Therapeutic effect and mechanism study of L-cysteine derivative 5P39 on LPS-induced acute lung injury in mice

Article abstract of DOI:10.1016/j.ejphar.2019.172893

Organosulfur compounds, such as L-cysteine, allicin and other sulfur-containing organic compounds in Allium species, have been proposed to possess many important physiological and pharmacological functions. A novel L-cysteine derivative, t-Butyl S-allylthio-L-cysteinate (5P39), was designed and synthesized by combining L-cysteine derivative and allicin pharmacophore through a disulfide bond. This study aimed to explore the effects and mechanisms of 5P39 on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice. At the experimental concentration (5, 10 and 20 μM), 5P39 suppressed the excessive secretion of nitric oxide (NO) and interleukin-6 (IL-6) in mice peritoneal macrophages stimulated by LPS. A mouse model of ALI was established by tracheal instillation of LPS for 2 h before 5P39 (30 and 60 mg/kg) administration. The results showed that 5P39 treatment down-regulated the wet/dry weight ratio (W/D ratio) of lungs and reduced the protein concentration, the number of total cells as well as the myeloperoxidase (MPO) activity in bronchoalveolar lavage fluid (BALF). 5P39 administration improved the histopathological changes of lungs in ALI mice with the decreased levels of pro-inflammatory cytokines in BALF. The inhibitory effects of 5P39 on the toll-like receptor 4 (TLR4) expression and macrophages accumulation in lung tissues were observed by immunohistochemistry. Additionally, 5P39 significantly attenuated the LPS-activated high expression of key proteins in TLR4/MyD88 signaling pathway. Taken together, the present study showed that 5P39 effectively alleviate the severity of ALI, and its mechanism might relate to the inhibition of LPS-activated TLR4/MyD88 signaling pathway, demonstrating a promising potential for further development into an anti-inflammatory drug candidate.

Full text of DOI:10.1016/j.ejphar.2019.172893

Journal Pre-proof
Therapeutic effect and mechanism study of L-cysteine derivative 5P39 on LPS-
induced acute lung injury in mice
Yanting He, Yalei Zhao, Yuchen Feng, Anqi Ren, Yunyi Zhang, Yang Wang, Hong Li
PII:
S0014-2999(19)30845-3
DOI:
https://doi.org/10.1016/j.ejphar.2019.172893
EJP 172893
Reference:
To appear in: European Journal of Pharmacology
Received Date: 2 September 2019
Revised Date: 11 December 2019
Accepted Date: 20 December 2019
Please cite this article as: He, Y., Zhao, Y., Feng, Y., Ren, A., Zhang, Y., Wang, Y., Li, H., Therapeutic
effect and mechanism study of L-cysteine derivative 5P39 on LPS-induced acute lung injury in mice,
European Journal of Pharmacology (2020), doi: https://doi.org/10.1016/j.ejphar.2019.172893.
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2019 Published by Elsevier B.V.

HL and YW funded the study; YW and YLZ designed and synthesized compound
P39; HL, YYZ and YTH conceived and designed the experiments; YTH, YCF and
5
AQR performed the in vitro and in vivo experiments; YTH and YLZ wrote the
original draft; HL, YW and YYZ revised the manuscript. All authors reviewed the
manuscript.

Therapeutic effect and mechanism study of L-cysteine derivative
P39 on LPS-induced acute lung injury in mice
5
a,1
b,1
a
a
a
b,
Yanting He , Yalei Zhao , Yuchen Feng , Anqi Ren , Yunyi Zhang , Yang Wang *,
a,
Hong Li *
a
b
Department of Pharmacology, Department of Medicinal Chemistry, School of
Pharmacy, Fudan University, Shanghai 201203, China
1
These authors contributed equally to this work.
*
Corresponding authors. E-mail addresses: lxzhang@shmu.edu.cn (H. Li);
wangyang@shmu.edu.cn (Y. Wang).
ABSTRACT
Organosulfur compounds, such as L-cysteine, allicin and other sulfur-containing
organic compounds in Allium species, have been proposed to possess many important
physiological and pharmacological functions. A novel L-cysteine derivative, t-Butyl
S-allylthio-L-cysteinate (5P39), was designed and synthesized by combining
L-cysteine derivative and allicin pharmacophore through a disulfide bond. This study
aimed to explore the effects and mechanisms of 5P39 on lipopolysaccharide
(
LPS)-induced acute lung injury (ALI) in mice. At the experimental concentration (5,
0 and 20 µM), 5P39 suppressed the excessive secretion of nitric oxide (NO) and
1
interleukin-6 (IL-6) in mice peritoneal macrophages stimulated by LPS. A mouse
model of ALI was established by tracheal instillation of LPS for 2 h before 5P39 (30
and 60 mg/kg) administration. The results showed that 5P39 treatment
down-regulated the wet/dry weight ratio (W/D ratio) of lungs and reduced the protein
concentration, the number of total cells as well as the myeloperoxidase (MPO)
activity in bronchoalveolar lavage fluid (BALF). 5P39 administration improved the
histopathological changes of lungs in ALI mice with the decreased levels of

pro-inflammatory cytokines in BALF. The inhibitory effects of 5P39 on the toll-like
receptor 4 (TLR4) expression and macrophages accumulation in lung tissues were
observed by immunohistochemistry. Additionally, 5P39 significantly attenuated the
LPS-activated high expression of key proteins in TLR4/MyD88 signaling pathway.
Taken together, the present study showed that 5P39 effectively alleviate the severity
of ALI, and its mechanism might relate to the inhibition of LPS-activated
TLR4/MyD88 signaling pathway, demonstrating a promising potential for further
development into an anti-inflammatory drug candidate.
Keywords: t-Butyl S-allylthio-L-cysteinate; Acute lung injury; Lipopolysaccharide;
Inflammation; Macrophages; Toll-like receptor 4
1
. Introduction
Acute lung injury (ALI) is an inflammatory disease with high morbidity and
mortality (Ye et al., 2019). ALI or its more severe form, acute respiratory distress
syndrome (ARDS), is characterized by severe pulmonary edema, pneumonic cell
aggregation and excessive production of inflammatory cytokines, leading to a strong
pulmonary inflammatory response (Xu et al., 2015; Matthay et al., 2017).
Epidemiological study has reported that ALI remains an important public health issue
worldwide and a major challenge for clinicians (Bellani et al., 2016). Although some
therapeutic strategies have been taken to improve functional outcomes, the mortality
in ALI patients remains high (Wang et al., 2019b). Therefore, there is an urgent need
to study the underlying mechanisms and novel therapeutic methods of ALI.
LPS is a major component of the cell wall of Gram-negative bacteria and has
been widely used to establish experimental models for ALI drug development (Yao et
al., 2017). The TLR family is the most important subgroup of pathogen recognition
receptors (PRRs) (Pandey et al., 2019). Toll-like receptor 4 (TLR4), as the most
primary response receptor of LPS (Zhou et al., 2019), is one of the important
members of the TLR family.
LPS activates TLR4, and then induces phosphorylation of nuclear transcription

factor-κB (NF-κB) via the adaptor protein (myeloid differentiation factor 88, MyD88)
and ubiquitin ligase (tumor necrosis factor receptor-associated factor 6, TRAF6) (Xia
et al., 2019). Subsequently, pro-inflammatory cytokines such as tumor necrosis
factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) were expressed in
large amounts (Xia et al., 2019; Liu et al., 2019). These inflammatory mediators
initiate and amplify the inflammatory response and then cause lung injury (Zhang et
al., 2014; Zhou et al., 2016).
Recently, ample publications (Kashfi and Olson, 2013; Song et al., 2014; Zheng
et al., 2017 and references therein) have probed organosulfur compounds, such as
L-cysteine, methionine, allicin and other sulfur-containing organic compounds in
Allium species (Fig. 1A), which have been proposed to possess many important
physiological and pharmacological functions. L-cysteine derivative
S-propargyl-cysteine was reported exerting anti-inflammatory (Gong et al., 2011) and
anti-oxidative effects (Pan et al., 2012). S-allylmercaptocysteine showed protective
effect on cisplatin nephrotoxicity (Zhu et al., 2017). Miron synthesized
S-allylthio-captopril (CPSSA) by combining captopril with allicin through a disulfide
bond, and the study of its biological effect proved that CPSSA not only possessed the
advantages of both captopril and allicin, but also showed more potent
antihypertensive effect than captopril through synergic effect (Miron et al., 2004;
Miron et al., 2010). In our previous study, a series of conjugates of
Danshensu-cysteine derivatives were reported to exert significant cytoprotective
effects by anti-oxidative and anti-apoptotic activities (Dong et al., 2009; Jia et al.,
2
012; Pan et al., 2013; Liu et al., 2013; Seetapun et al., 2013; Pan et al., 2015; Ren et
al., 2018).
In a continuing effort to identify the bioactivities of these sulfur-containing
organic compounds, tert-butyl S-allylthio-L-cysteinate (5P39, Fig. 1B) was designed
and synthesized by conjugating L-cysteine derivative and allicin pharmacophore
through a disulfide bond based on the combination principles. In this work, 5P39
demonstrated to be a promising potential anti-inflammatory agent with a mechanism
related to the inhibition of LPS-activated TLR4/MyD88 signaling pathway.

2
. Materials and Methods
2
.1. Chemical synthesis of 5P39
Target compound 5P39 was prepared via the synthetic route described in Fig. 1B.
The oxidation of diallyl disulfide (1) with 30% H O afforded allicin (2), which
2
2
followed by reaction with L-cysteine hydrochloride to give S-allylthio-L-cysteine (3)
in 90% yield for two steps. Compound 3 was then converted into 5P39 by
transesterification reaction with tert-butyl acetate in the presence of HClO in 24%
4
yield, and byproduct 4 was obtained meanwhile in yield of 12%.
To a rapidly stirred solution of compound 1 (973 mg, 6.65 mmol) in acetic acid
(
4.0 ml) was added dropwise a mixture of 30% hydrogen peroxide (1.0 ml, 9.81 mmol)
in acetic acid (3.0 ml) during 0.2 h at 0 � . Then the mixture was continually stirred at
� for 2 h and at r.t. for 4 h, and poured into ice-water (50 ml). The mixture was
extracted with CH Cl (4 × 50 ml), and the combined organic solvent was washed
0
2
2
with saturated aqueous Na CO solution (3 × 50 ml), dried over anhydrous Na SO ,
2
3
2
4
filtered and concentrated to give a residue (1.08 g) of crude product 2. To a solution of
the residue (450 mg) in water (40 ml) was added L-cysteine hydrochloride (2.0 g, 12.7
mmol) and enough sodium bicarbonate to adjust pH 6. White precipitate appeared
immediately and was filtered after standing for 0.33 h, and then washed with water
and ether successively to give a white solid. The obtained white solid was dissolved in
9
% HCl (aq.) and extracted with ether. To the aqueous layer was added NaHCO to
3
adjust pH 6 and white solid precipitated out. The white solid was filtered, washed
with water and ether, and dried to give compound 3 (480 mg, 90% for two steps) as a
white powder, mp: 187-190 � (Freeman et al., 1994. mp: 198-199 � ).
To a rapidly stirred solution of compound 3 (3.49 g, 18.0 mmol) in t-butyl
acetate (36 ml, 268 mmol) at 0 � was slowly added HClO (1.53 ml, 26.8 mmol). The
4
mixture was stirred at room temperature for 12 h then adjusted to pH 9 by 10 %
Na CO (aq.), and then extracted with CH Cl (3 × 100 ml). The combined organic
2
3
2
2
solvent was dried over anhydrous Na SO , filtered and concentrated to give an oil.
2
4
Silica gel flash column chromatography using CH Cl as the eluent gave t-butyl
2
2
S-allylthio-L-cysteinate (5P39, 1.10 g, 24%) and t-butyl S-(t-butyl)-L-cysteinate (4,

1
0
.50 g, 12 %) as pale yellow oil respectively. Compound 5P39: H NMR (400 MHz,
CDCl ) δ 5.80-5.95 (m, 1H), 5.25-5.16 (m, 2H), 3.69-3.66 (m, 1H), 3.35 (d, J = 7.3
3
Hz, 2H), 3.10 (dd, J = 13.4, 4.5 Hz, 1H), 2.84 (dd, J = 13.4, 8.0 Hz, 1H), 1.48 (s, 9H).
1
3
C NMR (CDCl , 100 MHz) δ 172.3, 132.7, 118.1, 81.1, 53.8, 43.7, 41.6, 27.4.
3
+
1
ESI-MS m/z: 250.1 (M + H ). Compound 4: H NMR (400 MHz, CDCl ) δ 3.54 (dd, J
3
=
7.4, 4.5 Hz,1H), 2.83 (ddd, J = 19.7, 12.3, 5.9 Hz, 2H), 1.48 (s, 9H), 1.33 (s, 9H);
+
ESI-MS (m/z): 234.2 (M + H ).
2
.2. Reagents and antibodies
LPS (Escherichia coli 055: B5), dexamethasone (DEX, used as the positive
control in the in vitro experiments) and 3-(4,5-dimethyl-2-thiazolyl)
-
2,5-diphenyl-2-H-tetrazolium bromide (MTT) were purchased from Sigma–Aldrich
St. Louis, MO, USA). Fetal bovine serum (FBS) and RPMI-1640 medium were
(
purchased from Biological Industries (Kibbutz Beit-Haemek, Israel).
Diaminobenzidine (DAB) assay kit was purchased from Zhongshan Jinqiao
Biotechnology Co., Ltd (Beijing, China). Dexamethasone sodium phosphate injection
(DEX, used as the positive control in LPS-induced ALI model) was purchased from
Chongqing Laimei Pharmaceutical Co., Ltd. (Chongqing, China). The enhanced
bicinchoninic acid (BCA) protein assay kit and horseradish peroxidase
(HRP)-conjugated IgG were purchased from Beyotime (Jiangsu, China).
Enzyme-linked immunosorbent assay (ELISA) kits for mouse IL-6, TNF-α and IL-1β
detection were purchased from Boatman Biotechnology Co., Ltd. (Shanghai, China).
Myeloperoxidase (MPO) determination kit was purchased from Nanjing Jiancheng
Bioengineering Institute (Jiangsu, China). Antibody specific to inducible nitric oxide
synthase (iNOS) was purchased from Cell Signaling Technology (Danvers, MA,
USA). Antibodies specific to TLR4, F4/80, MyD88, TRAF6, phospho-nuclear
factor-κB p65 (p-NF-κB p65), β-actin were purchased from Abcam (Cambridge, UK).
Antibodies specific to NF-κB p65 were purchased from Shanghai Abways
Biotechnology Co., Ltd (Shanghai, China). Other reagents used in Western blot were
purchased from Beyotime (Jiangsu, China).
2
.3. Animals and ethics statement

Male BALB/c mice (14-18g) were purchased from Shanghai Slaccas Laboratory
Animal Co., Ltd. (SPF II Certificate; No. SCXK2017-0005) and housed in the SPF
Laboratory Animal Room with a 12 h light/dark cycle.
All animals received humane care in accordance with the “Guide for the Care
and Use of Laboratory Animals” published by the National Institutes of Health. All
animal experiments were approved by the Animal Ethical Committee of School of
Pharmacy, Fudan University (No. 2018-03-YL-LH-01).
2
2
.4. Effect of 5P39 in vitro
.4.1. Harvest and culture of peritoneal macrophages
Male BALB/c mice were intraperitoneally injected with 1 ml of 5% sodium
thioglycollate medium to activate peritoneal macrophages. Mice were killed 5 days
after injection to harvest peritoneal macrophages. These cells were adjusted to 1×10
cells/ml with 10% FBS-RPMI-1640 and cultured at 200 µl/well in a 96-well plate.
The cells used for experiment were divided into 10 groups including normal group ,
6
5
P39 (5, 10, 20 µM) group, DEX (10 µM) group, LPS (1 µg/ml) group, LPS (1 µg/ml)
5P39 (5, 10, 20 µM) group, and LPS (1 µg/ml) + DEX (10 µM) group. The
+
DEX-treated groups were taken as positive control.
Peritoneal macrophages were cultured at 37 � in a humidified atmosphere
containing 5% CO . The whole experiment procedures were carried out in aseptic
2
conditions and all materials were previously sterilized and pyrogen-free (Xu et al.,
2
015; Wu et al., 2013).
2
.4.2. Detection of cell viability and inflammatory mediators
Peritoneal macrophages were treated by 5P39 with or without LPS stimulation
for 24 h, and the culture supernatants were collected for detection. Then culture
mediums containing 0.5 mg/ml MTT were added to the wells. After 4 h of incubation,
the mediums were discarded and the formazan crystals generated by the action of
mitochondrial enzymes in viable cells were dissolved in dimethyl sulfoxide (DMSO).
The absorbance of each well was measured at 570 nm using a microplate reader (Wu
et al., 2013). The change of absorbance measured by the MTT method reflected the
effect of 5P39 on cell activity.

Nitric oxide (NO) production in the peritoneal macrophages was detected by the
Griess reagent to test its end product, nitrite (Wu et al., 2013). Griess reagent
contained a mixture of equal volume of 0.1% N-(1-naphthyl) ethylenediamine
dihydrochloride in H O and 1% sulfanilamide in 5% H PO . Briefly, 50 µl culture
2
3
4
supernatant collected previously was mixed with 50 µl Griess reagent in a 96-well
plate. The absorbance was tested at 540 nm. The concentration of nitrite was
determined according to a standard curve formed with sodium nitrite.
IL-6 production in the peritoneal macrophages was tested by mouse IL-6 ELISA
kit according to the manufacturer’s instructions.
2
.4.3. Culture of Ana-1 cell
Ana-1 cells were obtained from ATCC (ATCCPTA-2662, Manassas, VA), which
was a continuous cell line of murine macrophages. The cells in this experiment were
6
adjusted to 2×10 cells/ml with 10% FBS-RPMI-1640 and cultured at 1 ml/well in a
6
-well plate. These cells were divided into 3 groups including normal group, LPS (0.5
µg/ml) group and 5P39 (20 µM) group. The culture conditions of Ana-1 cells were
similar to peritoneal macrophages in section 2.4.1. The cells treated by LPS with or
without 5P39 administration for 30 min or 24 h were collected for Western blot
analysis (in section 2.10).
2
.5. Experimental design of Animals
Male BALB/c mice were randomly divided into 5 groups (n = 6): Sham-operated
animals group (sham group), LPS (3 mg/kg) stimulated group (model group), LPS (3
mg/kg) + 5P39 group (5P39 30 mg/kg group and 5P39 60 mg/kg group), and LPS (3
mg/kg) + DEX (4 mg/kg) group. The doses of LPS and drugs were determined based
on our previous experiments and preliminary results.
5
P39 was ground and dissolved in PEG400. Then the 5P39 solution was diluted
with 1% carboxymethylcellulose sodium (CMC-Na) and administered intragastrically
i.g.). LPS was dissolved in sterilized normal saline (NS). The model of ALI was
(
established as previously described (Xu et al., 2015; Xie et al., 2013). The mice were
anesthetized with 20% urethane (1 g/kg) before intratracheal instillation of NS (sham
group, i.t.) or LPS (i.t.). 2 h after NS or LPS treatment, mice were administrated with

vehicle (1% CMC-Na-5% PEG400, i.g.), 5P39 (i.g.), or DEX (tail vein, i.v.). The
mice in sham group and model group were given vehicle intragastrically.
Mice were killed 24 h after LPS instillation. Superior lobe of left lung was used
to collect bronchoalveolar lavage fluid (BALF), which was flushed four times with
0
.5 ml NS. The BALF from each sample was centrifuged to isolate the cells. The
supernatant was collected for subsequent analysis of protein, MPO, and
pro-inflammatory cytokines, while the total cells were gathered and resuspended in
1
00 µl 0.01 mol/L phosphate buffered saline (0.01 M PBS, pH 7.4) for cell counts.
Inferior lobe of left lung and middle lobe of right lung were stored at -80 � for
subsequent analysis. Superior lobe of right lung was taken for histopathologic
examination. Inferior lobe of right lung of mice was separated and weighed for
wet/dry weight ratio (W/D ratio) count.
2
.6. Histopathologic evaluation of lung tissues
The superior lobe of right lung was immediately removed and fixed in 4%
formaldehyde in 0.01 M PBS (pH 7.4) for histopathological evaluation (Xu et al.
015). Then the tissues were successively dehydrated in graded alcohols and
2
embedded in paraffin. The paraffin sections (5 µm) mounted on glass slide were
stained with hematoxylin and eosin (H&E) to observe the histopathological changes
of lung under light microscopy 200 × magnification. Four pictures were randomly
taken in each pathological section (n = 6) to measure the cell-covered area fraction (%)
using the ImageJ system (Cheng et al., 2012). The area covered by cells was
considered as “relative cell area”. The area except the major vein and lung hilum was
considered as “overall area”. The cell-covered area fraction (%) = relative cell area /
overall area × 100%.
2
.7. Measurement of the lung wet/dry weight ratio, the protein concentration, the total
cells numbers, and the MPO activity in lung
The inferior lobe of right lung of mouse was weighed immediately to acquire the
“wet weight”, and then dried in an oven to constant weight to acquire the “dry
weight”. Finally, the ratio of wet weight to dry weight was calculated, called wet/dry
weight ratio (W/D ratio) (Xu et al. 2015).

The protein concentration and MPO activity in BALF were measured by BCA
protein assay kit and MPO assay kit respectively according to the manufacturer’s
instructions. The total cell numbers in BALF were counted via a hemocytometer.
2
.8. Immunohistochemistry
Immunohistochemical staining was performed as previously described (Xu et al.,
015). First, the high temperature antigen retrieval was carried out in the
2
deparaffinized slide and the endogenous peroxidase was quenched. The non-specific
binding sites of the sections were blocked with 5% BSA. Next, sections were
incubated with rabbit monoclonal anti-TLR4 antibody (1:100) or rat anti-F4/80
antibody (1:100) overnight at 4 � in a humid chamber. The next day, these sections
were incubated with HRP-conjugated goat anti-rabbit or anti-rat IgG antibodies for 1
h at 37 � . Finally, the sections were visualized with the chromogenic substrate
solution DAB and counterstained with hematoxylin. Then the sections were observed
under light microscopy (Zeiss LSM 710). Four pictures were randomly taken in each
pathological section (n = 6) and the average optical density (AOD) of positive area
was measure by the ImageJ system (Chen et al., 2018). AOD = Integrated Optical
Density (IOD) / Area.
2
.9. ELISA assay of the levels of TNF-α, IL-1β and IL-6 in BALF
The levels of TNF-α, IL-1β and IL-6 in BALF were detected by mouse TNF-α,
IL-1β and IL-6 ELISA kit according to the manufacturer’s instructions.
2
.10. Western blot analysis
Western blot analysis was performed as previously described (Feng et al., 2019).
Ana-1 cells and lung tissues were treated in radio immunoprecipitation assay (RIPA)
lysis reagent. Protein concentration was determined by the BCA protein assay kit. The
protein samples were fractionated on 8% sodium dodecyl sulfate-polyacrylaminde gel
electrophoresis (SDS-PAGE), blotted onto polyvinylidene difluoride (PVDF)
membranes. After blocking with Tris-buffered saline and Tween 20 (TBST)
containing 5% skim milk for 45 min at room temperature, the membranes were
incubated overnight at 4 � with the antibodies against TLR4, MyD88, TRAF6,
phospho-NF-κB p65, NF-κB p65, iNOS, and β-actin (1:3000 dilution for TRAF6, 1:

1
000 dilution for the others). On the next day, the membranes were washed three
times with TBST, and then incubated with horseradish peroxidase (HRP)-conjugated
IgG (1:2000 dilution) for 3 h. Afterwards, signals were detected by universal
electrochemiluminescence (ECL) reagent and captured with a camera-based imaging
system (Alpha Innotech, Santa Clara, CA, USA). The β-actin protein was served as
internal control. The NF-κB p65 protein was served as reference of p-NF-κB p65.
2
.11. Statistical analysis
Experimental data were presented as means ± standard deviation (S.D.).
One-way analysis of variance (ANOVA) was performed for multiple group
comparisons. If data were found to have significant changes, post-hoc comparisons
were performed using Fisher's PLSD. P < 0.05 was regarded as statistically
significant.
3
. Results
3
.1. Effects of 5P39 on cell viability and inflammatory responses in vitro
The peritoneal macrophages were isolated and cultured to evaluate the influences
of 5P39 on cell viability, NO and IL-6 production. As shown in Fig. 2A, 5P39 had no
adverse effect on cell viability with or without LPS stimulation. As shown in Fig. 2B
and C, stimulations with LPS 1 µg/ml increased the levels of NO and IL-6 in the
culture supernatants, and were significantly reduced with 5P39 treatment (P < 0.05).
The 5P39 treatment alone had no effect on NO and IL-6 secretion compared with
control group.
Ana-1 cells were incubated with 5P39 and LPS for 30 min or 24 h, then the cells
were harvested to test the expression of key proteins in TLR4/MyD88 signaling
pathway. As shown in Fig. 2D, 5P39 administration for 30 min significantly inhibited
the expression of TLR4, TRAF6, iNOS and p-NF-κB p65 (P < 0.05), while the
expression of MyD88 was not affected. As shown in Fig. 2E, 5P39 treatment for 24 h
significantly reduced the expression of MyD88, TLR4, TRAF6 and iNOS (P < 0.05),
but had no effect on the expression of p-NF-κB p65.

3
.2. Effects of 5P39 on the lung injury in LPS-induced ALI mice
To assess the effects of 5P39 (30 and 60 mg/kg) on the lung injury in
LPS-induced ALI mice, the histopathological changes of the lung tissues were
detected. As shown in Fig. 3A, compared with the sham group, the lung tissues of the
model group were characterized by thickening of the alveolar wall, interstitial
hemorrhage, infiltration of neutrophils and macrophages, and edema. 5P39 (30 and 60
mg/kg) administration alleviated these symptoms. The results of semi-quantitative
analysis also showed that the cell-covered area fraction of lung tissues in the model
group was significantly increased, while 5P39 (30 and 60 mg/kg) treatment markedly
inhibited the change.
To further evaluate the severity of lung edema, we measured lung wet/dry (W/D)
ratio (Fig. 3B), a standard index of lung edema (Ye et al., 2019). Treatment of 5P39
markedly lowered the lung edema induced by LPS stimulation (P < 0.001).
As vascular leakage is a feature of ALI, we detected the protein concentration
and the total cell number in BALF. As shown in Fig. 3C and D, compared with the
sham group, the challenge of LPS to mice markedly upregulated the protein
concentration and the number of total cells, while 5P39 obviously suppressed the
LPS-induced damage (P < 0.001).
MPO activity, a reliable marker of polymorphonuclear neutrophil infiltration and
activation (Ye et al., 2019), was also tested in this study. As shown in Fig. 4E, MPO
activity in BALF increased dramatically after LPS administration. 5P39 (30 and 60
mg/kg) obviously inhibited the increasing MPO activity challenged by LPS (P <
0
.001).
3
.3. Effects of 5P39 on the production of pro-inflammatory cytokines (TNF-α, IL-1β
and IL-6) in BALF of LPS-induced ALI mice
It was found that the levels of TNF-α (Fig. 4A), IL-1β (Fig. 4B) and IL-6 (Fig.
4
C) in BALF were significantly upregulated after LPS instillation when compared
with the sham group (P < 0.01). Administration of 5P39 clearly downregulated the
secretion of these pro-inflammatory cytokines (P < 0.05).

3
.4. 5P39 attenuated TLR4 expression and inflammatory cells accumulation in lung
tissues in LPS-induced ALI mice
The effect of 5P39 on TLR4 expression and on the infiltration of macrophages
(F4/80 expression) in lung tissues of mice was evaluated. The results showed intense
immunostaining for TLR4 expression (Fig. 5A) and macrophages infiltration (Fig. 5B)
in lung tissues of ALI model group. Compared with model group, ALI mice treated
with 5P39 and DEX showed relatively low levels of staining in the lung tissues. The
results of semi-quantitative analysis were consistent with the above observations.
3
.5. 5P39 inhibited the LPS-activated TLR4/MyD88 signaling pathway
As shown in Fig. 6A, LPS instillation markedly increased TLR4 expression in
lung tissues, whereas the increasing was inhibited by 5P39 (30 and 60 mg/kg)
administration. As shown in Fig. 6 (B and C), in comparison to the sham group, LPS
treatment dramatically upregulated the expression of MyD88 and TRAF6 in lung
tissues, while 5P39 clearly suppressed the expression (P < 0.05).
Similarly, LPS obviously upregulated the expression of p-NF-κB p65 and iNOS
(Fig. 6D and E) in lung tissues, whereas 5P39 (60 mg/kg) significantly inhibited the
expression caused by LPS (P < 0.01).
4
. Discussion
In this study, LPS stimulated an inflammatory like response in mouse peritoneal
macrophages. The result of cell viability assay showed that 5P39 had no adverse
effect on cell viability at the experimental concentration. In addition, the results of
NO and IL-6 detection showed that 5P39 effectively inhibit the secretion of
inflammatory mediators, indicating that 5P39 had obvious anti-inflammatory effect.
5
P39 inhibited the overexpression of key proteins in TLR4/MyD88 signaling pathway
when Ana-1 cells were stimulated with LPS for 30 min or 24 h. These results showed
an interference of 5P39 with the immediate TLR4 signaling axis.
Based on the results of in vitro experiments, the anti-inflammatory effect and
mechanism of 5P39 were further studied by establishing a mouse ALI model with

LPS. ALI is a common clinical problem with high morbidity and mortality in some
emergency infectious diseases (Li et al., 2018; Jiang et al., 2018). Pulmonary edema,
inflammatory cell infiltration and secretion of a large number of inflammatory
cytokines are the main pathological features of ALI (Tan et al., 2018; Lin et al., 2019).
In the present study, we found that 5P39 significantly suppressed lung W/D ratio, the
protein concentration and the number of total cells in BALF. H&E staining results
also showed that 5P39 administration clearly improved lung tissues hemorrhage,
inflammatory cell infiltration and alveolar wall thickening caused by LPS. The above
results indicated that 5P39 could obviously alleviate LPS-induced ALI.
During LPS-induced ALI, neutrophils are the earliest immune cells that are
recruited to the site of inflammation and are essential for host defense (Grommes and
Soehnlein, 2011; Peng et al., 2019). However, excessive activation of neutrophils
leads to massive release of pro-inflammatory cytokines, destruction of the alveolar
basement membrane, and formation of pulmonary edema (Peng et al., 2019; Zhang et
al., 2018). MPO is an enzyme mainly found in neutrophils, which is proportional to
the number of neutrophils in lung tissues and served as an indicator of neutrophil
influx into tissues (Jiang et al., 2018; Niu et al., 2015). Experimental results
demonstrated that LPS markedly upregulated the activity of MPO, while 5P39
observably inhibited the upregulation. Additionally, previous study suggested that
pro-inflammatory cytokines such as TNF-α, IL-1β and IL-6 could activate neutrophils
and macrophages accumulation, induce lung tissues injury, then lead to pulmonary
edema (Zhao et al., 2017; Peng et al., 2019). After LPS challenge, TNF-α is
considered to be the first to amplify the inflammatory response through triggering
leukocyte activation and release of other factors (Li et al., 2018). IL-1β, another
important pro-inflammatory cytokine, induces the production of secondary
inflammatory cytokines (Veerdon et al., 2011). IL-6 promotes the release of
vasoactive substances and neutrophils, causing a cascade of inflammation and
aggravating tissues injury (Shin et al., 2015). Our study showed a beneficial effect of
5
P39 on protecting against ALI by reducing these pro-inflammatory cytokines
production, which were consistent with the results of the in vitro experiments.

Macrophages are widely distributed in various organs and tissues of the body,
and are non-specific immune cells of the body. Macrophages have
immunomodulatory effects such as antigen presentation and phagocytic necrosis
(Benedetto et al., 2019). However, when the body is seriously infected and the
inflammatory reaction in the tissues is too strong, the hyperactive macrophages
release a series of inflammatory mediators and chemokines (Niesle et al., 2014).
Subsequently, a large number of inflammatory cells such as neutrophils and
macrophages adhere to the lung tissues, causing microcirculatory disturbances in the
lungs and aggravating the occurrence of lung injury (Silva, 2010). F4/80 is a cell
surface glycoprotein expressed in a variety of mature macrophages and is a mouse
macrophage marker (Dos Anjos Cassado, 2017). In this study, the expression of F4/80
was detected in lung tissues by immunohistochemistry. Histopathological observation
revealed that 5P39 significantly inhibited macrophages infiltration.
LPS has been reported to be a commonly used stimulator to induce ALI in mice
(
Lin et al., 2019). After invading the body, LPS bind to the specific receptor TLR4 on
surface of cells such as macrophages and tracheal epithelial cells, which will lead to
cells activation (Liu et al., 2019). Subsequently, a series of downstream adaptor
proteins and protein kinases such as MyD88 and TRAF6 are activated, resulting in the
activation of transcription factor NF-κB and the production of a large number of
chemokines and inflammatory mediators, causing lung injury (Wang et al., 2019a;
Zhang et al., 2018).
In the present study, it showed that LPS instillation resulted in a significant
increase of TLR4 expression in lung tissue, 5P39 notably inhibited this increase. The
testing results of key proteins in lung tissues revealed that 5P39 down-regulated the
levels of TLR4, MyD88, TRAF6 and p-NF-κB p65 activated by LPS, suggesting that
5
P39 might inhibit LPS-activated TLR4/MyD88 signaling pathway, which was
consistent with the in vitro results. All these results showed that the anti-inflammatory
effect of 5P39 might be related with down-regulating TLR4/MyD88 signaling
pathway.
Furthermore, the expression of iNOS was investigated in lung tissue by Western

blot. Activation of the TLR4 signal arouses the expression of many genes associated
with pre-immune and inflammation, such as iNOS (Hu et al., 2017; He et al., 2017).
iNOS is one of the key enzymes catalyzes the production of NO from arginine in
inflammatory reaction (Wang et al., 2017). NO, a short-lived gaseous signaling
molecule, protects against invading pathogens and is therefore critical for the
inflammatory response and innate immune system (Li et al., 2018; Cinelli et al., 2019).
However, high level of NO is released into the blood circulation, stimulating
inflammatory cell recruitment and activation in the ALI (Li et al., 2015). Besides,
+
under pathological conditions, NO inhibits the Na channel, thereby reducing the fluid
transport capacity of alveolar epithelial cells, leading to pulmonary edema (Althaus et
al., 2011). The experimental results displayed that the expression of iNOS in lung
tissue was significantly increased in the LPS-induced group and clearly decreased
after 5P39 treatment, which was consistent with the in vitro results. The results
indicated that 5P39 might limit the secretion of NO by decreasing the expression of
iNOS, thereby relieving lung injury caused by LPS.
DEX is a corticosteroid that is widely used in the treatment of various
inflammatory diseases (Caceres-del-Carpio et al., 2016; Giuliano et al., 2012). It is
also often selected as a positive control in the study of acute lung injury models (Xu et
al., 2015; Xie et al., 2012). However, there are many adverse effects or limited
therapeutic effects for the existing corticosteroids (Mokra et al., 2019). In this
experiment, 5P39 showed a similar effect to DEX in improving LPS-induced lung
injury and aimed to become an anti-inflammatory drug candidate in the future.
In conclusion, the present research showed that 5P39 significantly improved
lung injury in LPS-induced ALI mice by relieving lung tissue edema, reducing
inflammatory cells infiltration, and inhibiting excessive expression of inflammatory
factors. The anti-inflammatory mechanism of 5P39 might relate to inhibition of the
LPS-TLR4/MyD88 pathway. Consequently, 5P39 demonstrated a potential
therapeutic agent for the treatment of ALI.
Acknowledgments
This work was supported by grants from National Natural Science Foundation of

China (21472025 and 21877015 to Y. W.; 81274165 to H. L.).
Conflict of interests
The authors declare that there are no conflicts of interest.

Figure legends
Fig.1. (A) The structures of L-cysteine, allicin and their conjugated derivatives. (B)
The synthesis of compound 5P39.
Fig.2. Effects of 5P39 on LPS-induced inflammation in vitro. Peritoneal macrophages
were treated by 5P39 (5, 10, 20 µM) with or without LPS (1 µg/ml) stimulation for 24
h, then the cell viability (A) was detected by MTT assay and the culture supernatant
was collected for NO (B) and IL-6 (C) detection. Ana-1 cells were treated by LPS (0.5
µg/ml) with or without 5P39 (20 µM) for 30 min (D) or 24 h (E), then the key
proteins in TLR4/MyD88 signaling pathway were detected by Western blot. Data
*
**
***
were expressed as means ± S.D., n = 3. P < 0.05, P < 0.01 and P < 0.001
#
##
###
compared with the control group, P < 0.05, P < 0.01 and P < 0.001 compared
with the LPS group, tested by the One-way ANOVA and the Fisher’s PLSD.
Fig.3. Effects of 5P39 on the lung injury in LPS-induced ALI mice. Mice were
administrated with vehicle (i.g.), 5P39 (i.g.), or DEX (4 mg/kg, i.v.) 2 h after
instillation of NS or LPS (3 mg/kg). Lung sections were stained with H&E to observe
the histopathological changes of lung under light microscopy 200 × magnification.
The semi-quantitative analysis was done by measuring the cell-covered area fraction
(A). The inferior lobe of the right lung was collected to get the lung W/D ratio (B).
The protein concentration (C), the total cells numbers (D) and the MPO activity (E) in
BALF were measured at 24 h after LPS challenge. Data were expressed as means ±
*
**
***
S.D., n = 6. P < 0.05, P < 0.01 and P < 0.001 compared with the model group,
tested by the One-way ANOVA and the Fisher’s PLSD.
Fig.4. Effects of 5P39 on the production of pro-inflammatory cytokines (TNF-α,
IL-1β and IL-6) in BALF of LPS-induced ALI mice. Mice were administrated with
vehicle (i.g.), 5P39 (i.g.), or DEX 4 mg/kg (i.v.) 2 h after instillation of NS or LPS (3
mg/kg). The BALF was collected to detect the levels of TNF-α (A), IL-1β (B) and
*
IL-6 (C) 24 h after LPS challenge. Data were expressed as means ± S.D., n = 6. P <

**
***
0
.05, P < 0.01 and P < 0.001 compared with the model group, tested by the
One-way ANOVA and the Fisher’s PLSD.
Fig.5. 5P39 attenuated TLR4 expression and inflammatory cells accumulation in lung
tissues in LPS-induced ALI mice. Mice were administrated with vehicle (i.g.), 5P39
(i.g.), or DEX (i.v.) 2 h after instillation of NS or LPS (3 mg/kg). TLR4 expression (A)
and macrophages infiltration (B) were detected by immunohistochemistry analysis
under light microscopy 400 × magnification. The semi-quantitative analysis was done
by measuring the AOD.
Fig.6. 5P39 inhibited the LPS- activated TLR4/MyD88 signaling pathway and iNOS
production. Mice were administrated with vehicle (i.g.), 5P39 (i.g.), or DEX 4 mg/kg
(i.v.) 2 h after instillation of NS or LPS (3 mg/kg). Lung homogenates were prepared
and analyzed by Western blot with antibodies specific to TLR4 (A), MyD88 (B),
TRAF6 (C), p-NF-κB p65 (D) and iNOS (E). The β-actin protein was served as
internal control. The NF-κB p65 protein was reference of p-NF-κB p65. Data were
*
**
***
expressed as means ± S.D., n = 4. P < 0.05, P < 0.01 and P < 0.001 compared
with the model group, tested by the One-way ANOVA and the Fisher’s PLSD.

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