Anti-inflammatory effect of pyroglutamyl-leucine on lipopolysaccharide-stimulated RAW 264.7 macrophages

Article abstract of DOI:10.1016/j.lfs.2014.08.017

Aims: Food-derived peptides have been reported to yield a variety of health promoting activities. Pyroglutamyl peptides are contained in the wheat gluten hydrolysate. In the present study, we investigated the effect of pyroglutamyl dipeptides on the lipopolysaccharide (LPS)-induced inflammation in macrophages.

Full text of DOI:10.1016/j.lfs.2014.08.017

Life Sciences 117 (2014) 1–6
Contents lists available at ScienceDirect
Life Sciences
journal homepage: www.elsevier.com/locate/lifescie
Anti-inflammatory effect of pyroglutamyl-leucine on
lipopolysaccharide-stimulated RAW 264.7 macrophages
Shizuka Hirai a, Sho Horii a, Yoshiaki Matsuzaki a, Shin Ono b, Yuki Shimmura c, Kenji Sato d, Yukari Egashira a,
⁎
a
Laboratory of Food and Nutrition, Division of Applied Biochemistry, Graduate School of Horticulture, Chiba University, Matsudo, Chiba 271-8510, Japan
Graduate School of Science and Engineering, University of Toyama, 3190 Gofuku, Toyama 930-8555, Japan
Health Care Research Center, R&D Division, Nisshin Pharma Inc., 5-3-1 Tsurugaoka, Fujimino-City, Saitama 356-8511, Japan
b
c
d
Division of Applied Life Sciences, Kyoto Prefectural University, Shimogamo, Kyoto 606-8522, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Aims: Food-derived peptides have been reported to yield a variety of health promoting activities. Pyroglutamyl
peptides are contained in the wheat gluten hydrolysate. In the present study, we investigated the effect of
pyroglutamyl dipeptides on the lipopolysaccharide (LPS)-induced inflammation in macrophages.
Main methods: RAW 264.7 macrophages were treated with LPS and various concentrations of pyroglutamyl-leucine
(pyroGlu-Leu), -valine (pyroGlu-Val), -methionine (pyroGlu-Met), and -phenylalanine (pyroGlu-Phe). Cell viabili-
ty/proliferation and various inflammatory parameters were measured by the established methods including
ELISA and western blotting. The binding of fluorescein isothiocyanate-labeled LPS to RAW 264.7 cells was also
measured fluorescently.
Received 25 April 2014
Accepted 29 August 2014
Available online 16 September 2014
Keywords:
Pyroglutamyl-leucine
Wheat gluten hydrolysate
Inflammation
Key findings: All the tested dipeptides significantly inhibited the secretion of nitric oxide, tumor necrosis factor
(TNF)-α, and interleukin (IL)-6 from LPS-stimulated RAW 264.7 macrophages. Above all, pyroGlu-Leu inhibited
the secretion of all these inflammatory mediators even at the lowest dose (200 μg/ml). PyroGlu-Leu dose-
dependently suppressed IκBα degradation and MAPK (JNK, ERK, and p38) phosphorylation in LPS-stimulated
RAW 264.7 cells. On the other hand, it did not affect the binding of LPS to the cell surface.
Significance: Our results indicated that pyroGlu-Leu inhibits LPS-induced inflammatory response via the blocking
of NF-κB and MAPK pathways in RAW 264.7 macrophages.
Macrophage
Pyroglutamyl peptide
© 2014 Elsevier Inc. All rights reserved.
Introduction
act as a cytotoxic agent in pathological processes, particularly in inflam-
matory disorders (Alderton et al., 2001; Bogdan, 2001). NO is produced
Inflammation is a host response to tissue injury and infections that
leads to the release of a large amount of inflammatory mediators.
Over-activated or chronic inflammation contributes to the pathogenesis
of many diseases such as bronchitis (Vernooy et al., 2002), chronic renal
disease (Sean Eardley and Cockwell, 2005), and cancer in various tissues
(Macciò and Madeddu, 2012; Santiago et al., 2007; Szabo et al., 2010).
Macrophages play a critical role in the process of inflammation in
many different tissues (Martinez et al., 2009). There are several pro-
inflammatory mediators involved during an inflammatory response.
Lipopolysaccharide (LPS), which is one of the most potent initiators of
inflammation derived from the outer membrane of gram negative
bacteria, activates several signaling pathways including inhibitory
κB (IκB)/nuclear factor-κB (NF-κB) and mitogen-activated protein
kinases (MAPKs) by acting on toll-like receptor (TLR) 4 to induce the
expression of inflammatory genes and the release of mediators such as
nitric oxide (NO), tumor necrosis factor-α (TNF-α), and interleukin-6
(IL-6) (Guha and Mackman, 2001). NO is a free oxygen radical and can
from L-arginine by inducible nitric oxide synthase (iNOS) during inflam-
mation. TNF-α and IL-6 are pivotal pro-inflammatory cytokines which
are involved in a variety of immune responses leading to inflammation
(Beutler and Cerami, 1989; Delgado et al., 2003).
In the past several decades, many researchers have reported that
food-derived peptides yield a variety of health promoting activities, in-
cluding a reduction of blood pressure (Karaki et al., 1993; Sipola et al.,
2001), modulation of immune cell functions (Lahov and Regelson,
1996; Miyauchi et al., 1997), and regulation of nerve functions (Yang
et al., 2001; Yoshikawa et al., 1984). Wheat gluten has a unique amino
acid composition: glutamyl residues account for about 40% of the amino
acids (Kasarda et al., 1984). It is demonstrated that wheat gluten hydroly-
sate (WGH) contains various functional peptides which have opioidergic
activity (Morley et al., 1983; Schusdziarra et al., 1981) or inhibitory effect
on angiotensin-I converting enzyme (Motoi and Kodama, 2003). More-
over, WGH is reported to have the inhibitory effect on muscle damage
after exercise (Sawaki et al., 2004) and liver cirrhosis (Horigushi et al.,
2004; Suzuki et al., 2012). Higaki-Sato et al. (2006) reported that signifi-
cant amounts of free pyroglutamic peptides were observed in the portal
veins of rats administered with WGH. Furthermore, Sato et al. (2013)
⁎
Corresponding author. Tel.: +81 47 308 8861; fax: +81 47 308 8720.
E-mail address: egashira@faculty.chiba-u.jp (Y. Egashira).
http://dx.doi.org/10.1016/j.lfs.2014.08.017
0024-3205/© 2014 Elsevier Inc. All rights reserved.

2
S. Hirai et al. / Life Sciences 117 (2014) 1–6
recently demonstrated that pyroglutamyl-leucine (pyroGlu-Leu) is the
active component in WGH that exerts hepatoprotective activity. These
results suggest that pyroglutamyl peptides, especially pyroGlu-Leu, may
be the effective components that act on these inflammatory diseases;
however, the underlying mechanism has not been clarified.
In the present study, we examined the effect of some pyroglutamyl
dipeptides (Fig. 1) on inflammatory responses in vitro, and determined
the underlying mechanism of the action of pyroglutamyl-leucine
(pyroGlu-Leu) in LPS-stimulated RAW 264.7 macrophages.
white product was precipitated by petroleum ether to give 850 mg of
protected dipeptide in 96% yield. The protected dipeptide was treated
with 4 M HCl/dioxane at room temperature for 3 h to remove the
protected groups. After the removal of excess HCl/dioxane, white solid
was precipitated by diethyl ether to give 430 mg of desired product in
85% yield. MS (ESI) m/z: 243.09 [M + H]+ (calcd. 243.13).
Pyroglutamyl-valine (pyroGlu-Val)
To a solution of HCl·Val-OtBu (210 mg, 1.0 mmol) in DMF (10 ml)
was added Et3N (0.15 ml, 1.1 mmol) and Boc-pyroGlu-OH (229 mg,
1.0 mol) at 0 °C. HOBt (271 mg, 2.0 mmol) and EDC·HCl (287 mg,
1.5 mmol) were added and the reaction mixture was stirred for 12 h
at room temperature. After the removal of the solvent under reduced
pressure, the residue was dissolved in ethyl acetate, and washed with
5% NaHCO3 and 10% citric acid, and dried over anhydrous sodium
sulfate. After filtration, the resulting filtrate was concentrated under
reduced pressure and white product was precipitated by petroleum
ether to give 325 mg of protected dipeptide in 85% yield. The protected di-
peptide was treated with 4 M HCl/dioxane at room temperature for 3 h to
remove the protected groups. After the removal of excess HCl/dioxane,
white solid was precipitated by diethyl ether to give 202 mg of desired
product in 85% yield. MS (ESI) m/z: 229.13 [M + H]+ (calcd. 229.11).
Materials and methods
Peptide synthesis
All the dipeptides were synthesized by a conventional solution meth-
od as described below. Reagents and solvents were purchased from
Wako Pure Chemical Ind., Ltd. (Osaka, Japan) and used without further
purification. All the amino acid derivatives for peptide synthesis were
purchased from Watanabe Chemical (Hiroshima, Japan). Reagents for
peptide synthesis, N-hydroxybenzotriazole (HOBt), and 1-ethyl-3-(3-
dimethylaminopropyl) carbodiimide monohydrochloride (EDC·HCl),
were purchased from Peptide Institute Inc. (Osaka, Japan). The final
products were analyzed by reversed-phase HPLC using a Shimazu
high-pressure gradient system consisting of two LC-10ADvp pumps
and a SPD-10Avp UV–Vis detector and a Tosoh analytical C18 column
(TSKgel ODS-100S, 3.0 × 150 mm). Molecular ion masses of the synthe-
sized peptides were determined by electrospray ionization time-of-flight
mass spectrometry (ESI-TOFMS) on a Hitachi NanoFrontier LC–MS
system (Tokyo, Japan).
Pyroglutamyl-methionine (pyroGlu-Met)
To a solution of HCl·Met-OtBu (242 mg, 1.0 mmol) in DMF (10 ml)
was added Et3N (0.15 ml, 1.1 mmol) and Boc-pyroGlu-OH (229 mg,
1.0 mol) at 0 °C. HOBt (271 mg, 2.0 mmol) and EDC·HCl (287 mg,
1.5 mmol) were added and the reaction mixture was stirred for 12 h
at room temperature. After the removal of the solvent under reduced
pressure, the residue was dissolved in ethyl acetate, and washed with
5% NaHCO3 and 10% citric acid, and dried over anhydrous sodium
sulfate. After filtration, the resulting filtrate was concentrated under
reduced pressure and white product was precipitated by diethyl ether
to give 248 mg of protected dipeptide in 59% yield. The protected dipep-
tide (198 mg) was treated with 4 M HCl/dioxane at room temperature
for 3 h to remove the protected groups. After the removal of excess
HCl/dioxane, white solid was precipitated by ether to give 90 mg of
desired product in 73% yield. MS (ESI) m/z: 261.09 [M + H]+ (calcd.
261.09).
Pyroglutamyl-leucine (pyroGlu-Leu)
To a solution of HCl·Leu-OtBu (500 mg, 2.2 mmol) in N,N-
dimethylformamide (DMF) (15 ml) was added triethylamine (Et3N)
(0.31 ml, 2.2 mmol) and Boc-pyroGlu-OH (510 mg, 2.2 mol) at 0 °C.
HOBt (600 mg, 4.4 mmol) and 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide hydrochloride (EDC·HCl) (640 mg, 3.3 mmol) were
added and the reaction mixture was stirred for 12 h at room tempera-
ture. After the removal of the solvent under reduced pressure, the resi-
due was dissolved in ethyl acetate, and washed with 5% NaHCO3 and
10% citric acid, and dried over anhydrous sodium sulfate. After filtration,
the resulting filtrate was concentrated under reduced pressure and
B
A
C
D
Fig. 1. Structures of pyroglutamyl-leucine (A), pyroglutamyl-valine (B), pyroglutamyl-methionine (C), and pyroglutamyl-phenylalanine (D).

S. Hirai et al. / Life Sciences 117 (2014) 1–6
3
Pyroglutamyl-phenylalanine (pyroGlu-Phe)
SDS-PAGE and transferred to an Immobilon-P membrane (Millipore,
MA, USA). After blocking, the membrane was incubated with anti-
IκBα (1/200) (sc-371: Santa Cruz Biotechnology, CA, USA), anti-JNK
(1/1000) (#9258: Cell Signaling Technology, MA, USA), anti-pJNK
(Thr183/Tyr185) (1/1000) (#9251: Cell Signaling Technology),
anti-ERK (1/1000) (#4695: Cell Signaling Technology), anti-pERK
(Thr202/Tyr204) (1/1000) (#9101: Cell Signaling Technology),
anti-p38 (1/1000) (#9212: Cell Signaling Technology), or anti-p-p38
(Thr180/Tyr182) (1/200) (#9211: Cell Signaling Technology) antibody
overnight, and then with a secondary antibody conjugated to horserad-
ish peroxidase; anti-rabbit IgG (1/2000) (#W4011: Promega) for 1 h.
The secondary antibody staining was visualized by chemiluminescence
immunoassay using a chemiluminescent HRP substrate (Millipore).
To a solution of HCl·Phe-OtBu (1.29 g, 5.0 mmol) in DMF (50 ml) was
added Et3N (0.77 ml, 5.5 mmol) and Boc-pyroGlu-OH (1.15 g, 5.0 mol) at
0 °C. HOBt (1.35 g, 10 mmol) and EDC·HCl (1.44 g, 7.5 mmol) were
added and the reaction mixture was stirred for 12 h at room temperature.
After the removal of the solvent under reduced pressure, the residue was
dissolved in ethyl acetate, and washed with 5% NaHCO3 and 10% citric
acid, and dried over anhydrous sodium sulfate. After filtration, the
resulting filtrate was concentrated under reduced pressure and white
product was precipitated by petroleum ether to give 2.03 g of protected
dipeptide in 94% yield. The protected dipeptide (502 mg, 1.2 mmol)
was treated with 4 M HCl/dioxane at room temperature for 3 h to remove
the protected groups. After the removal of excess HCl/dioxane, white
solid was precipitated by ether to give 285 mg of desired product in
89% yield. MS (ESI) m/z: 277.13 [M + H]+ (calcd. 277.11).
Measurement for the binding of FITC-conjugated LPS to RAW 264.7 cells
RAW 264.7 cells were seeded on 96 well black plates (1 ×
106 cells/ml) and pretreated with 400 or 800 μg/ml of pyroGlu-Leu for
4 h, and then incubated with 5 μg/ml of FITC-conjugated LPS (Sigma)
and pyroGlu-Leu at the same doses as the pretreatment for 1 h. After
washing the cells with PBS, the binding of FITC-LPS to the cell surface
was measured on the basis of fluorescent intensity at 485/535 nm
using Infinite F200, fluorescence plate reader (Tecan Group Ltd.,
Männedorf, Switzerland).
Cell culture
RAW 264.7 macrophages (RIKEN BioResource Center, Tsukuba,
Japan) were cultured in Dulbecco's modified Eagle's medium (DMEM;
Nissui, Tokyo, Japan) with 10% fetal bovine serum (FBS) and 100 U/ml
penicillin/100 μg/ml streptomycin (Gibco BRL, NY, USA) at 37 °C in a
humidified 5% CO2 atmosphere. The cells were seeded on 24 well-plates
(5 × 105 cells/ml), and treated with 2 ng/ml LPS (Sigma, MO, USA)
and various concentrations of pyroglutamyl dipeptides (pyroGlu-Leu,
pyroGlu-Val, pyroGlu-Met, and pyroGlu-Phe) in a serum free medium.
Statistical analysis
The data are presented as mean SE of four replicants. Statistical
comparison among groups were carried out using ANOVA and Williams'
or Shirley–Williams' multiple comparison tests. Differences were
considered significance at P b 0.05.
Cell viability assay
CellTiter 96® AQueous One Solution Cell Proliferation Assay
(Promega, WI, USA) was used to evaluate the cytotoxity of each dipep-
tides. RAW 264.7 macrophages were seeded in 96-well plates (5 × 105
cells/well), and treated with various concentrations of pyroglutamyl
dipeptides in the presence or absence of LPS for 24 h. Then, 20 μl of
CellTiter 96® AQueous One Solution Reagent was added into each
well. After 20 min, the absorbance at 490 nm was measured.
Results
Anti-inflammatory effect of pyroglutamyl dipeptides
First, we measured the cytotoxicity of each pyroglutamyl dipeptides.
As shown in Fig. 2A, less than 90% of survival rate was observed in the
cells treated with pyroGlu-Val, -Met, and -Phe even at 200 μg/ml.
PyroGlu-Leu showed the lowest effect on the cell viability; no significant
cytotoxic effect was observed in the cells treated with pyroGlu-Leu at
the concentration below 400 μg/ml.
Next, we examined the effect of pyroglutamyl dipeptides on the LPS-
induced inflammation in RAW 264.7 macrophages. PyroGlu-Leu,
pyroGlu-Val, pyroGlu-Met, and pyroGlu-Phe were all suppressed LPS-
induced secretion of NO (Fig. 2B), TNF-α (Fig. 2C), and IL-6 (Fig. 2D)
from RAW 264.7 cells in a dose-dependent manner. Above all, pyroGlu-
Leu significantly inhibited the secretion of all these inflammatory media-
tors even at the lowest dose (200 μg/ml) (Fig. 2B, C, D).
Measurement of nitric oxide release
The amount of nitrite in the cell-free culture supernatants was
measured using a Griess reagent (Granger et al., 1996). Briefly, 100 μl
of supernatant was mixed with an equivalent volume of Griess reagent
[1:1 (v/v) of 0.1% N-1-naphthyl-ethylenediamine in distilled water and
1% sulfanilamide in 5% phosphoric acid] on a 96-well flat bottom plate.
After 10 min, the absorbance at 550 nm was measured, and the amount
of nitrite was calculated from the NaNO2 standard curve.
Measurement of TNF-α and IL-6 by ELISA
The concentrations of TNF-α and IL-6 in the culture supernatants
and plasma in mice were determined by ELISA using Mouse TNF-α or
IL-6 ELISA MAX Set Deluxe (Biolegend, CA, USA) in accordance with
the manufacturer's instructions.
Suppression of IκBα degradation and MAPK phosphorylation by pyroGlu-Leu
in LPS-stimulated RAW 264.7 cells
As pyroGlu-Leu most markedly inhibited the secretion of inflamma-
tory mediators, we focused on the mechanism by which pyroGlu-Leu
inhibits LPS-induced inflammation in macrophages. NF-κB and MAPK
are known as the major signaling pathways that regulate the induction
of TNF-α and IL-6 (Guha and Mackman, 2001). Therefore, the degrada-
tion of IκBα, which leads to the activation of NF-κB, and phosphoryla-
tion of MAPK: c-Jun N-terminal kinase (JNK), extracellular signal-
regulated kinase (ERK), and p38, were examined in RAW 264.7 cells
treated with LPS. Although LPS treatment for 30 min markedly promot-
ed IκBα degradation in RAW 264.7 macrophages, the degradation was
inhibited by pyroGlu-Leu in a dose-dependent manner (Fig. 3). Further-
more, LPS-induced phosphorylation of JNK, ERK, and p38 were all
Western blotting
Western blotting was performed as previously described (Hirai et al.,
2007; Takahashi et al., 2002). In brief, RAW 264.7 cells were washed
with PBS and placed immediately in a lysis buffer containing 20 mM
Tris–HCl (pH 7.5), 15 mM NaCl, and a protease inhibitor cocktail set III
(Calbiochem, CA, USA). The lysate was centrifuged at 15,000 rpm for
5 min, and the supernatant was stored for subsequent analysis. Protein
concentration was determined using a Protein Assay Dye Reagent
Concentrate (BioRad Laboratories, CA, USA) on the basis of the method
of Bradford (1976). Fifteen micrograms of protein was separated by 10%

4
S. Hirai et al. / Life Sciences 117 (2014) 1–6
A
B 30
120
100
80
60
40
20
0
25
*
*
20
*
*
*
*
*
**
*
*
*
pyroGlu-Leu
pyroGlu-Val
pyroGlu-Met
pyroGlu-Phe
*
pyroGlu-Leu
pyroGlu-Val
pyroGlu-Met
pyroGlu-Phe
15
10
5
*
*
*
*
*
*
*
*
0
-
+
0
+
+
+
LPS
-
+
0
+
+
+
LPS
peptide
(μg/ml)
peptide
0
200
400
800
(μg/ml)
0
200
400
800
D
6
5
4
3
2
1
0
25
C
20
15
10
5
*
*
*
*
*
*
*
*
*
*
pyroGlu-Leu
pyroGlu-Val
pyroGlu-Met
pyroGlu-Phe
pyroGlu-Leu
pyroGlu-Val
pyroGlu-Met
pyroGlu-Phe
*
*
*
*
*
*
*
*
*
*
*
0
-
+
0
+
+
+
-
+
0
+
+
+
LPS
peptide
(μg/ml)
LPS
peptide
0
200
400
800
0
200
400
800
(μg/ml)
Fig. 2. Effect of pyroglutamyl dipeptides on the cell viability and secretion of inflammatory mediators in LPS-stimulated RAW 264.7 macrophages. RAW 264.7 macrophages were treated
with LPS (2 ng/ml) and various concentrations of each pyroglutamyl dipeptides for 24 h. Cell viability was measured (A). NO level in the culture supernatant was measured by griess
reaction (B). TNF-α (C) and IL-6 (D) levels were also measured by ELISA. Values are means SE of four replicants. *P b 0.05 vs culture treated with only LPS.
inhibited by pyroGlu-Leu treatment in a dose-dependent manner
(Fig. 3).
IκB
PyroGlu-Leu has no effect on the binding of LPS to RAW 264.7 macrophages
p-JNK
JNK
Some kinds of peptides are reported to inhibit the cellular binding
of LPS to macrophages (Huang et al., 2010; Nagaoka et al., 2001). There-
fore, we examined the effect of pyroGlu-Leu on the binding of FITC-LPS
to RAW 264.7 cells. Incubation of pyroGlu-Leu with FITC-LPS did not
affect fluorescent intensity of cell surface (Fig. 4).
p-ERK
ERK
120
100
80
60
40
20
0
p-p38
p38
-
+ + +
100 200 400
LPS
+
0
-
+
0
+
+
FITC-LPS
pyroGlu-Leu
(μg/ml)
pyroGlu-Leu
0
0
400
800
(μg/ml)
Fig. 4. Effect of pyroglutamyl-leucine on the binding of FITC-LPS to RAW 264.7 cells. RAW
264.7 macrophages were pretreated with 400 or 800 μg/ml of pyroGlu-Leu for 4 h, and
then incubated with 5 μg/ml of FITC-LPS and pyroGlu-Leu for 1 h. After washing the cells,
Fig. 3. Effect of pyroglutamyl-leucine on the expressions of LPS signaling molecules. RAW
264.7 macrophages were treated with LPS (2 ng/ml) and various concentrations of
pyroGlu-Leu for 30 min for IκBα, p-JNK, JNK, p-ERK, and ERK, and for 5 min for p-p38 and
p38 by western blotting.
the binding of FITC-LPS was measured fluorescently. Values are means
SE of four
replicants.

S. Hirai et al. / Life Sciences 117 (2014) 1–6
5
Discussion
Peptides play an important role in the biochemical and physiological
required to clarify the availability and the effectiveness of pyroGlu-Leu
in vivo.
functions of our body. Recently, various functional peptides are found in
plants (Lico et al., 2012), animals (Redwan, 2009), or marine organisms
(Aneiros and Garateix, 2004), and their health-promoting effects are
expected. In the present study, focusing on pyroglutamyl peptides
which had been found in the plasma of rats administered with WGH
(Higaki-Sato et al., 2006), we evaluated their effects on inflammatory
responses.
Conclusion
Our study demonstrated that pyroGlu-Leu suppresses LPS-induced
inflammation in RAW 264.7 macrophages. As pyroGlu-Leu did not affect
the binding of LPS to the cell membrane, this dipeptide was considered
to be incorporated into the cells and directly inhibit NF-κB and MAPK
activation.
Inflammation is a host response to injury related to chemical or
microbiological toxins. LPS is expressed in the outer membrane of
gram-negative bacteria, and plays a key role in the initiation of inflam-
mation by producing inflammatory mediators including NO, TNF-α, and
IL-6 (Guha and Mackman, 2001). Our results indicated that all kinds of
pyroglutamyl dipeptides we used in this study reduced the production
of inflammatory mediators stimulated by LPS. Especially, pyroGlu-Leu
showed the most significant suppression of these mediators at the low-
est dose (200 μg/ml). These results suggest that pyroGlu-Leu may have
a most potent therapeutic effect on the LPS-induced inflammation.
Focusing on the pyroGlu-Leu, we examined its anti-inflammatory
mechanism in LPS-stimulated RAW 264.7 cells. LPS activation of mam-
malian cells occurs through its binding to TLR-4 and activation of its
downstream signals including IκB/NF-κB and MAPK pathways (Kawai
and Akira, 2006). In the unstimulated state, NF-κB is retained in the
cytosol by the association with the inhibitory protein, IκB. After LPS
stimulation, IκB becomes phosphorylated and this triggers its proteolyt-
ic degradation, leading to the activation and translocation of NF-κB to
nucleus (Yamamoto and Gaynor, 2004). MAPKs are protein Ser/Thr
kinases, which are also known to play major roles in the transmission
of inflammatory signals to the nucleus (Guha and Mackman, 2001;
Xiao et al., 2007). LPS stimulation induces the phosphorylation and acti-
vation of three types of MAPKs; JNK, ERK, and p38 MAPK (Guha and
Mackman, 2001). JNK signaling regulates the expression of iNOS (Uto
et al., 2005), whereas ERK signaling and p38 signaling upregulate iNOS
expression and the production of proinflammatory cytokines including
TNF-α and IL-6 in LPS-stimulated macrophages (Ajizian et al., 1999;
Chan and Riches, 2001). On the other hand, Caivano (1998) reported
that ERK and p38 are not rate-limiting for iNOS induction in RAW 264
macrophages. Our data showed that pyroGlu-Leu dose-dependently
inhibited the degradation of IκBα and phosphorylation of three types of
MAPKs. These findings suggest that the anti-inflammatory properties of
pyroGlu-Leu are probably due to the inhibition of LPS-stimulated activa-
tion of NF-κB via the suppression of IκBα degradation and partly the
phosphorylation of MAPKs in RAW 264.7 cells. On the other hand,
pyroGlu-Leu did not interfere the binding of LPS to the cell surface of
macrophages in the present study. Several anti-inflammatory peptides
are reported to suppress LPS-induced activation of macrophages through
the interaction with LPS, which results in the neutralization of LPS action
(Nagaoka et al., 2001; Rosenfeld et al., 2008; Srivastava et al., 2012). On
the other hand, it is also reported that relatively low molecular-weight
peptides such as dipeptides or tripeptides can be imported into the
cells by peptide transporters (Yang et al., 2002). Therefore, pyroGlu-
Leu, a dipeptide, is considered to be transported into the cells and directly
affect downstream signaling of TLR4 in macrophages.
Conflict of interest statement
We have no conflict of interest other than that Shimmura Y is an employee of Nisshin
Pharma Inc.
Acknowledgments
We appreciate Dr. Yoshio Suzuki, associate professor at Juntendo
University, for providing valuable advices. This work was supported in
part to Y.E. by the grant from the Iijima Memorial Foundation for the
Promotion of Food Science and Technology (H23).
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