang, L., Zhang, F., Hu, X., Zhang, W., et al., 2014. Omega-3
polyunsaturated fatty acids enhance cerebral angiogenesis and provide
long-term protection after stroke. Neurobiol. Dis. 68, 91103.
Wang, Y., Gao, A., Xu, X., Dang, B., You, W., Li, H., et al., 2015. The neuroprotection
of lysosomotropic agents in experimental subarachnoid hemorrhage probably
involving the apoptosis pathway triggering by cathepsins via chelating
intralysosomal iron. Mol. Neurobiol. 52, 6477.

Yoneda, H., Shirao, S., Kurokawa, T., Fujisawa, H., Kato, S., Suzuki, M., 2008. Does
eicosapentaenoic acid (EPA) inhibit cerebral vasospasm in patients after
aneurysmal subarachnoid hemorrhage. Acta Neurol. Scand. 118, 5459.
Yoneda, H., Shirao, S., Nakagawara, J., Ogasawara, K., Tominaga, T., Suzuki, M.,
2014. A prospective, multicenter, randomized study of the efcacy of
eicosapentaenoic acid for cerebral vasospasm: the EVAS study. World
Neurosurg. 81, 309315.