protective effects against silicainduced
lung injury and �brosis remain to be elucidated. The aim of the
present study was to investigate the effects of OA on oxidative
stress, and the expression of cytokines and collagen in silicotic
rats. Male rats were induced by intratracheal instillation of
silicosis (250mg
/
kg), with the exception of the control group
(NS). The rats in the OA group were intragastrically admin
-
istered with OA (60mg
/
kg
/
d). The rats in the solvent control
cellulose (10ml
/
kg) solution for 56 consecutive days. The
data showed that OA signi�cantly attenuated the extent of
silicosis �brosis by histopathologic analysis of the lung tissues.
In addition, oxidative stress activated by silica exposure, as
evidenced by increasing of malondialdehyde content, and
activities of superoxide dismutase and glutathione peroxidase
in the lung, was regulated by treatment with OA. Furthermore,
enzyme‑linked immunosorbent assay analysis showed that OA
signi�cantly decreased the levels of tumor necrosis factor‑
and transforming growth factor‑
analysis showed that OA significantly decreased collagen
types I and III. In investigating the mechanisms underlying
the action of OA, it was found that OA decreased the level
of phosphorylated AKT1, which in turn inactivated the tran
-
scriptional of nuclear factor (NF)‑
B in the development and
progress of silicosis. In conclusion, these results suggested that
the protective effects of OA were due, at least in part, to its
antioxidant activity and its ability to decrease the expression
of cytokines and collagen by modulating the AKT
/
NF‑
B
pathway.
Introduction
Silicosis, a form of occupational lung disease caused by the
inhalation of crystalline silica dust, is characterized by sili
-
cotic nodule formation and pulmonary interstitial �brosis
1
).
Occupational exposure to respirable crystalline silica dust
particles occurs in sand blasting, drilling, pulverizing, cutting
bricks and concrete blocks, grinding concrete and use of other
pneumatic equipment
2
) every year, therefore, silicosis is
an occupational health concern in developing and developed
countries
3
,
4
). In China, ~20,000cases of pneumoconiosis are
diagnosed each year, and silicosis is the most common, fastest
progressing and serious type
5
). The pathological process of
silicosis includes progressive in�ammation, �broblast prolif
eration and collagen deposition. In the initial in�ammatory
responses, alveolar macrophages, the �rst cells responded to
stimuli of the body, are important. Following exposure to silica,
a number of macrophages undergo apoptosis, resulting in the
production of reactive oxygen species (ROS), which include
hydroxyl radicals, superoxide anions, hydrogen peroxide,
singlet oxygen
6
,
7
) and nitric oxide (NO)
8
). The generation
of oxidants results in cell and lung damage; increase the expres
-
sion of inflammatory cytokines, including tumor necrosis
factor (TNF)‑
, and transforming growth
factor (TGF)‑
; activate cell signaling pathways, including the
mitogen‑activated protein kinase pathways; and phosphorylate
and activate speci�c transcription factors, including nuclear
factor (NF)‑
B, which intensi�es chronic in�ammation and
promotes pulmonary �broblasts to proliferate and synthesize
excess collagen
1
).
Oleanolic acid (3
‑hydroxyolean‑12‑en‑28‑oic acid; OA) is
a plantderived pentacyclic terpenoid, which exists naturally
free acid, or as an aglycone of triterpenoid saponins
9
). It has
Protective effects of oleanolic acid on oxidative
stress and the expression of cytokines and collagen
by the AKT
/
NF-
B pathway in silicotic rats
HAI
1
XUN WANG
2
, HAI
3
, YONGHENG WANG
1
,
DONG TANG
4
5
and
HUI WANG
2
1
2
3
Department of Pathogen and Microbiology, School of Basic Medical Sciences;
4
5
Tangshan, Hebei 063210, P.R. China
Received February 19, 2016; Accepted February 6, 2017
DOI: 10.3892/mmr.2017.6402
Correspondence to
: Mrs. Hai‑Bing Peng, Medical Experimental
Center of Jitang College, North China University of Science and
Technology, 20 Bohai Road, Tangshan, Hebei 063210, P.R.China
E‑mail: leizi19760728@sina.com
Key words
: oleanolic acid, silicosis,
oxidative stress, tumor necrosis
factor
, tumor growth factor‑
1, Akt
/
nuclear factor
B, collagen
PENG
etal:
PROTECTIVE EFFECTS OF OA IN SILICOSIS DURING LUNG INJURY AND FIBROSIS
3122
been shown to exhibit numerous pharmacological properties,
including hepatoprotective, anti‑oxidative, anti‑inammatory
and anticancer activities. Therefore, OA and its derivatives
possess a wide range of applications
10
). It has been reported
that OA may exert bene�cial effects on renal and liver �brosis
by activating nuclear factor, erythroid 2 like 2
11
) and has
been used as an oral treatment for human liver dysfunction
in China
10
). However, the effectiveness of OA in the treat
-
ment of �brotic lung diseases, including silicosis, remains to
be elucidated. It has been reported that the phosphatidylino
-
sitolkinase (PI3K)
/
Akt pathway is the most important
pathway for the �broblast to myo�broblast differentiation of
normal and diseased primary human lung �broblasts
12
). The
present study aimed to investigate the protective effects of
OA in an experimental model of silica‑induced in�ammation
and �brosis by examining the oxidation
/
antioxidant system,
TNF‑
, TGF‑
1, Akt
/
NF‑
B and collagen.
Materials and methods
Materials.
OA (purity >99.9%; cat. no.110742‑200513) was
purchased from the National Institutes for Food and Drug
Control (Beijing, China) and was suspended in 0.6% sodium
carboxymethyl cellulose for use. Crystalline SiO
2
(~95%;
15
µ
m), obtained from the National Institute of Occupational
Health and Poison Control, Chinese Center for Disease Control
and Prevention (Beijing, China), were subjected to grinding,
and heating for at 180˚C for 6h, followed by dilution with
sterile saline to a concentration of 50mg
/
ml in suspension,
autoclaved and stored at 4˚C. Enzyme‑linked immunosorbent
assay (ELISA) kits, malondialdehyde (MDA), superoxide
dismutase (SOD) and glutathione (GSH) test kits, and Masson's
stain were purchased from Nanjing Jiancheng Bioengineering
Institute (Nanjing, China). Phosphorylated (p)AKT1 (phospho
S473; cat. no.ab81283) and AKT (cat. no.ab28422) antibody
were purchased from Abcam (Cambridge, MA, USA). NF‑
B
antibody (cat. no.sc‑8008) was purchased from Santa Cruz
Biotechnology, Inc. (Dallas, TX, USA).
Animalsand treatment.
A total of
96
male adult Wistar rats
(aged 6‑8weeks and weighing 180‑200g) were purchased from
Vital River Laboratory Animal Technology Co., Ltd. (Beijing,
China). The animal experiments were reviewed and approved
by the Institutional Animal Care and Use Committee at the
North China University of Science and Technology (Tangshan,
China). The animals received food and water according to
guidelines set by the National Institutes of Health (Bethesda,
MD, USA) and were housed in an air‑conditioned room with a
h light
/
dark cycle at constant temperature (21˚C) and 55%
humidity for at least 1week prior to the experiments.
The rats were divided into four groups according to the
randomized block design, namely, a control group, model
group, solvent control group and OA group, with 24 rats in
each group. With the exception of the rats in the control group,
the rats were induced by intratracheal instillation of SiO
2
(250mg
/
kg). The rats in the OA group were intragastrically
administered with OA (60mg
/
kg) daily from the second day
following SiO
2
administration. The rats in the solvent control
group were gavaged daily with 0.6% sodium carboxymethyl
cellulose (10ml
/
kg) solution, whereas the rats in the control
group were gavaged with physiological saline in the same
conditions for 56 consecutive days. The rats (n=6
/
group)
were sacri�ced on days 7, 14, 28 and 56. Blood samples were
collected into heparinized tubes via the abdominal aorta at the
four distinct time points. The blood samples were immediately
centrifuged at 3,000
g
for 10min at room temperature, and the
serum was frozen at ‑80˚C for subsequent analyses. The lung
tissues were immediately perfused with physiological saline
solution to removed blood cells. Then right lower lobe tissues
were removed and stored at ‑80˚C for western blot analysis,
and the right upper lobes were used for the determination of
hydroxyproline. The left lung tissues were �xed in parafor
-
maldehyde for the assessment of morphological changes using
hematoxylin and eosin (HE) staining and Masson's staining
with immunohistochemical analysis.
Analysis of the oxidation
/
antioxidant system.
The oxida
-
tion
/
antioxidant system includes MDA, SOD and GSH
peroxidase (GHSPx). The MDA content and activities of
SOD
/
GSH‑Px in serum samples were assayed using MDA
and SOD
/
GSH‑Px test kits according to the manufacturer's
protocol (Nanjing Jiancheng Bioengineering Institute). MDA,
the end product of lipid peroxidation of cells, condenses with
thiobarbituric acid to form a product with a maximum absorp
-
tion at 532nm, therefore, the MDA content in the samples
was determined by comparing the optical density (O.D.) of
the samples to the standard substance. SOD and GSH‑Px, two
important antioxidant enzymes, can scavenge free radicals
and inhibit lipid peroxidation. The activities of SOD and
GSH‑Px activity are expressed in units, with 1 unit dened
as 50% inhibition of nitrite formation. SOD can inhibit the
hydroxylamines of superoxide radical anions oxidized to form
nitrite, and appear violet with a color reagent; the change in
absorbance was recorded at 550nm. The activity of GSH‑Px
was determined using the disul�de‑nitrobenzoic acid direct
method and compared at the O.D. at 412nm with the standard
substance.
Analysis of the levels of TNF-
and TGF-
1.
The serum
contents of TNF‑
and TGF‑
1 were detected using ELISA,
according to the manufacturer's protocol (Wuhan Boster
Biological Technology, Ltd., Wuhan, China). The antirat
TNF‑
(cat. no.EK0526) or TGF‑
1 (cat. no.EK0514) speci�c
antibody was coated on the ELISA plates. The standards and
samples were pipetted into the wells and the TNF‑
/
TGF‑
1
present in the sample bound to the wells by the immobilized
antibody. The liquid was removed from the wells and bioti
-
nylated anti‑rat TNF‑
or TGF‑
1 antibody (1:100 dilution,
at 37˚C for 60min) was added. Following washing away
unbound biotinylated antibody, HRPconjugated streptavidin
was pipetted into the wells for 30min at 37˚C. The wells were
again washed, and TMB color liquid was added to the wells
for 20min at 37˚C, with color developing in proportion to the
level of TNF‑
(TGF‑
1) bound. The stop solution alters the
color from blue to yellow, and the intensity of the color was
measured at 450nm. The placental TNF‑
/
TGF‑
1 levels
were calculated as pg
/
ml using the standard curve.
Determination of collagen content.
The content of collagen
in the right upper lobe lung tissue was determined using a
MOLECULAR MEDICINE REPORTS 15: 3121-3128, 2017
3123
hydroxyproline assay according to the manufacturer's protocol
(Nianjing Jiancheng Bioengineering Institute.). Brie�y, the
lung tissue samples (80mg) were hydrolyzed in boiling water
for 5h. Following addition of the immobilized reagents, the
mixture was placed in 60˚C water for 15min and centrifuged
at 3,000
g
for 15min at room temperature after cooled down.
The hydroxyproline content of the supernatant was quanti�ed
by spectrophotometry at 550nm. The data are expressed as
collagen (
µ
g)
/
wet weight (mg).
Immunohistochemical analysis of AKT1-phospho S473,
NF-
B, and collagen types I and II.
The immunohistochem
-
istry used Streptavidinperoxidase Histostain TMPlus kits
(cat. no.SP‑9000; OriGene Technologies, Inc., Beijing, China).
The important stages were as follows: Paraffin‑embedded
sections (5µm) were deparaf�nized, rehydrated and underwent
removal of endogenous peroxidase with 3% H
2
O
2
. Following
incubation with goat serum working solution, the working
solution was discarded and the tissue sections were incubated
with primary antibodies against AKT1‑phospho S473 (1:200;
Abcam), NF‑
B (1:200; Santa Cruz Biotechnology, Inc.), and
collagen type I (cat. no.BA0325) and III (cat. no.BA0326;
1:200; Wuhan Boster Biological Technology, Ltd.) overnight
at 4˚C, followed by the biotinylated secondary antibody at
37˚C for 15min and streptavidin‑peroxidase at 37˚C for
15min. Immunoreactivity was visualized with DAB (Fuzhou
Maixin Biotech. Co., Ltd., Fuzhou, China). Brown color
staining was considered a positive result. Sections were coun
-
terstained with hematoxylin and images were captured from
six separate randomly selected �elds using Olympus FV1000
(magni�cation x400; Olympus Inc., Center Valley, PA, USA).
Quantitative analysis was performed in a blinded‑manner
using an automatic image analysis system at Beijing University
of Aeronautics and Astronautics (Beijing, China), with the
average O.D. values as quantitative indicators.
Western blot analysis of AKT1 and AKT1-phospho S473.
The middle lobe of the right lung (100mg) was lysed with
RIPA lysis buffer (1ml), and then centrifuged at 10,000
g
for 15min at 4˚C. The supernatant was collected and protein
content was determined using a protein assay kit (Beyotime
Institute of Biotechnology, Tianjin, China). The proteins (10µg)
were separated by 10% SDSPAGE under a constant voltage
of 120V for 2h, and then transferred onto a polyvinylidene
�uoride membrane at a constant electric current of 250mA
for 30min (Bio‑Rad Laboratories, Inc., Hercules, CA, USA).
Following blocking with 5% non‑fat dry milk, the membranes
were incubated at 4˚C overnight with primary antibodies at the
following dilution ratios: GAPDH antibody (cat. no.sc25778,
1:2,000; Santa Cruz Biotechnology, Inc.); anti‑AKT1‑phospho
S473 antibody (1:5,000; Abcam); anti‑AKT1 antibody (1:5,000;
Abcam). The membranes were then washed three times with
PBST and incubated with HRPconjugated antirabbit IgG
antibody (cat. no.074‑1506, 1:5,000; KPL, Inc., Gaithersburg,
MD, USA) for 2h at room temperature, followed by washing
with PBST. The proteins were visualized using chemilumines
-
cence (ECL; Beyotime Institute of Biotechnology).
Statistical analysis.
All data are expressed as the
meanstandard
deviation. SPSS 17.0 software (SPSS, Inc.,
Chicago, IL, USA) was used to perform statistical analyses.
Multiple group comparisons were performed using one‑way
analysis of variance followed by pair‑wise comparison with
the Student's
t
test. P0.05 was considered to indicate a statis
-
tically signi�cant difference.
Results
Effect of OA on histopathologic changes in the lungs.
Pathological changes in the lung tissues of the rats were
observed by light microscopy with HE and Masson's staining.
As shown in
Fig.
d, the lungs of the rats in the control group,
which received physiological saline, had a thin alveolar septum
without signi�cant in�ammation and no obvious abnormalities
shown by the HE stain. However, in the model group and solvent
control group, at day 14 post‑instillation, there was marked in�l
-
tration of in�ammatory cells and alveolar septum thickening
in the lungs, with occasional small numbers of cellular nodules
(stageI; and TableI). At 28days, primarily cellular nodules
(stageI+) and �brotic cellular nodules (stageII+) were observed.
At 56days, �brous nodules were integrated with each other,
there were more �brotic cellular nodules (stagesII+ and III).
By contrast, OA treatment signi�cantly reduced in�ammatory
cell in�ltration and alveolar septum thickening at 14 days; the
size and number of cellular nodules (stageI+) were decreased
at 28 and 56days. Masson's staining (
Fig.
Bac) showed: blue
collagen fibers, red muscle fibers, cytoplasm and red blood
cells, and brown nuclei. In the control group, there was a small
quantity of blue collagen �ber around the bronchial and alveolar
septum area during the investigation. In the model group and
solvent control group, diffuse collagen �bers were increased
and arranged irregularly in the nodules at 28 days; increased
collagen deposition was present, which was arranged in
concentric circles, at 56 days; lung �brosis was aggravated. OA
treatment signi�cantly reduced collagen �bers, in small sections
or small bundles. These results indicated that the silicosis model
was successfully constructed and that OA exerted a signi�cant
protective effect.
Effect of OA on oxidative stress in the lungs.
As shown in
TableII, compared with the control group, the content of MDA
in sera of the model group and solvent control group increased,
peaking at 14days, followed by a marginal decrease, although
signi�cant differences were found in the statistical analysis
(P<0.05). However, OA treatment signi�cantly decreased the
Table I. Silicotic nodule grades in the lungs of rats in each
group (n=6).
Nodule grade following instillation
---------------------------------------------------------------------------------------------
Group7 days14 days28 days56 days
Control 00
OA
OA, oleanolic acid.
PENG
etal:
PROTECTIVE EFFECTS OF OA IN SILICOSIS DURING LUNG INJURY AND FIBROSIS
3124
content of MDA, compared with the content in the model
group and solvent control groups at corresponding time points
(P0.05).
The activities of SOD
/
GSH‑Px increased in the sera from
the model group and solvent control group, compared with
the control group at corresponding time points (P<0.05),
Table II. Effect of OA on oxidative stress in sera of the rats with silicosis.
MDA content (
µ
/
l)SOD activity (U
/
/
--------------------------------------------------------------------------------------------
----------------------------------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------------------
Group
7 d14 d28 d56 d7 d14 d28 d56 d7 d14 d28 d56 d
100.94±11.08
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
143.69±20.11
a
a
a
a
a
a
a
b,c
b,c
b,c
b,c
b,c
b,c
b,c
b,c
b,c
b,c
b,c
b,c
Data are presented as the mean ± standard deviation (n 6).
a
P<0.05, compared with
control group;
b
P<0.05, compared with silicosis group;
c
P
compared with solvent control group. OA, oleanolic acid; MDA,
Figure 1. Morphological observation of lung tissue in each group.
(A)Morphological observation of lung tissue with hematoxylin and eosin
stain (magni�cation x 100) on (a) day 7; (b) day 14; (c) day 28; (d) day 56.
(B)The morphological observations of lung tissue using Masson's stain
(magni�cation, x100) on (a) day 14; (b) day 28; (c) day 56.
A
B
MOLECULAR MEDICINE REPORTS 15: 3121-3128, 2017
3125
however, the activities of SOD
/
GSHPx increased with
time in the sera from the model group and solvent control
group. Compared with the model group and solvent control
group, the activities of SOD
/
GSH‑Px were signicantly
increased in the OA group at the corresponding time points
(P0.05).
Effect of OA on collagen changes in the lungs.
Hydroxyproline
content is an important indicator of total collagen in lung
�brosis, which are predominantly composed of collagen types
I and III. In the present study, we detected the content of
hydroxyproline using a hydroxyproline kit, and the expression
levels of collagen types I and III using immunohistochemistry
(
Fig.
A‑C). No signi�cant differences in the hydroxyproline
content or levels of collagen types I and III were found in the
control group during the investigation. However, the total
collagen
/
hydroxyproline content and the expression levels of
collagen I and III in the model group and solvent control group
were signi�cantly higher, compared with those in the control
group at corresponding time points (P<0.05). As expected, OA
treatment signi�cantly reduced these levels, compared with
those in the model group and solvent control group (P<0.05),
but remained higher, compared with those in the control group
(P0.05).
Effect of OA on cytokines in the lungs.
TNF‑
and TGF‑
1
are important cytokines and are involved in in�ammation and
pulmonary �brosis. As shown in
Fig.
A and B, no differences
in the serum contents of TNF‑
and TGF‑
1 were found in
rats of the control group at the four time points (P>0.05). The
serum contents of TNF‑
in the model group and solvent
control group were signi�cantly increased, peaking at day
14day post‑instillation, with a subsequent marginal decrease,
but with statistically signi�cant differences at each time point,
compared with those in the control group (P<0.05), whereas
TGF‑
1 increased following instillation (P<0.05). No signi�
-
cant differences between the model group and solvent control
group were found at different time points. OA treatment had
inhibitory effects on the contents of TNF‑
and TGF‑
1,
compared with the model group and solvent group at the
corresponding time points (P<0.05).
Effect of OA on p-AKT1
/
NF-
B in the lungs.
Immuno
-
histochemical methods were used to observe the
expression of pAKT1
/
NF‑
B in the rat lung tissues. As shown
in
Fig.
, the levels of NF‑
B p65 in the nuclear fractions were
signi�cantly increased in the model group and solvent control
group, compared with that in the control group (P<0.05);
whereas positive staining for pAKT1 (
Fig.
) was primarily
present in the nuclei of interstitial cells in the lung tissues of the
model group and solvent control group, but not in the control
group. OA treatment markedly reduced this positive staining
(P<0.05). The immunohistochemical results were further
con�rmed using western blot analysis (
Fig.
), which showed
that the model group and solvent control groups exhibited
increased expression levels of ph‑AKT1, compared with levels
in the control group at the corresponding time points (P<0.05)
and that OA treatment signi�cantly weakened the expression
of p‑AKT1 in the rat lungs, compared with levels in the model
group and solvent control group at the corresponding time
points (P0.05).
Discussion
In the present study, the effects of OA on silicainduced
lung injury and �brosis were examined for the �rst time, to
the best of our knowledge. Firstly, successful establishment
of the silicosis model in rats was confirmed by observing
morphology and pathological changes in lung tissues with HE
and Masson's staining. Subsequently, biochemical indicators,
including changes in cytokines, collagen and the AKT
/
NF‑
B
pathway were measured in the pathogenesis ofsilicosis, and
the effects of OA on these changes were determined. The
resulting data suggested that OA possessed protective effects
against silica‑induced lung injury and �brosis.
It is known that silicosis is a preventable occupational
disease with no effective treatments available. In silica‑induced
in�ammation and �brosis, various mediators, including ROS,
cytokines and growth factors released from activated alveolar
macrophages, are key in the development and progression of
the disease
13
-
15
). ROS, which include hydroxyl radicals,
superoxide anions, hydrogen peroxide and singlet oxygen, are
generated not only at the particle surface, but also by phagocytic
cells attempting to digest the silica particle. Particle‑derived
ROS can also react with cellderived ROS and RNS, yielding
novel toxic moieties, including peroxynitrite from NO and
superoxide anions (O
2
16
). Therefore, oxidative stress is
Figure 2. Effect of OA on changes in collagen in the lung. (A)Collagen;
(B)collagen type I; (C)collagen type III.
*
P<0.05, compared with control
group;
#
P<0.05, compared with silicosis (model) group;
P<0.05, compared
with solvent control group. OA, oleanolic acid.
PENG
etal:
PROTECTIVE EFFECTS OF OA IN SILICOSIS DURING LUNG INJURY AND FIBROSIS
3126
caused by an imbalance between the production of ROS and the
ability of the biological system to repair the resulting damage.
It has been demonstrated that oxidative stress is involved
in the development of silica‑induced pulmonary disease in
rats
8
). In humans, silica exposure also activates oxidative
stress in the development and progression of silicosis
17
). The
quanti�cation of oxidative stress can also be assessed by the
measurement of aldehydes, including MDA, the end product
of lipid peroxidation of cells. The plasma levels of MDA are
correlated with the severity of silicosis
18
). Oxidative stress
is also evidenced by increased the expression of antioxidant
enzymes, including SOD and GSH‑Px. SOD, a superoxide
anion radical scavenger, can inhibit the peroxidation of
free radicals and indirectly re�ect the level of free radicals
scavenged. GSH‑Px can promote the removal of hydrogen
peroxide (H
2
O
2
) and free radicals by GSH, and is important
in the integrity of cell membrane structure and function. This
was supported by a study by Zhang
etal
(
19
), which found that
the mean serum levels of GSH and MDA, and activity of SOD
in the silicosis group were signi�cantly higher, compared with
those in control subjects without silicosis (P0.05). Consistent
with these results, the present study found that MDA content
and the activity of SOD
/
GSH‑Px in the sera of the model
group and solvent control group were increased, however, OA
modulated these changes by downregulating MDA content
and increasing the activity of SOD
/
GSHPx, which suggested
that OA may have an antioxidant role in the development and
progression of silicosis.
Intracellular ROS has a fundamental role in the
silicainduced transduction pathway leading to the produc
-
tion of TNF‑
. Scarfì
etal
found that H
2
O
2
and OH• radicals
were key signals in the enhanced production of TNF‑
in
QA‑stimulated RAW 264.7 murine macrophages
20
). TNF‑
is a pleiotropic cytokine, shown to be involved in in�ammation
and �brosis
(
21
,
22
).
In several studies, serum levels of TNF‑
in silicosis groups were reported to be signi�cantly higher,
compared with those in control groups
23
,
24
). The released
TNF‑
increases the function of neutrophils and eosinophils,
which generates increased superoxide and lysosomal enzyme
release, and produces toxic effects to surrounding tissues to
further increase the in�ammatory response. TNF‑
can also
aggregation secrete abundant human TGF‑
1, promotes the
proliferation of �broblasts and secretes increased collagen
25
).
The results of the present study suggested that TNF‑
was
produced at an early stage of the in�ammatory process in
the silicosis model, peaked 14days following instillation and
remained at a higher level, compared with levels in the control
group, which was consistent with the literature. Of note, OA
treatment inhibited the levels of TNF‑
, compared with those
Figure 3. Effect of OA on cytokines in the lung. (A)Effect of OA on TNF‑
(B)Effect of OA on TGF‑
.
*
P<0.05, compared with control group;
#
P0.05,
compared with silicosis (model) group;
P<0.05, compared with solvent
control group. OA, oleanolic acid. TNF‑
, tumor necrosis factor‑
; TGF‑
,
transforming growth factor‑
.
Figure 4. Effect of OA on NF‑
B
/
p65 in the lung.
*
P<0.05, compared with
control group;
#
P<0.05, compared with silicosis (model) group;
P0.05,
compared with solvent control group. OA, oleanolic acid; NF‑
B, nuclear
factor
B; OD, optical density.
Figure 6. Effect of OA on p‑AKT
/
AKT in the lung. Levels of
AKT
/
AKT were determined using western blot analysis. OA, oleanolic
acid; p‑, phosphorylated.
Figure 5. Effect of OA on p‑AKT
/
AKT in the lung.
*
P<0.05, compared with
control group;
#
P<0.05, compared with silicosis (model) group;
P0.05,
compared with solvent control group. OA, oleanolic acid; p‑, phosphorylated.
MOLECULAR MEDICINE REPORTS 15: 3121-3128, 2017
3127
in the model group and solvent group at the corresponding
time points.
TGF‑
1, a multifunctional cytokine, regulates the prolif
-
eration and differentiation of cells
26
) and is known to
promote the pathogenesis of lung �brosis. The mRNA and
protein expression of TGF‑
1 have been shown to increase in
the lungs of the silicotic animals
27
,
28
). Consistent with these
results, the present study found that, 56days postSiO
2
instil
-
lation, the pulmonary expression of TGF‑
1 increased almost
2.13‑fold, compared with that in the control group, which was
markedly inhibited by chronic administration of OA. These
data suggested that OA suppressed the expression of TNF‑
and TGF‑
1, accordingly reducing the rate of the development
and progression ofsilicosis.
As is already known, NF‑
B, a critical transcription factor
in modifying the production of inflammatory cytokines,
growth factors and ROS, is activated by silica in macrophages
and other types of lung cells, and is important in the initiation
and progression of silica‑induced pulmonary �brosis
29
-
32
).
Several studies have conrmed that the PI3K
/
Akt signaling
pathway can be an upstream activator of the NF‑
B signaling
cascade
33
) and adjust cells physiological function
34
). AKT,
a serine
/
threonine protein kinase, is activated via the PI3K
pathway. Activated AKT can promote the transcriptional
activity of NF‑
B by accelerating the degradation of IKB
and phosphorylating NF‑
B
/
p65
35
). OA has been reported
to inhibit the activation and nuclear translocation of NF‑
B,
resulting in suppression of the TNF‑
‑induced in�ammatory
response
36
). In the present study, the data showed that levels
of p‑Akt Ser473 and NF‑
B
/
p65 were increased in the progres
-
sion of silica‑induced pulmonary �brosis in rats, and that OA
treatment downregulated the phosphorylation of AKT‑Ser473
and decreased the level of NF‑
B
/
p65. These results indicated
that OA inhibited silica‑induced pulmonary in�ammation and
�brosis in rats, possibly through regulating the AKT
/
NF‑
B
pathway.
In conclusion, these results of the present study suggested
that OA restored the oxidant
/
antioxidant balance, and
decreased pulmonary cytokines and collagen through
regulating the AKT
/
NF‑
B pathway in silicainduced lung
injury and �brosis. The protective effects of OA in silicosis
may be relevant, not only during the �rst steps of the lung
in�ammatory response, but also subsequently. OA, a penta
-
cyclic triterpenoid extensively found in a variety of plants
and medicinal herbs, may be an effective option for treating
silicosis, for which there are no other specific treatment
options.
Acknowledgements
This study was supported by the Tangshan Science and
Technology Bureau Foundation of China (grant no.14130262B).
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