Journal of Functional Foods 34 (2017) 248253

Contents lists available at ScienceDirect

Journal of Functional Foods

journal homepage: www.elsevier.com/locate/jff

Soy protein-derived ACE-inhibitory peptide LSW (Leu-Ser-Trp) shows

anti-inflammatory activity on vascular smooth muscle cells
Qinlu Lin a,1, Wang Liao b,1, Jie Bai a, Wei Wu a, Jianping Wu b,
a
College of Food Engineering, Central South University of Forestry and Technology, Changsha 410004, PR China
b
Department of Agricultural, Food & Nutritional Science, University of Alberta, Edmonton, AB, Canada

a r t i c l e i n f o a b s t r a c t

Article history: Soy proteins are a rich source of various bioactive peptides. LSW (Leu-Ser-Trp) was previously identified
Received 22 January 2017 from thermolysin-digested soy protein hydrolysate as a potent ACE-inhibitory peptide; however, its bio-
Received in revised form 19 March 2017 logical effects on vascular cells have not been elucidated. The present study evaluated anti-oxidant and
Accepted 24 April 2017
anti-inflammatory activities of LSW on vascular smooth muscle cells (VSMCs). Ang II promoted oxidative
Available online 4 May 2017
stress and inflammation in VSMCs; adding LSW did not show anti-oxidant activity, while COX-2, but not
iNOS, was down-regulated in Ang II-stimulated VSMCs, suggesting its anti-inflammatory activity. AT1R
Keywords:
mediates most of the pathological effects of Ang II in VSMCs; LSW down-regulated Ang II-stimulated
Soy protein
ACE-inhibitory peptide
AT1R expression, via decreased phosphorylation of both Src and ERK1/2. Furthermore, LSW could also
LSW reduce the phosphorylation of nuclear transcription factor p50, but not p65. Results of this study sug-
VSMC gested a novel function of LSW as an anti-inflammatory agent on VSMCs, which might broaden its uses
Inflammation as functional food ingredients.
2017 Elsevier Ltd. All rights reserved.

1. Introduction ACE-inhibitory peptides characterized from food proteins have

As a major health challenge, cardiovascular disease (CVD)
accounts for 30% of global deaths, which is equivalent to
17.3 million people deaths in 2008, or 23.3 million deaths
expected by 2030 (Mendis, Puska, & Norrving, 2011). Hypertension
is a well-established risk factor for CVD (Vasan et al., 2001). The
pathophysiology of hypertension is complicated, while angiotensin
II (Ang II) is the key component responsible for vascular tone and
blood pressure regulation (Atlas, 2007). Inhibition of angiotensin
converting enzyme (ACE), leading to reduced formation of Ang II,
is a common strategy to develop antihypertensive agents (Lvy,
2004). A number of ACE inhibitory drugs have been successfully
applied as the first-line of antihypertensive drugs but are associ-
ated with various side effects (Atkinson & Robertson, 1979). Hence,
Abbreviations: CVD, cardiovascular disease; ACE, angiotensin converting
enzyme; Ang II, angiotensin II; LSW, Leu-Ser-Trp; VSMC, vascular smooth muscle
cell; IRW, Ile-Arg-Trp; COX-2, cyclooxygenase 2; iNOS, inducible nitric oxide
synthase; AT1R, angiotensin type I receptor; ERK1/2, extracellular signal regulated
kinases 1 and 2; ROS, reactive oxygen species; DHE, dihydroethidium; IQW, Ile-Gln-
Trp.
Corresponding author at: 4-10 Ag/For Centre, Department of Agricultural, Food
& Nutritional Science, University of Alberta, Edmonton, AB T6G 2P5, Canada.
E-mail address: jwu3@ualberta.ca (J. Wu).
1
Contributed equally to this work.
been extensively explored as alternative to blood pressure reduc-
tion (Majumder & Wu, 2015; Martinez-Maqueda, Miralles, Recio,
& Hernandez-Ledesma, 2012).
Soybean is an important economic plant in the world and it is
consumed in Asia for thousands of years as a traditional food. Soy-
bean is an inexpensive and abundant protein source. During the
past decades, there is a growing interest in developing functional
values of soy protein for health benefit purposes. Antihypertensive
peptides have also been characterized from soy proteins (Chiang,
Tsou, Tsai, & Tsai, 2006; Kodera & Nio, 2006; Wu & Ding, 2001)
or fermented soybean products (Rho, Lee, Chung, Kim, & Lee,
2009; Shin et al., 2001). Recently our lab has characterized a new
peptide LSW (Leu-Ser-Trp) with an IC50 value of 2.7 mM, from soy
protein by using LC-MS/MS coupled with quantitative structure
and activity relationship (QSAR) modeling approach (Gu & Wu,
2013).
Ang II is a multi-functional peptide in blood vessels. Apart from
vasoconstrictive effect, Ang II can also activate inflammatory cas-
cade in vasculature, inducing vascular inflammation and develop-
ing atherosclerosis (Dandona, Dhindsa, Ghanim, & Chaudhuri,
2006). Hence, reduced generation of Ang II via inhibiting ACE is
considered as a strategy to reduce Ang II-associated inflammation.
ACE-inhibitory compounds were reported to show anti-oxidant
and anti-inflammatory effects in vasculature (Agha & Mansour,

http://dx.doi.org/10.1016/j.jff.2017.04.029
1756-4646/ 2017 Elsevier Ltd. All rights reserved.
 Q. Lin et al. / Journal of Functional Foods 34 (2017) 248253
2000; Evangelista & Manzini, 2005). We recently reported that IRW
(Ile-Arg-Trp), an egg white ovotransferrin-derived ACE-inhibitory
peptide, exerted anti-proliferative, anti-oxidant and anti-
inflammatory effects on vascular smooth muscle cells (VSMCs)
against Ang II stimulation (Liao, Chakrabarti, Davidge, & Wu,
2016), suggesting possible new functions of food protein-derived
ACE-inhibitory peptides in retarding vascular lesion during devel-
opment of hypertension. Given its strong ACE-inhibitory activity,
the present study aims to investigate the anti-oxidant and anti-
inflammatory activities, as well as relevant cell signaling pathways
of soy protein derived peptide LSW on VSMCs.
2. Materials and methods
2.1. Reagents
Dulbeccos phosphate buffered saline (PBS) and dithiothreitol
(DTT) were purchased from Sigma Aldrich (St Louis, MO, USA). Dul-
beccos modified Eagle medium (DMEM) and fetal bovine serum
(FBS) were bought from Gibco/ Invitrogen (Carlsbad, CA, USA).
The antibiotics used in cell culture (Penicillin-Streptomycin and
Gentamicin) were from Life Technologies (Carlsbad, CA, USA).
Triton-X-100 was from VWR International (West Chester, PA,
USA). Ang II was purchased from Sigma Aldrich. The tripeptide
IRW was synthesized by Genscript (Piscataway, NJ, USA with a pur-
ity of 95% (validated by HPLC-MS/MS). The peptide was dissolved
in PBS at a stock concentration of 10 mM, aliquoted and stored at
20 C until use. In western blot, primary antibodies of cyclooxy-
genase 2 (COX-2), inducible nitric oxide synthase (iNOS) and a-
tubulin were purchased from Abcam (Cambridge, MA, USA). Pri-
mary antibodies of angiotensin type I receptor (AT1R), Src, p50,
phospho-p50 (p-p50) and p65 were from Santa Cruz Biotechnology
(Dallas, TX, USA). Primary antibodies of phospho-Src (p-Src), extra-
cellular signal regulated kinases 1 and 2 (ERK1/2), phospho-ERK1/2
(p-ERK1/2) and phospho-p65 (p-p65) were from Cell Signaling
(Danvers, MA, USA).
2.2. Cell culture
VSMCs used in the present study were from a commercially
available A7r5 vascular smooth muscle cell-line derived from rat
aorta (ATCC, CRL-1444, Manassas, VA, USA). Cells between passage
4 and 12 were grown in the growth medium (DMEM + 10% FBS
+ antibiotics) until they reached 80% of confluence. For each exper-
iment, the growth medium was replaced by the quiescing medium
(DMEM + 1% FBS + antibiotics). Afterwards, cells were treated with
50 mM of LSW 1 h prior to the addition of 1 mM of Ang II for differ-
ent time periods in different experiments. Concentrations of both
the peptide and Ang II were based on our previous study (Liao
et al., 2016).
2.3. Oxidative fluorescence assay
The reaction between intracellular reactive oxygen species
(ROS) and dihydroethidium (DHE) could yield ethidium, a com-
pound binding to nuclear DNA and release nuclear fluorescence
(Peshavariya, Dusting, & Selemidis, 2007). DHE staining has been
used as a cell-based assay in a number of studies evaluating the
anti-oxidant potential of bioactive peptides (Huang et al., 2010;
Jahandideh, Chakrabarti, Davidge, & Wu, 2016; Liao et al., 2016;
Majumder, Chakrabarti, Davidge, & Wu, 2013). A7r5 cells in the
quiescing medium were treated with 50 mM of LSW for 1 h, fol-
lowed by the addition of 1 mM of Ang II. The co-treatment of LSW
and Ang II was 30 min. Afterwards, 20 mM of DHE was added, fol-
lowed by incubation in dark for 30 min. Then, the cells were
249
washed 3 times and imaged under Olympus IX81 fluorescent
microscope (Carson Scientific Imaging Group). For each data point,
3 pictures were randomly taken for measuring mean fluorescence
intensity (MFI) by ImageJ software (http://imagej.net/Welcome).
MFI/cell was calculated based on the cell number in each field.
Results were presented as% of the untreated group.
2.4. Western blot
At the end of each experiment, cell lysates were obtained by
adding boiling Laemmles buffer (50 mM DTT + 0.2% Triton-X-
100) for western blot. The total protein from cells was then sepa-
rated by 9% sodium dodecyl sulfate polyacrylamide gel elec-
trophoresis (SDS-PAGE), transferred to nitrocellulose membranes
and immunoblotted with specific primary antibodies and sec-
ondary antibody (Goat anti-rabbit IRDye 680RD or Donkey anti-
mouse 800CW from Licor Biosciences, Lincoln, NE, USA). Protein
bands were detected by Licor Odyssey BioImager (Licor Bio-
sciences) and quantified by Image Studio Lite 5.2 (Licor Bio-
sciences). Protein bands of COX-2, iNOS and AT1R were
normalized to the loading control a-tubulin. In the signaling path-
way study, protein bands of phospho-Src, phospho-ERK1/2,
phospho-p50 and phospho-p65 were normalized to the corre-
sponding total form. Cell lysates from untreated cells were loaded
on every gel and all data were expressed as% of the corresponding
untreated.
2.5. Statistical analysis
All data are presented as mean SEM (standard error of mean)
from 4 to 7 independent experiments. Data analysis were per-
formed by one way analysis of variance (ANOVA) coupled with
Tukeys post hoc test using the PRISM 5 statistical software (Graph
Pad Software, San Diego, CA). P < 0.05 was considered to be
significant.
3. Results
3.1. LSW did not show antioxidant activity in VSMCs upon Ang II
stimulation
As expected, Ang II increased superoxide production in VSMCs.
Despite the slight decrease in ROS production with LSW treatment,
the antioxidant activity of this peptide is mild. Besides, LSW had no
effect on the basal superoxide level in VSMCs (Fig. 1). The above
results indicated the non-significant antioxidant activity of LSW
on VSMCs.
3.2. LSW down-regulated the expression of COX-2 but not iNOS in Ang
II-stimulated VSMCs
In addition to increased oxidative stress, inflammation is
another cellular events in VSMCs in response to Ang II stimulation.
We next examined the effect of LSW on inflammatory response in
VSMCs, in which, COX-2 and iNOS were selected as the biomarkers
(Liao et al., 2016). Although a significant effect of LSW was only
observed on COX-2 but not iNOS (Fig. 2), down-regulation of
COX-2 by the peptide treatment indicated the role of LSW in reduc-
ing inflammation by Ang II in VSMCs.
3.3. LSW down-regulated Ang II-stimulated AT1R expression in VSMCs
AT1R is responsible for Ang II-stimulated inflammation in
VSMCs. We then examined the effect of LSW on expression of
AT1R in VSMCs. Increased expression of AT1R was observed after
250 Q. Lin et al. / Journal of Functional Foods 34 (2017) 248253
Fig. 1. LSW did not show antioxidant activity in Ang II-stimulated VSMCs A7r5.
cells were pre-treated with 50 mM of LSW for 1 h prior to 30 min stimulation with
1 mM of Ang II. Cells were treated with 20 mM of DHE for 30 min, then visualized by
fluorescent microscopy. MFI per cell was then calculated and the data were
expressed as% of the untreated from 4 independent experiments. Mean SEM are
shown. * Indicates p < 0.05 as compared to the untreated group. NS indicates not
significant (p > 0.05) as compared to the Ang II-stimulated group.
Ang II stimulation, but was abolished by the peptide treatment
(Fig. 3). Down-regulation of AT1R by LSW indicated AT1R, at least
partially, was involved in the anti-inflammatory role of LSW to
reduce the expression of COX-2 against Ang II stimulation.
3.4. LSW reduced the phosphorylation of the Src family of protein
tyrosine kinases in Ang II-stimulated VSMCs
In order to further elucidate the possible mechanisms underly-
ing anti-inflammation activity of LSW in VSMCs, its possible asso-
Fig. 2. LSW down-regulated the expression of COX-2 but not iNOS in Ang II-
stimulated VSMCs. A7r5 cells were pre-treated with 50 mM of LRW for 1 h prior to
23 h stimulation with 1 mM of Ang II. Total protein from cells were extracted and
immunoblotted for COX-2 (A) or iNOS (B) and a-tubulin (loading control). Protein
bands were quantified by densitometry and normalized to their respective loading
controls. Data are expressed as mean SEM of 4 and 7 independent experiments in
A and B, respectively. * and *** indicate p < 0.05 and p < 0.001, respectively, as
compared to the untreated group. # Indicates p < 0.05 as compared to the Ang II-
stimulated group. NS indicates not significant (p > 0.05) as compared to the Ang II-
stimulated group.
ciated intracellular signaling was investigated. Phosphorylation of
tyrosine kinase proteins is one of the major upstream intracellular
signal transduction events regulated by AT1R. In VSMCs, the Src
family kinases are likely the candidates of tyrosine kinases to
mediate AT1R signaling events (Touyz & Schiffrin, 2000). Down-
regulation of phosphorylation ratio of Src by LSW (Fig. 4) indicated
the potential of this peptide in inhibiting phosphorylation of tyro-
sine kinase.
3.5. LSW inhibited phosphorylation of ERK1/2 in Ang II-stimulated
VSMCs
ERK1/2 is a key intermediate kinase signaling connecting the
upstream tyrosine kinase signaling and down-stream effectors
 Q. Lin et al. / Journal of Functional Foods 34 (2017) 248253
Fig. 3. LSW down-regulated AT1R expression in Ang II-stimulated VSMCs. A7r5
cells were pre-treated with 50 mM of LRW for 1 h prior to 23 h stimulation with
1 mM of Ang II. Cells were lysed and immunoblotted for AT1R and the loading
control a-tubulin. Protein bands were quantified by densitometry and normalized
to their respective loading controls. Data are expressed as mean SEM of 4
independent experiments. *** Indicates p < 0.001, as compared to the untreated
group. #indicates p < 0.05 as compared to the Ang II-stimulated group.
Fig. 4. LSW reduced the phosphorylation of Src in Ang II-stimulated VSMCs. A7r5
cells were pre-treated with 50 mM of IRW for 1 h prior to 1 min stimulation with
1 mM of Ang II. Cells were lysed and immunoblotted for phospho-Src and total Src.
Phosphorylated protein bands of Src were quantified by densitometry and
normalized to their respective total form. Data are expressed as Mean SEM of 6
independent experiments, normalized to the untreated control. ** Indicates p < 0.01,
as compared to the untreated group. # indicates p < 0.05 as compared to the Ang II-
stimulated group.
(Touyz & Schiffrin, 2000). Followed by tyrosine kinase, we then
focused on regulatory roles of LSW in ERK1/2 activation. Phospho-
rylation of ERK1/2 was augmented by Ang II stimulation, but was
reduced by LSW treatment (Fig. 5).
251
Fig. 5. LSW reduced the phosphorylation of ERK1/2 in Ang II-stimulated VSMCs.
A7r5 cells were pre-treated with 50 mM of IRW for 1 h prior to 15 min stimulation
with 1 mM of Ang II. Cells were lysed and immunoblotted for phospho-ERK1/2 and
total ERK1/2. Phosphorylated protein bands of ERK1/2 were quantified by densit-
ometry and normalized to their respective total form. Data are expressed as
Mean SEM of 6 independent experiments, normalized to the untreated control.
*
Indicates p < 0.05, as compared to the untreated group. # Indicates p < 0.05 as
compared to the Ang II-stimulated group.
3.6. LSW could exert its anti-inflammatory effect through NF-jB
signaling
As COX-2 is in the down-stream of NF-jB signaling, we further
examined LSWs effect on two transcription factors (p50 and p65)
of NF-jB signaling. Ang II stimulated phosphorylation of both tran-
scription factors. LSW treatment significantly reduced the phos-
phorylation of p50, but not p65 (Fig. 6). These results indicated
the effect of LSW on NF-jB signaling in Ang II-stimulated VSMCs,
but this peptide has differentially regulatory effects on different
transcription factors.
4. Discussions
During the past three decades, food protein-derived anti-
hypertensive peptides gained substantial interests as a promising
alternative to anti-hypertensive drugs (Martnez-Maqueda,
Miralles, Recio, & Hernndez-Ledesma, 2012). Soybean is rich in
food protein. Several anti-hypertensive peptides have been identi-
fied from soy protein hydrolysates or fermented products (Shin
et al., 2001; Wu & Ding, 2001). For these peptides, they were ini-
tially characterized as ACE-inhibitory peptides. However, results
from various mechanistic study indicated that ACE might not be
the only target for food protein-derived ACE-inhibitory peptides
(Majumder & Wu, 2015).
Ang II is known as a potent vasoconstrictor. While, it also func-
tions in other ways in controlling the diameter and peripheral
resistance of blood vessel (Touyz & Schiffrin, 2000). In VSMCs, over
stimulation of Ang II is always coupled with oxidative stress and
inflammatory responses, which would promote vascular remod-
elling and increase blood pressure ultimately (Griendling,
Minieri, Ollerenshaw, & Alexander, 1994; Touyz & Schiffrin,
2000). Previously, our group found that IRW, an ACE-inhibitory
252 Q. Lin et al. / Journal of Functional Foods 34 (2017) 248253
Fig. 6. LSW attenuated NF-jB activation in VSMCs upon Ang II stimulation through
decreasing p50 phosphorylation but not p65. A7r5 cells were pre-treated with
50 mM of IRW for 1 h prior to 15 min stimulation with 1 mM of Ang II. Cells were
lysed and immunoblotted for phospho-p50 and total p50, or phosphop-p65 and
total p65. Phosphorylated protein bands were quantified by densitometry and
normalized to their respective total form. Data are expressed as Mean SEM of 4
and 6 independent experiments in A and B, respectively, normalized to the
untreated control. * and ** Indicate p < 0.05 and p < 0.01, respectively, as compared
to the untreated group. # Indicates p < 0.05 as compared to the Ang II-stimulated
group. NS indicates not significant (p > 0.05) as compared to the Ang II-stimulated
group.
peptide derived from egg white ovotransferrin, could modulate the
effects of Ang II in VSMCs (Liao et al., 2016), indicating the poten-
tial of food protein-derived ACE-inhibitory peptides participating
in the events of VSMCs against Ang II.
In the present study, the anti-oxidant and anti-inflammatory
activities of a soybean protein-derived ACE-inhibitory peptide
LSW were evaluated in Ang II-stimulated VSMCs. Although, LSW
did not show anti-oxidant activity, COX-2 was down-regulated,
indicating its anti-inflammatory activity. In VSMCs, over produc-
tion of superoxide by Ang II stimulation may increase the expres-
sion of some proinflammatory molecules including COX-2. The
inconsistent results of anti-oxidant and anti-inflammatory tests
of LSW suggested down-regulation of COX-2 by LSW was indepen-
dent of suppression of superoxide generation. Besides, as com-
pared to IRW, an egg white-derived ACE-inhibitory peptide (Liao
et al., 2016), LSW could only down-regulate the expression of
COX-2, but not iNOS, indicating LSW may be more selective to
COX-2 in VSMCs.
Given the anti-inflammatory activity of LSW in Ang II-
stimulated VSMCs, we further explored signaling pathways regu-
lated by LSW. AT1R is a membrane G-protein-coupled receptor,
which mediates most of the pathological effects of Ang II in VSMCs
(Touyz & Schiffrin, 2000). It was found in this study that LSW could
down-regulate AT1R expression in VSMCs, suggesting its addi-
tional mechanism of action other than ACE inhibition. Along with
our recent finding that egg white hydrolysate could attenuate
hypertension in spontaneously hypertensive rats via down-
regulating AT1R expression in aorta (Jahandideh et al., 2016), these
results further supported that AT1R is another target for anti-
hypertensive agents derived from food proteins.
In VSMCs, the detrimental effects of AT1R upon Ang II stimula-
tion are activated through various kinases (Touyz & Schiffrin,
2000). Src family kinases and ERK1/2 are two most well-studied
kinases associated with the action of AT1R. As expected, Ang II
can rapidly phosphorylate Src, and then transduces the signaling
for ERK1/2 phosphorylation (Ishida, Ishida, Thomas, & Berk,
1998). Adding LSW decreased phosphorylation of both Src and
ERK1/2, supporting the regulatory roles of this peptide in these
intracellular pathways. However, whether the reduced phosphory-
lation of Src and ERK1/2 is dependent on the down-regulation of
AT1R requires further investigation. The investigation of possible
intracellular signaling kinases regulated by LSW in VSMCs deep-
ened our view on the mechanisms of this peptides.
Followed by ERK1/2 activation, NF-jB activation could directly
activate the transcription of several pro-inflammatory molecules
including COX-2 and iNOS (Dinarello, 2000). p50 and p65 are major
transcription factors of NF-jB (Viatour, Merville, Bours, & Chariot,
2005). We previously found IRW could reduce the phosphorylation
of p65 in Ang II-stimulated VSMCs (Liao et al., 2016). Interestingly,
LSW could reduce the phosphorylation of p50, but not p65. The
structure differences may affect the anti-inflammatory activity of
peptides. In a previous study, it was found IRW could inhibit
nuclear translocations of both p65 and p50 on vascular endothelial
cells, while IQW, which is only different in one amino acid residue,
could only affect the nuclear translocation of p50. Such a difference
might account for the more pronouncing anti-inflammatory effect
of IRW on vascular endothelial cells (Majumder et al., 2013).
Results from the current study suggested differentially regulatory
roles of IRW and LSW in NF-jB pathway (Liao et al., 2016) on
VSMCs, further suggesting the importance of peptide structure in
regulatory pathway of anti-inflammatory activity.
In conclusion, the current study demonstrated the anti-
inflammatory activity of soybean protein-derived ACE-inhibitory
peptide LSW in Ang II-stimulated VSMCs, which may help to mod-
ulate vascular abnormalities during hypertension development.
Furthermore, Ang II receptor, the Src family kinases, ERK1/2 and
NF-jB are involved in the anti-inflammatory action of LSW in
VSMCs against Ang II stimulation. Results of this study indicated
a novel function of LSW in addition to ACE inhibition, which might
broaden the applications of ACE inhibitory peptides as functional
food ingredients.
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