Anti‐inflammatory potential of Lactobacillus reuteri LM 1071 in LPS‐stimulated RAW264.7 cells

Abstract

Aims:
To investigate anti-inflammatory effects of Lactobacillus reuteri LM1071 in lipopolysaccharides (LPS)-induced inflammation RAW264.7 cells.

Methods and results:
To evaluate anti-inflammatory activities of L. reuteri LM1071, LPS-stimulated RAW264.7 cells were used. Gene expression levels of eight immune-associated genes including IL-1β, IL-6, and TNF-α and protein production levels of COX-1 and COX-2 were analyzed. Moreover, the production of eicosanoids as important biomarkers for anti-inflammation was determined.

Conclusions:
The current study demonstrates that L. reuteri LM1071 has anti-inflammatory potential by inhibiting the production of inflammation mediators such as NO, eicosanoids such as PGE1 & PGE2, pro-inflammatory cytokines, and COX proteins. It can also enhance the production of inflammatory associated genes such as IL-11, BMP4, LEFTY2, and EET metabolite.

Significance and impact of the study:
Lactobacillus reuteri is one of crucial bacteria for food fermentation. It can be found in the gastrointestinal system of human and animals. Several studies have shown that L. reuteri has valuable effects on host health. The current study firstly demonstrated that L. reuteri has beneficial effect on the inflammation containing the variation of eicosanoids which are the one of the most important biomarkers and moreover eicosanoid-associated genes as well as proteins.

Full text

J Appl Microbiol. 2021;00:1–9. wileyonlinelibrary.com/journal/jam
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© 2021 The Society for Applied Microbiology
INTRODUCTION
Inflammation is a primary host defence mechanism
against many stimuli such as external infections, chem-
ical damage and physical damage that can lead to tissue
injury (Yu et al., 2019). Macrophages are among import-
ant innate immune cells that respond to these stimuli by
releasing of pro- inflammatory mediators and phagocy-
tosis for injured tissue reparation (Ran & Montgomery,
2012; Watanabe et al., 2019). During inflammation, mac-
rophages regulate the production of pro- inflammatory
cytokines such as interleukin (IL)- 1β, IL- 6 and tumour
necrosis factor- α (TNF- α) (Dinarello, 2006) as well as the
Received: 10 June 2021
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Revised: 13 October 2021
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Accepted: 18 October 2021
DOI: 10.1111/jam.15331
ORIGINAL ARTICLE
Anti- inflammatory potential of Lactobacillus reuteri
LM1071 via eicosanoid regulation in LPS- stimulated
RAW264.7 cells
A- yeongJang1
|
WeerawanRod- in1
|
ChaiwatMonmai1
|
MinnSohn2
|
Tae- rahkKim2
|
Min- GyuJeon2
|
Woo JungPark1
A- yeong Jang and Weerawan Rod- in are contributed equally to this
research.
1Department of Marine Food Science
and Technology, Gangneung- Wonju
National University, Gangneung,
Gangwon, Korea
2Center for Research and Development,
LACTOMASON, Jinju, Korea
Correspondence
Woo Jung Park, Department of
Marine Food Science and Technology,
Gangneung- Wonju National University,
Gangneung, Gangwon 25457, Korea.
Email: pwj0505@gwnu.ac.kr
Funding information
LACTOMASON
Abstract
Aims: To investigate anti- inflammatory effects of Lactobacillus reuteri LM1071 in
lipopolysaccharides (LPS)- induced inflammation RAW264.7 cells.
Methods and Results: To evaluate anti- inflammatory activities of L. reuteri
LM1071, LPS- stimulated RAW264.7 cells were used. Gene expression levels
of eight immune- associated genes including IL- 1β, IL- 6 and TNF- α and
protein production levels of COX- 1 and COX- 2 were analysed. Moreover, the
production of eicosanoids as important biomarkers for anti- inflammation was
determined.
Conclusions: The current study demonstrates that L. reuteri LM1071 has anti-
inflammatory potential by inhibiting the production of inflammation mediators such
as NO, eicosanoids such as PGE1 & PGE2, pro- inflammatory cytokines and COX
proteins. It can also enhance the production of inflammatory associated genes such
as IL- 11, BMP4, LEFTY2 and EET metabolite.
Significance and Impact of the Study: Lactobacillus reuteri is one of the crucial
bacteria for food fermentation. It can be found in the gastrointestinal system of
human and animals. Several studies have shown that L. reuteri has valuable effects
on host health. The current study firstly demonstrated that L. reuteri has a benefi-
cial effect on the inflammation containing the variation of eicosanoids (PGE1 and
PGE2) which are one of the most important biomarkers and moreover eicosanoid-
associated genes as well as proteins (COX- 2).
KEYWORDS
anti- inflammation, COX, eicosanoid, Lactobacillus reuteri LM1071, RAW264.7 cells

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JANG  .
production of pro- inflammatory mediators such as cyclo-
oxygenase- 2 (COX- 2) and inducible nitric oxide synthase
(iNOS) (Choi et al., 2019).
Lactobacillus spp. exist in various food products in
every part of the world (Greifová et al., 2017). They
are probiotic bacteria that can provide good effects
on several physiological processes including immune
regulation (Ding et al., 2017). Several studies have
reported the potential of Lactobacillus spp. to regula-
tion immunity by minimizing inflammatory responses
through T- lymphocytes (Jang et al., 2012; Shah et al.,
2012; Smits et al., 2005), B- lymphocytes (Hosoya et al.,
2014; Lee et al., 2013), nature killer (NK) cells (Makino
et al., 2016; Matsusaki et al., 2016; Shida et al., 2017)
and macrophages (Sohn et al., 2015; Ukibe et al.,
2015). In addition, Lactobacillus rhamnosus and L.
plantarum can remarkably induce the production of
pro- inflammatory cytokines such as IL- 6 and TNF- α
(Jorjão et al., 2015; Lee et al., 2016a). However, L. gas-
seri and L. rhamnosus can decrease the production
of IL- 6 and TNF- α (Mortaz et al., 2015; Ukibe et al.,
2015).
Lactobacillus reuteri is one of crucial bacteria for
food fermentation. It can be found in the gastrointes-
tinal system of human and animals (Duar et al., 2017).
Several studies have shown that L. reuteri has valuable
effects on host health. It can protect host against gas-
trointestinal infections by bacteria such as Clostridium
difficile (Cherian et al., 2015), Salmonella (Abhisingha
et al., 2018) and Escherichia coli (Genís et al., 2017).
Many strains of L. reuteri have anti- inflammatory po-
tential effects. For example it has been reported that
L. reuteri GMNL- 263 can decrease serum levels of
TNF- α, IL- 6 and monocyte chemoattractant protein- 1
(MCP- 1) in high- fat diet administrated rats (Hsieh
et al., 2016). The production of TNF- α in lipopoly-
saccharides (LPS)- induced THP- 1 cells is suppressed
by L. reuteri BM36301 (Lee et al., 2016b). L. reuteri
DSM12246 can suppress the production of IL- 12, IL- 6
and TNF- α in dendritic cells of mice (Christensen
et al., 2002). Recently, it has been reported that L. re-
uteri LM1071 isolated from human breast milk exhib-
its anti- inflammatory effects on mRNA expression of
IL- 6, TNF- α and IL- 4 in IL- 1β- stimulated HT- 29 cells
and that it can be used as an effective and safe pro-
biotic for humans (Kim et al., 2020). However, anti-
inflammatory effects of L. reuteri based on the evidence
of eicosanoid production have not been reported yet.
Thus, the objective of the current study was to firstly
investigate the beneficial effects of L. reuteri LM1071
on inflammation including the production of eicosa-
noids known to be important biomarkers. Effects of
L. reuteri LM1071 on eicosanoid- associated genes and
proteins in LPS- induced inflammation RAW264.7 cells
were also determined.
MATERIALS AND METHODS
Lactic acid bacteria strain
L. reuteri LM1071 was obtained from LACTOMASON at
a concentration of 4.8×109 cells g1 (Kim et al., 2020).
L. reuteri LM1071 was prepared in stock solution at
1 mg ml1 (w/v) that dissolved in dimethyl sulfoxide
(DMSO), and diluted various concentrations using cul-
ture medium for experiment. Treatment groups included
10, 20, 30 and 40 μg ml1 corresponding to 4.8 × 104,
9.6×104, 1.44×105 and 1.92×105 cells ml1 respectively.
Animal cell culture
RAW264.7 cells were purchased from Korean Cell Line
Bank (KCLB, South Korea). Cells were maintained in RPMI-
1640medium (Gibco™, USA) supplemented with 10% foetal
bovine serum (FBS) and 1% penicillin/streptomycin (Welgene,
South Korea) at 37°C in a humidified 5% CO2 incubator.
Nitric oxide (NO) production and cell
proliferation assay
RAW264.7 cells were seeding at a density of 1×105 cells
well1 in a 96- well plate. After 24h incubation, the culture
medium was removed. Cells were pretreated with vari-
ous concentrations (10, 20, 30 and 40 μg ml1) of L. reu-
teri LM1071 for 1h. Cells were stimulated with or without
1μgml1 of LPS (from Escherichia coli, Sigma- Aldrich, USA)
and incubated at 37°C for 24h. NO production in culture su-
pernatant was determined using Griess reagent (Promega,
USA). The culture supernatant was mixed with the same
volume of the Griess reagent following the manufacture's
instruction. The absorbance at 540nm was measured using
an EL800 Absorbance Microplate Reader (BioTek, USA). A
standard curve generated using sodium nitrite (provided in
the kit) was used for nitrite concentration determination.
Stimulated cells were used to evaluate the cytotoxicity
of L. reuteri LM1071 to RAW264.7 cells using an EZ- cytox
cell viability assay kit (Daeil Labservice, Korea) accord-
ing to the manufacturer's instructions. Cell proliferation
ratio was calculated according to the following formula:
Cell proliferation ratio (%)=
The absorbance at 450 nm of test group
The absorbance at 450 nm of control group
×
100

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ANTI- INFLAMMATORY EFFECT OF L. REUTERI
RNA extraction and cDNA synthesis
After treatment with L. reuteri LM1071 and LPS, total
RNA was extracted from RAW264.7 cells using Tri rea-
gent® (Molecular Research Center, Inc., USA). RNA was
precipitated using absolute isopropanol alcohol at 4°C for
30min. The RNA pellet was collected by centrifugation
and washed with 70% ethanol thrice. Total RNA was dis-
solved in nuclease- free water. Its quantity was measured
using a nanophotometer (Implen, Germany). Then, 1g
of RNA was transcribed into cDNA using a high- capacity
cDNA reverse transcription kit (Applied biosystems,
USA) according to the manufacturer's recommended
instructions.
Quantification of immune- associated
gene expression
To evaluate inflammatory gene expression, 5 ng
of cDNA from each treatment group was used as a
template. The qPCR was performed with TB Green®
Premix Ex Taq™ II (Takara Bio Inc., Japan) in a
QuantStudio™ 3 FlexReal- Time PCR System (Applied
Biosystem, USA). The PCR reaction contained
0.4μmoll1 of specific primer set (Table 1). Relative
mRNA expression was normalized against β- actin
as a reference gene and analysed using QuantStudio
3software.
Eicosanoid level determination
Levels of prostaglandin E1 (PGE1: Enzo Life Science,
USA), prostaglandin E2 (PGE2: Enzo Life Science, USA),
leukotriene B4 (LTB4: Enzo Life Science, USA) and epox-
yeicosatrienoic acids (EET: MyBioSource, USA) were
measured using ELISA kits following to the manufactur-
er's instructions.
Western blot
RAW264.7 cells were placed in a six- well plate and treated
with different concentrations of L. reuteri LM1071 and LPS
as described in the NO production section. Cells were har-
vested and lysed with RIPA buffer (Tech & Innovation,
China) supplemented with 0.1% of protease inhibitor
(Thermo Scientific, USA). Protein concentration was
evaluated using Pierce™ BCA Protein Assay Kit (Thermo
Scientific, USA). The same amount of protein from each
treatment group (30 μg) was separated on 10% SDS-
polyacrylamide gel electrophoresis (SDS- PAGE). These
separated proteins were transferred onto a polyvinylidene
fluoride (PVDF) membrane. Western blot was carried out
as described by Narayanan et al. (2003). Briefly, the mem-
brane was incubated with antibodies specific to COX- 1
(Cell Signaling Technology, USA), COX- 2 (Cell Signaling
Technology, USA) and α- tubulin (Abcam, United Kingdom)
at 4°C overnight. The membrane was then incubated with
TABLE  List of primers used in quantitative real- time PCR experiment
Gene Accession No. Primer sequence
iNOS BC062378.1 Forward: 5’- TTCCAGAATCCCTGGACAAG3’
Reverse: 5’- TGGTCAAACTCTTGGGGTTC3’
COX−2 NM_011198.4 Forward: 5’- AGAAGGAAATGGCTGCAGAA3’
Reverse: 5’- GCTCGGCTTCCAGTATTGAG3’
IL−1βNM_008361.4 Forward: 5’- GGGCCTCAAAGGAAAGAATC3’
Reverse: 5’- TACCAGTTGGGGAACTCTGC3’
IL−6 NM_031168.2 Forward: 5’- AGTTGCCTTCTTGGGACTGA3’
Reverse: 5’- CAGAATTGCCATTGCACAAC3’
TNF- αD84199.2 Forward: 5’- ATGAGCACAGAAAGCATGATC3’
Reverse: 5’- TACAGGCTTGTCACTCGAATT3’
IL−11 BC134354.1 Forward: 5’- TCCCCTCGAGTCTCTTCAGA3’
Reverse: 5’- TCTCCGTCAGCTGGGAATTT3’
BMP4 D14814.1 Forward: 5’- CTTCAACCTCAGCAGCATCC3’
Reverse: 5’- GATGAGGTGTCCAGGAACCA3’
LEFTY2 NM_177099.4 Forward: 5’- CAGCTGCAGCTCAGCCAGGCCC3’
Reverse: 5’- AGCGGTCAGCGTGACTTCCC3’
β- actin NM_007393.5 Forward: 5’- CCACAGCTGAGAGGGAAATC3’
Reverse: 5’- AAGGAAGGCTGGAAAAGAGC3’

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JANG  .
goat anti- rabbit IgG- HRP secondary antibody at room tem-
perature for 1h. Expression levels of proteins were detected
using Pierce® ECL Plus Western Blotting Substrate (Thermo
Scientific, USA). ChemiDoc XRS + imaging system (Bio-
Rad, USA) was used for blot imaging and ImageLab soft-
ware (version 4.1; Bio- Rad, USA) was used to determine
the quantity of protein expression.
Statistical analysis
All statistical analyses were performed using the Statistix
8.1software. Data are presented as means ± standard de-
viation. One- way analysis of variance (ANOVA) with a
Tukey's honest significance test was then performed to
determine differences among treatment groups. Statistical
significance was considered at p <0.05. All experiments
were performed three times independently.
RESULTS
Effect of L. reuteri LM1071 on NO
production and cell proliferation
Anti- inflammatory activities of L. reuteri LM1071
were evaluated using LPS- stimulated RAW264.7 cells.
Different concentrations of L. reuteri LM1071 cells were
used to treat cells to determine the production of NO and
cell proliferation. As shown in Figure 1a, RAW264.7 (- )
cells (cells cultured with RPMI medium without stimula-
tion by LPS) showed very low NO production level, while
FIGURE  Effect of L. reuteri
LM1071 in LPS- stimulated RAW264.7
cells. (a) effect on NO production and
(b) effect on cell proliferation. Data
are presented as mean ± SD of three
independent experiments (n=3).
Different letters (a, b, c and d) indicate
statistical differences between treatment
groups at p<0.05. (- ), RPMI medium as
the negative control
50
(a)
40
30
20
10
0
Nitric oxide production (µmol l-1)
(-) DMSO 10 20
L. reuteri concentration (µg ml-1)
30 40LPS
(-) DMSO 10 20
L. reuteri concentration (µg ml
-1
)
30 40LPS
a
b
c
dddd
140
(b)
120
100
80
60
40
20
0
Cell proliferation ratio (%)
cc
b
aa
cc

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ANTI- INFLAMMATORY EFFECT OF L. REUTERI
FIGURE  The immune- associated gene expression effect of L. reuteri LM1071 in LPS- stimulated RAW264.7 cells. (a) iNOS, (b) COX-
2, (c) IL- 1β, (d) IL- 6, (e) TNF- α, (f) IL- 11, (g) BMP4 and (h) LEFTY2. Data are presented as mean ± SD of three independent experiments
(n=3). Different letters (a, b, c, d, e and f) indicate statistical differences between treatment groups at p<0.05. (- ), RPMI medium as the
negative control
25
(a)
20
15
10
iNOS expression (fold)
5
0
e
(-) DMSO LPS 10 20 30 40
L. reuteri concentration (µg ml-1)
e
b
a
c
d
e
140
120
100
80
60
40
20
0
(b)
COX-2 expression (fold)
(-)DMSO LPS 10 20 30 40
L. reuteri concentration (µg ml-1)
e
d
c
ff
b
a
(c)
IL-1
β
expression (fold)
0
10
20
30
40
50
60
(-) DMSO LPS 10 20 30 40
L. reuteri concentration (µg ml-1)
e
d
ff
c
b
a
20
15
10
5
0
(d)
IL-6 expression (fold)
(-)DMSO LPS 10 20 30 40
L. reuteri concentration (µg ml-1)
(-) DMSO LPS 10 20 30 40
L. reuteri concentration (µg ml-1)
(-) DMSO LPS 10 20 30 40 (-)DMSO LPS 10 20 30 40
L. reuteri concentration (µg ml
-1
)
L. reuteri concentration (µg ml-1)
(-)DMSO LPS 10 20 30 40
L. reuteri concentration (µg ml-1)
a
b
c
ff
d
e
TNF-
α
expression (fold)
40
(e)
30
20
10
0
a
b
c
d
ff
e
IL-11 expression (fold)
2·5
2·0
1·5
1·0
0·5
0·0
(f)
(g) (h)
e
f
c
b
a
dd
BMP4 expression (fold)
3·0
2·5
2·0
1·5
1·0
0·5
0·0
f
dc
b
a
ee
LEFTY2 expression (fold)
3·0
2·5
2·0
1·5
1·0
0·5
0·0
d
c
b
a
dd
e

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JANG  .
LPS- stimulated cells generated 42.10±0.45μmoll1NO.
Treatments with various concentrations of L. reuteri
LM1071 significantly decreased the production of NO
in LPS- stimulated RAW264.7 cells. However, L. reuteri
LM1071 was not cytotoxic to LPS- stimulated RAW264.7
cells at any concentration up to 40μgml1 (Figure 1b).
Effect of L. reuteri LM1071 on expression of
immune- associated genes
LPS obviously increased gene expression levels of inflam-
matory markers such as iNOS (Figure 2a) and COX- 2 in
RAW264.7 cells (Figure 2b). It also induced the expression
of pro- inflammatory cytokine genes such as IL- 1β, IL- 6 and
TNF- α (Figure 2c– e). However, L. reuteri LM1071 effectively
suppressed the expression of pro- inflammatory cytokines
known to be inflammatory markers in a concentration-
dependent manner in RAW264.7 cells (Figure 2a– e).
Conversely, LPS significantly reduced expression levels
of IL- 11, bone morphogenic protein 4 (BMP4) and left-
right determination factor 2 (LEFTY2) (Figure 2f– h). Gene
expression of IL- 11, BMP4 and LEFTY2 significantly in-
creased depending on the concentration of L. reuteri.
Effect of L. reuteri LM1071 on
eicosanoid level
To investigate the effect of L. reuteri LM1071 on LPS-
induced eicosanoid production, RAW264.7 cells were pre-
treated with various concentrations of L. reuteri LM1071
along with 1μgml1 of LPS. As shown in Figure 3, LPS
significantly increased the production of PGE1, PGE2
and LTB4, but significantly reduced the production of
EET. Different concentrations of L. reuteri LM1071 dose-
dependently decreased the production of PGE2 in LPS-
stimulated RAW264.7 cells. Production levels of PGE1
and LTB4 were also significantly declined when LPS-
stimulated cells were pretreated with L. reuteri LM1071
at high concentrations (30– 40μgml1 for PGE1 and 20–
40μgml1 for LTB4). Contrarily, the production of EET
remarkably was raised according to the concentration of
L. reuteri LM1071 in RAW264.7 cells.
FIGURE  Effect of L. reuteri LM1071 on eicosanoid productions. (a) PGE1, (b) PGE2, (c) LTB4 and (d) EET. Data are presented as mean
± SD of three independent experiments (n=3). Different letters (a, b, c, d, e and f) indicate statistical differences between treatment groups
at p<0.05. (- ), RPMI medium as the negative control
4000
aaa
bb
c
(-) DMSO LPS 2010 30 40
L. reuteri concentration (µg ml-1)
(-) DMSO LPS 2010 30 40
(-) DMSO LPS 2010 30 40
(-) DMSO LPS 2010 30 40
L. reuteri concentration (µg ml-1)
L. reuteri concentration (µg ml
-1
)
L. reuteri concentration (µg ml
-1
)
c
(a)
(c) (d)
(b)
3000
2000
PEG1 production (pg ml-1)
1000
0
PEG2 production (pg ml-1)
EET production (µg ml-1)
350
300
250
200
150
100
50
0
a
b
c
d
ff
e
300
250
200
150
100
50
0
ee
f
d
c
b
a
LTB4 production (pg ml-1)
250
200
150
100
50
0
a
a
b
c
ccc

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ANTI- INFLAMMATORY EFFECT OF L. REUTERI
Effect of L. reuteri LM1071 on COX- 1 and
COX- 2 production
Protein production levels of COX- 1 and COX- 2 in LPS-
treated RAW264.7 cells were greatly up- regulated than
those in the untreated group (Figure 4). However, L.
reuteri LM1071 dose- dependently suppressed such in-
creases of production levels of COX- 1 and COX- 2 in
LPS- stimulated cells. Moreover, treatment with high con-
centrations of L. reuteri LM1071 (30 and 40μgml1) dra-
matically depressed the production of COX- 1 and COX- 2.
DISCUSSION
Inflammation response is a process of preventing host
cells against external intrusion (Mariathasan & Monack,
2007). Macrophages are native immune cells associ-
ated with innate immunity. They play a key role in in-
flammatory responses (Fujiwara & Kobayashi, 2005).
However, the imbalance of immune system caused
by excessive production of inflammatory mediators
and long period of inflammation may lead to chronic
inflammatory disorders (Shanura Fernando et al., 2018).
The present study was performed to investigate the anti-
inflammatory potential of L. reuteri LM1071 in LPS-
stimulated RAW264.7 cells.
Cytokines are critical mediators coordinating in-
flammatory processes. The production of TNF- α, IL-
1β and IL- 6 cytokines may increase inflammation and
tissue injury (Oishi & Manabe, 2018). Conversely,
BMP4, IL- 11 and LEFTY2have been indicated as anti-
inflammatory molecules (Baraban et al., 2016; Ma et al.,
2013). Therefore, reducing the production of IL- 1β,
IL- 6 and TNF- α and preserving the activity of BMP4,
IL- 11 and LEFTY2might be an effective way to bal-
ance inflammatory disease defence. Figure 2showed
that mRNA expression levels of pro- inflammatory cy-
tokines (TNF- α, IL- 1β and IL- 6) were down- regulated
by L. reuteri in LPS- stimulated RAW264.7 cells in a
concentration- dependent manner. Similarly, it has
been reported that L. reuteri can decrease mRNA
expression levels of IL- 6, TNF- α and IL- 4 in IL- 1β-
induced HT- 29 cells (Kim et al., 2020). Conversely,
treatment with various concentrations of L. reuteri
LM1071 significantly increased mRNA expression
FIGURE  The effect of L. reuteri
LM1071 on proteins associated COXs
production in LPS- stimulated RAW264.7
cells. (a) Western blot and (b) relative
band intensity. Data are presented
as mean ± SD of three independent
experiments (n=3). Different letters (a,
b, c and d) indicate statistical differences
between treatment groups at p<0.05. (- ),
RPMI medium as the negative control
-Tubulin
COX-2
COX-1
(-) DMSO LPS 10
L. reuteri concentration (µg ml-1)
20 30 40
Relative protein expression (fold)
400
300
200
100
0d
d
10 µg ml
-1
L. reuteri
20 µg ml-1 L. reuteri
30 µg ml-1 L. reuteri
40 µg ml-1 L. reuteri
1 µg ml-1 LPS
1% DMSO
(-) RPMI
a
b
ccd cd
cc
c
c
b
b
a
COX-1 COX-2
(a)
(b)

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JANG  .
levels of BMP4, IL- 11 and LEFTY2 dose dependently.
These results indicate that L. reuteri LM1071 possesses
biological activities to regulate inflammation- related
cytokines and other genes.
Prostaglandins are known to be important mediators of
inflammation (Bamba et al., 2003). During inflammation,
production levels of PGE2 and LTB4 as pro- inflammatory
metabolites derived from arachidonic acid are up-
regulated, whereas EET as an anti- inflammatory metab-
olite derived from arachidonic acid is down- regulated
(Zhang et al., 2018). Like previous references, our results
also showed that treatment with L. reuteri LM1071signifi-
cantly decreased the production of PGE2 and LTB4 but
increased the production of EET.
Excessive production of NO and pro- inflammatory cy-
tokines might cause a wide range of severe cell injuries and
induce inflammation (Tak & Firestein, 2001). LPS is rec-
ognized by macrophage TLR- 4, leading to the activation of
immune- associated pathways such as MAPK and NF- κB
pathways (Shanura Fernando et al., 2018). Such activation
can lead to increased production of NO, PGE2 and pro-
inflammatory cytokines (Yu et al., 2019). LPS can activate
inflammatory responses, resulting in up- regulated pro-
duction of inflammatory mediators such as NO, COX- 1,
COX- 2 and pro- inflammatory cytokines (Hu et al., 2008).
Our results showed that L. reuteri LM1071 exhibited an
anti- inflammatory activity through down- regulating the
production of NO in LPS- stimulated RAW264.7 cells. The
expression of iNOS, a gene for the generation of NO, was
also significantly decreased depending on the concen-
tration of L. reuteri. In addition, L. reuteri LM1071 sup-
pressed the production of COX- 2 at both gene and protein
levels (Figure 4). COX- 2 is one of the most important bio-
markers to confirm the variation of inflammation in LPS-
stimulated RAW264.7 cells (Lee et al., 2008; Wang et al.,
2018).
In conclusion, the current study demonstrates that L.
reuteri LM1071has an anti- inflammatory potential by in-
hibiting the production of inflammation mediators such as
NO, eicosanoids such as PGE1 & PGE2, pro- inflammatory
cytokines and COXs protein. It can also enhance the pro-
duction of inflammation- associated genes such as IL- 11,
BMP4, LEFTY2 and EET metabolite.
ACKNOWLEDGEMENT
This study was supported by a Research Program funded
by LACTOMASON in Korea.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
ORCID
Woo Jung Park https://orcid.org/0000-0001-9804-3444
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How to cite this article: Jang, A.- Y., Rod- in, W.,
Monmai, C., Sohn, M., Kim, T.- R., Jeon, M.- G. &
et al. (2021) Anti- inflammatory potential of
Lactobacillus reuteri LM1071 via eicosanoid
regulation in LPS- stimulated RAW264.7 cells.
Journal of Applied Microbiology, 00, 1– 9. https://
doi.org/10.1111/jam.15331