Transplantation of adipose-derived mesenchymal stem cells attenuates pulmonary fibrosis of silicosis via anti-inflammatory and anti-apoptosis effects in rats
Chen et al. Stem Cell Research & Therapy
Transplantation of adipose-derived mesenchymal stem cells attenuates pulmonary fibrosis of silicosis via anti-inflammatory and anti-apoptosis effects in rats
Shangya Chen 0 3
Guanqun Cui 2
Cheng Peng 0 1 3
Martin F. Lavin 0 3 6
Xiaoying Sun 4
Enguo Zhang 0 3 5
Ye Yang 0 3 5
Yingjun Guan 0 3
Zhongjun Du 0 3
Hua Shao 0 3
0 Department of Toxicology, Shandong Academy of Occupational Health and Occupational Medicine, Shandong Academy of Medical Sciences , Jinan, Shandong , People's Republic of China
1 Queensland Alliance for Environmental Health Sciences (QAEHS), the University of Queensland , Brisbane, QLD , Australia
2 Department of Respiratory Medicine, Qilu Children's Hospital of Shandong University , Jinan, Shandong , People's Republic of China
3 Department of Toxicology, Shandong Academy of Occupational Health and Occupational Medicine, Shandong Academy of Medical Sciences , Jinan, Shandong , People's Republic of China
4 Department of Dermatology, Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai University of Traditional Chinese Medicine , Shanghai , People's Republic of China
5 School of Medicine and Life Sciences, University of Jinan-Shandong Academy of Medical Sciences , Jinan, Shandong , People's Republic of China
6 University of Queensland Centre for Clinical Research (UQCCR), the University of Queensland , Herston, Brisbane, QLD , Australia
Background: Silicosis has been topping the list of high-incidence occupational diseases in developing countries and cannot be completely cured. Recent advances in stem cell research have made possible the treatment of various diseases including lung fibrosis. The application of stem cell therapy in occupational diseases, in particular the use of adipose-derived mesenchymal stem cells (AD-MSCs) in treatment of silicosis, has not yet been reported. The aim of the study is to explore the intervening effect of silica-induced lung fibrosis in rats. Methods: In this study, we investigated the anti-pulmonary fibrosis effects of the transplantation of AD-MSCs in rats in which lung fibrosis was induced by oral tracheal intubation with silica suspension. Twenty rats were divided into four groups: control group (n = 5), exposure group (n = 5), vehicle group (n = 5) and treatment group (n = 5). AD-MSCs were given to rats after exposure to silica for 24 h. Twenty-eight days after AD-MSC transplantation, we examined the organ coefficient, inflammatory cytokines, apoptosis, pathological and fibrotic changes in lung tissue. Results: Results showed that exposure to silica for 28 days induced an increase of the lung coefficient with significant pulmonary fibrosis. Treatment with AD-MSC transplantation led to a remissive effect on pulmonary fibrosis. We found that after AD-MSC transplantation the inflammatory response decreased and Caspase-3 protein expression significantly decreased with a significant increase of the Bcl-2/Bax ratio. Conclusions: Anti-inflammatory and anti-apoptosis of AD-MSCs may play important roles in their anti-pulmonary fibrosis effect. Our data suggest that transplantation of AD-MSCs holds promise for potential interference in the formation of silicosis through regulating inflammatory and apoptotic processes.
Adipose-derived mesenchymal stem cells; Silicosis; Pulmonary fibrosis; Animal model; Transplantation; Cell therapy
Silicosis refers to long-term inhalation and retention of
silica dust in the lungs, mainly due to occupational
exposure. The silicosis process involves the formation of
silicon nodules, diffusion of pulmonary fibrosis and
resultant diffused fibrotic pneumoconiosis [
characteristics of silicosis include alveolar epithelial cell
injury and the formation of pulmonary fibrosis, but the
mechanisms of silicosis are not fully clear. It is believed
that inflammatory-related and apoptosis-related
mechanisms play important roles in lung injury induced by
silica dust and intervening/therapeutic routes targeting
these pathways have been explored. For example,
extensive attempts have been made to promote the treatment
of silicosis through repairing injured cells but limited
effects have been obtained. The use of stem cells as
transplant tissue shows particular promise since stem cells
have shown potency to repair damaged pulmonary tissue
by replacing the endogenous damaged cells through cell
regeneration and changing the microenvironment [
Recently, much progress has been made in
understanding the mechanisms of mesenchymal stem cells (MSCs)
in the aspects of regulating immunity and tissue
remodeling in animal models of lung fibrosis [
]. In lung tissue
rescue and repair, adult MSCs have been proposed to be
a valuable therapeutic option due to their availability,
immunomodulatory effects and anti-apoptotic and
antiinflammatory properties [
As mesodermal-derived cells, bone marrow-derived
mesenchymal stem cells (BM-MSCs) are primarily
obtained through bone marrow aspiration. Mature adipose
tissue collection can help extract primary AD-MSCs to
obtain autologous AD-MSCs. Later, these
mesodermalderived cells were proved to be pluripotent stem cells with
multilineage differentiation potential and horizontal
differentiation ability [
]. AD-MSCs have similar biological
characteristics, immunosuppressive effects and
multilineage differentiation potential to BM-MSCs which can
differentiate into adipocytes, osteocytes, chondrocytes,
myocytes and neural precursor cells [
because of the low content of BM-MSCs in bone marrow,
prolonged time and complicated procedures for cell
collection, culture and purification are required before
transplantation. In contrast, AD-MSCs are plentiful in adipose
tissue which is easy to access. To obtain the same amount
of MSCs through bone marrow aspiration, the donor may
only need to provide a smaller amount of adipose tissue,
with relatively less pain and feasibility. These factors
ensure AD-MSCs have a wider range of applications in stem
In recent years, a number of studies have tried to use
MSCs for treatment of pulmonary injury. BM-MSCs
transfected with HGF have been reported to be effective
in improving pulmonary fibrosis in patients with
silicosis. Researchers transfused autologous
BM-MSCsHGF into silicosis patients, which reduced pulmonary
small nodules significantly, with pulmonary function and
inflammation of patients gradually ameliorated [
MSCs-HGF may be a potential treatment option for
pulmonary injury. AD-MSC transplantation was also found
to promote angiogenesis in the injured lung tissue by
enhancing the expression of HGF [
]. The mechanism of
action of MSCs in pulmonary diseases is thought to
include regulating inflammation and promoting
], and is affected by the quality of MSCs used
]. AD-MSCs have been shown to be effective in
repairing and regenerating lung tissue [
ameliorating idiopathic pulmonary fibrosis [
]. A recent
study found that AD-MSCs can mitigate
bleomycininduced lung fibrosis in aged mice [
silicosis-induced pulmonary fibrosis is different from
bleomycin-induced pulmonary fibrosis in etiology. So
far, there are currently no reported studies on AD-MSC
treatment of silica-induced pulmonary fibrosis.
Therefore, the aim of this study is to explore the preventive
effect and mechanism of AD-MSC treatment for silicosis.
To this end, we cultivated AD-MSCs for transplantation
in rats with oral tracheal intubation with silica
suspension and evaluated organ coefficients, pathological and
fibrotic changes in lung tissue and inflammatory
cytokines in lung tissue, and measured expression of the
protein involved in the mitochondria-associated
apoptotic pathway. Data from the study will provide evidence
for the effects of AD-MSCs on silica-induced fibrosis
and helpful information in the application of AD-MSCs
for therapeutic purposes in the future.
Experimental animals and experimental design
Male Sprague Dawley (SD) rats weighting 180–200 g
were purchased from Beijing Vital River Laboratory
Animal Technology Co. Ltd (certificate No. SCXK (Jing)
2012–0001) and maintained under standard housing
conditions (temperature 18–24 °C; relative humidity 45–
60%; light and dark cycle 12 h:12 h). Food and water
were provided ad libitum. All animals were treated
according to the protocols evaluated and approved by the
experimental animal ethical committee of Shandong
Academy of Medical Sciences.
Animal treatment and experimental design are shown in
Fig. 1. Twenty adult male SD rats were randomly divided
into four groups: control group (n = 5), which were
normally fed; exposure group (n = 5), which were exposed to
silica; vehicle group, (n = 5), in which DMEM culture
medium was administered by intravenous injection 24 h
after silica exposure; and treatment group (n = 5), which
received 5 × 105 AD-MSCs (suspended in DMEM culture
medium) by intravenous injection 24 h after exposure to
silica. On the 28th day after transplantation, the animals
from each group were evaluated for different parameters
Culture and identification of stem cells
AD-MSCs from adipose tissue of 4-week-old healthy male
adult SD rats (n = 3) were isolated and cultured according
to a previous report with slight modifications [
the SD rats were sacrificed by 3% pentobarbital sodium
anesthetic overdose. After being placed in 75% ethanol for
about 5 min, rats were dissected on a super-clean bench.
The abdominal skin was cut along the midline of the
abdomen until adipose tissue of the groin was exposed.
Adipose tissue of the groin was collected and washed three
times with phosphate buffered solution (PBS) containing
100 IU/ml penicillin and 100 μg/ml streptomycin
(ThermoFisher Scientific, USA). After being transferred
into a dry Petri dish, the tissue was cut quickly by
sterilized ophthalmic scissors for 10 min. The tissue pieces
were collected and digested with collagenase dissolved in
Dulbecco’s modified Eagle’s medium (DMEM) (with 5%
BSA, 5 mg/ml) (ThermoFisher Scientific, USA) at 37 °C
for 60 min. After centrifugation at 1000 rpm (Sorvall
ST8R; ThermoFisher Scientific, USA), the cell pellet was
suspended in complete medium (DMEM with 10%
embryonic stem cell-qualified fetal bovine serum, 100 IU/ml
penicillin and 100 μg/ml streptomycin) (Biological
Industries, USA). The cell suspension was filtered through a cell
filter with 70-μm pores. A cell suspension with 104 cells/
ml was obtained and incubated in CO2 incubator (Labserv
CO-150; ThermoFisher Scientific, USA) after being
washed with DMEM and centrifuged as already described.
The cells were observed daily and photographs were taken
under inverted microscope (IX70; Olympus, Japan).
AD-MSCs were identified and selected by flow
cytometry (FCM) with CD44, CD45, CD90, CD73 and CD11b
antibodies. After being subcultured to the third
generation, AD-MSCs at 80% confluence were washed twice
with PBS followed by digestion with 0.25% trypsin–
EDTA (ThermoFisher Scientific, USA). The cells were
then centrifuged at 1000 rpm and washed with PBS.
After incubation with antibodies and their isotype
controls (1:100) (Becton Dickinson and Co., USA) at 4 °C
for 30 min, the cells were flowed through the cytometer
at about 1000 cells per second. Results of FCM were
analyzed by FlowJo software (FlowJo, LLC, USA).
AD-MSCs were also identified by adipogenic,
osteogenic and chondrogenic differentiation. After being
subcultured to the third generation, AD-MSCs at 80%
confluence were induced in adipogenesis, osteogenic and
chondrogenic differentiation complete medium (using
adipogenesis, osteogenic and chondrogenic
differentiation kits from ThermoFisher Scientific, USA). After
induction for 21 days, cells were fixed with 4%
paraformaldehyde for about 20 min. The intracellular lipids
accumulated in the induced AD-MSCs in adipogenic
cultures were stained with Oil Red O (Sigma-Aldrich,
USA). The mineralized osteogenic cultures were stained
with Alizarin Red S (Sigma-Aldrich, USA) to detect
calcium deposition. The mucopolysaccharide accumulated
in chondrogenic cultures were stained with Toluidine
Blue (Sigma-Aldrich, USA). Photographs were taken
under the inverted microscope.
Silica-induced silicosis in rats
We improved the method of oral tracheal intubation
with silica suspension to induce silicosis in rats. The
micron-sized silica (approximately 80% between 1 and 5
μm; Sigma-Aldrich, USA) was autoclaved and made into
a suspension at 50 mg/ml in normal saline. Rats were
fixed on the operating table after 3% pentobarbital
sodium anesthesia. Incisors of rat were pulled down by
surgical suture and the tongue of the rat was pulled out
by sterile forceps with the light of exceeded luminosity
lamp-house toward the neck of the rat. The glottis was
the bright spot opening and closing with breath which
could be seen through the mouth. Then we wiped the
mucus near the glottis with a cotton swab. When the
glottis was open, the indwelling venous needle was
inserted into the trachea. Silica suspension (1 ml) was
injected quickly into the cannula. Then the rat was
gently shaken for about 5 min to distribute the suspension
uniformly in the lungs. The rats in the control group
were perfused with 1.0 ml of sterile normal saline.
AD-MSCs were resuspended at a concentration of 5 × 105
cells/ml for transplantation which was performed on rats
exposed to silica for 24 h. The cell dosage was 1 × 106
cells/kg. Briefly, rats were anesthetized with 3%
pentobarbital sodium. The rat tail was sterilized by 75%
ethanol. Then 1 ml of cell suspension with 5 × 105 cells/
ml was injected over 2 min using a disposable vein
infusion needle which was attached to a disposable
syringe of 1 ml. In order to minimize cell leakage, the
needle was left in place for 2 min after injection before
withdrawal. The vehicle group had DMEM culture
medium “transplanted” in the same way.
Inflammatory cytokine measurement
TNF-α, IL-1β, IL-6 and IL-10 were detected by ELISA
using different ELISA kits (ThermoFisher Scientific,
USA) according to the manufacturer’s protocols. Briefly,
rats from each group were euthanized 28 days post
transplantation. The lungs of rats were rapidly removed,
weighed and frozen at −80 °C for further experiment.
After being diluted, detection A solution was added into
the plate at 100 μl per well and placed at 37 °C for 1 h.
After the plate was washed three times, detection B
solution was added as detection A and placed at 37 °C for
30 min. The plate was then washed five times, and
3,3′,5,5′-tetramethylbenzidine (TMB) was added at 90 μl
per well and placed at 37 °C for 20 min. Finally, sulfuric
acid was added at 50 μl per well as the termination
solution. The optical density (OD) of each well of a 96-well
plate at 450 nm was analyzed using a microplate reader
(SpectraMax 190; Molecular Devices, USA).
Histopathological and fibrotic examination
Rats were deeply anesthetized with 3% pentobarbital
sodium 28 days post transplantation. The body weight was
measured and the heart, liver, spleen and lungs of rats
were isolated and excised with a razor blade and
weighed, and the organ coefficient (the ratio of the organ
weight to the body weight) was calculated. The organ
coefficient is an important indicator to evaluate toxic
effects of test substances in toxicology research. Then, the
lungs of rats were collected and some parts were fixed
in 4% (v/v) paraformaldehyde. After embedding in
paraffin blocks, tissues were sectioned into 5 μm slice, and
then mounted onto glass slides. Tissues on the glass
slides were stained with hematoxylin and eosin (H&E)
for histopathological evaluation, and stained with
Masson’s trichrome stain for fibrotic examination.
Photographs were taken using an optical microscope (BX51;
Olympus, Japan). The slides were examined by
pathologists who were blinded to the identity and analysis of
pathology sections. The results were peer-reviewed by
other certified veterinary pathologists. Fibrotic changes
were quantified by modified Ashcroft score with a grade
of 0–8 to represent alveolar structure from normal lung
to total fibrous obliteration of lung fields [
The lungs of rats were processed as already described
for terminal deoxynucleotide transferase
(TdT)-mediated dUTP nick end labeling (TUNEL) using an in-situ
cell death detection kit (Roche, Switzerland) according
to the manufacturer’s instructions. Briefly, lung slides of
each group were fixed with 4% paraformaldehyde in 0.1
M phosphate buffer (pH 7.4). After incubation with
proteinase K (100 μg/ml), sections were washed with PBS,
and permeabilized with 0.1% Triton X-100. The sections
were then washed with PBS twice and incubated in
TUNEL reaction mixture. After sections were rinsed
again, the converter-POD was used to visualize sections
with 0.02% 3,3′-diaminobenzidine (DAB). After
counterstaining with Mayer’s hematoxylin, sections were
mounted on the slides which were gelatin-coated and
placed at room temperature overnight to be air-dried.
Positive results were brown-stained nucleus. An
apoptotic index was evaluated to analyze differences between
groups in apoptosis detection. The apoptosis index was
determined from 10 blindly selected high-power fields
for each slice by counting the number of positive cells in
500–1000 cells per field to calculate the percentage of
Western blot assay
We used western blot analysis to detect the expression
of Bax, Bcl-2 and Caspase-3 proteins in lung tissue of
rats from each group. The lungs were processed as
already described and lung tissues were homogenized
for protein extraction. After being resuspended in
homogenization buffer, the tissues were centrifuged for
10 min (12,000 rpm, 4 °C). Supernatants were collected
as protein suspension. SDS-PAGE (12% resolving gels at
120 V, 5% stacking gels at 75 V) was used to separate
protein samples. Then protein samples were transferred
to PVDF membranes (200 mA, 1 h). The membranes
were blocked with 5% nonfat dry milk followed by
incubation with different primary antibodies and β-actin
(Santa Cruz, USA) overnight at 4 °C. After being washed
with TBST, the membranes were incubated with
secondary antibodies (Santa Cruz, USA) at 1:1000
dilutions for 1.5 h at room temperature. After being washed
with TBST, the membranes were treated with enhanced
chemiluminescence (ECL detection kit; Pierce) and
exposed to the Odyssey CLx near-infrared fluorescence
imaging system (LI-COR, USA). Results were quantified
by AlphaEaseFC software (Alpha Innotech, USA). We
performed experiments in triplicate to ensure
Data are presented as means ± SD. Statistical analysis
was performed using one-way analysis of variance
(ANOVA). Data for each group were compared with
other groups by least-significant difference (LSD) for
significance. All statistical analyses used SPSS software
(IBM SPSS Statistics 20.0, USA). Results were
considered statistically significant at p < 0.05.
Culture and identification of AD-MSCs
Primary cells were mainly spindle-shaped and partially
polygonal, and showed rapid proliferation in the first 3
days. With the cell expansion progressed, most cells
were long spindle-shaped and fibroblast-like with the
number of hybrid cells decreased significantly, and the
proliferation rate of AD-MSCs decreased gradually.
After expansion to the third generation, the cells were
long spindle-shaped and evenly distributed, and cells
were whirlpool-like or chrysanthemum-like aggregated
at a density of 100%. The cell morphology was more
consistent with a faster growth rate. Flow cytometry
measurement of cells at the third generation showed
that expression of CD44, CD73 and CD90 was positive
(99.15%, 96.30% and 99.88%), with negative expression
of CD11b and CD45 (0.26% and 0.82%). Meanwhile,
adipogenic, osteogenic and chondrogenic differentiation of
cells at the third generation all succeeded. Thus, these
results confirmed that the vast majority of cells in the
third generation were AD-MSCs (Fig. 2).
Organ coefficients of rats
The organ coefficient is considered a commonly used
indicator of animal status. After rats were sacrificed,
organs were removed and rinsed with normal saline.
The organs were then blotted dry on filter paper and
immediately weighed. The ratio of organ weight/body
weight was considered the organ coefficient. There
was no significant difference among the heart
coefficient of rats in different groups. Compared to the
control group, the lung coefficients of rats
significantly increased in the treatment group, exposure
group and vehicle group (p < 0.05) (Table 1).
AD-MSC transplantation recovers the silica-induced inflammatory cytokines
Inflammatory cytokines of lung tissue were evaluated
after rats were sacrificed (Table 2). Compared to the
control group, the TNF-α, IL-1β, IL-6 and IL-10 levels
of rats significantly increased in rats in the exposure
group and vehicle group (p < 0.05). There was no
significant difference between the exposure and vehicle groups
for each inflammatory cytokine. Notably, the TNF-α,
IL1β, IL-6 and IL-10 levels in the treatment group
significantly decreased compared to the vehicle group (p < 0.
05). The expression levels of TNF-α, IL-6 and IL-10
significantly decreased in the treatment group, with the
IL1β level decreased compared to the exposure group (p <
0.05). There was also a significant increase of IL-1β in
rats from the treatment group compared to the control
AD-MSCs mitigate the lung pathology changes by silica
The alveolar structure of rats from the control group was
intact with no inflammatory cell infiltration or fibrosis, as
shown in Fig. 3Aa1, Bb1. Lung alveolar structures in rats
from the exposure and vehicle groups were severely
destroyed with a large amount of inflammatory cell
infiltration, phagocytic cells and silicon nodules (Fig. 3Aa2, a3).
Telangiectasia, interstitial lymphocytic infiltration, alveolar
epithelium disarrangement and pulmonary vascular wall
thickening were observed in rats from the exposure and
vehicle groups (Fig. 3Bb2, b3). Alveolar structure destruction,
scattered fusion of silicon nodules, inflammatory cells and
lymphocytes were significantly reduced in the treatment
group compared with the exposure group (Fig. 3Aa4, Bb4).
Collagen fibers, mucus and cartilage were stained blue,
while cytoplasm, muscle, cellulose and glial were stained
red in Masson staining. After Masson staining, normal
collagen fiber stent could be observed in the lung tissue of the
control group (Fig. 4Aa1, Bb1). There was a large amount of
collagen fibers deposited in the pulmonary mesenchyme,
especially around bronchi and vessels in the exposure
group and vehicle group (Fig. 4Aa2, a3, Bb2, b3). Collagen
fiber deposition could also be found in the treatment group,
but the area of deposition was decreased compared to the
exposure group or vehicle group (Fig. 4Aa4, Bb4). The
result of modified Ashcroft score evaluation was consistent
with the result of the slice observation, as shown in Fig. 5.
AD-MSCs reduced the silica-induced cellular apoptosis
We used the TUNEL method to detect the apoptosis level
in lung tissues (Fig. 6). TUNEL-positive cells were
observed to distribute in rats of the exposure, vehicle and
treatment groups 28 days after transplantation. The
apoptosis index showed much difference among the control
group and the three other groups. An increased apoptosis
index in the exposure, vehicle and treatment groups was
Table 1 Organ coefficients of rats after surgery
(mean ± SD, n = 5)
ap < 0.05, vs control group
observed compared with control group. The apoptosis
index in the treatment group was significantly decreased
compared with that in the exposure group (p < 0.05). No
significant difference of the apoptosis index was seen
between the vehicle group and the exposure group.
Expressions of Bax, Bcl-2 and Caspase-3
We found that silica exposure led to upregulation of
Caspase-3 protein and downregulation of Bax and Bcl-2.
165.63 ± 32.09
334.45 ± 77.70a
After oral tracheal intubation with silica suspension,
expression of Caspase-3 protein was significantly
upregulated, while expression of Bax and Bcl-2 proteins was
significantly downregulated (Fig. 7). In rats with
ADMSC transplantation, compared with those in the
exposure and vehicle groups, Caspase-3 protein significantly
decreased while Bax and Bcl-2 protein expression
increased. Further analysis showed that the ratio of Bcl-2/
Bax in the exposure and vehicle groups was significantly
decreased, and the Bcl-2/Bax ratio in the treatment
group was significantly increased when compared with
the exposure group and vehicle group.
Recently, much attention has been paid to stem cell
therapy in experimental silicosis since it appears to be
susceptible to therapeutic intervention. Transplantation
of BM-MSCs has been demonstrated to be a promising
treatment for therapy of silicosis [
]. Clinical study has
shown that transplantation of autologous BM-MSCs can
effectively reduce pulmonary fibrosis with and improve
lung function in patients with silicosis [
with current techniques, it is hard to obtain stem cells
from bone marrow and this hinders the use of
BMMSCs. In contrast, AD-MSCs have a wider range of
sources and are more readily available. In addition,
liposuction is more likely to be accepted by patients
compared with bone marrow puncture in clinical practice.
To take these advantages, we selected AD-MSCs for
intervening in experimental silicosis. The effect of
ADMSCs on silica-induced silicosis has not been reported.
In this study, we successfully obtained AD-MSCs from
adipose tissue of rats and cultured them in vitro.
Primary AD-MSCs showed rapid proliferation in the first 3
days. After transfer to the third generation, the cells with
a long spindle-shaped morphology were more uniform
with a faster growth rate (Fig. 2). Then, we verified the
cells by checking the expression of surface molecules
and their adipogenic, osteogenic and chondrogenic
differentiation ability (Fig. 2).
Pulmonary fibrosis is characterized by a certain degree
of lung inflammation and abnormal tissue repair involving
a complex network of cytokines. These events result in
increased collagen gene expression and abnormal collagen
deposition in the lungs that eventually produce fibroblast
]. In order to observe the distribution of
inflammatory cells and detect collagen deposition in lung
interstitium, we used H&E staining and Masson staining to
evaluate differences in the progression of pulmonary
fibrosis between groups. Rats from the exposure groups
showed positive staining, which suggested the formation
of lung fibrosis (Figs. 3 and 4). The quantitative score of
pathological analysis of pulmonary fibrosis was then
conducted by estimation of the Modified Ashcroft scale [
We found that inflammatory cells aggregated and collagen
was deposited in the lung, accompanied by severe
distortion of pulmonary structure and large fibrous areas in rats
exposed to silica suspension (Figs. 3 and 4). After
ADMSC transplantation, decreased inflammatory cells and
collagen deposition in the lung tissue, reduced damage to
the lung structure and formation of small fibrous masses
were found in the lung interstitium (Fig. 4). The results
showed that early intervention of AD-MSCs could reduce
inflammatory reaction and slow down the process of
Researchers hypothesized that the inflammatory
reaction mechanisms underlying MSC therapy of
pulmonary fibrosis were mediated by paracrine signaling
(Reference). The therapeutic effect of BM-MSCs was
found to trigger IL-1RA secretion to suppress the
upregulated IL-1 and TNF-α proteins to protect the lung
against damage and fibrosis [
]. Consistent with several
previous studies using fetal membrane-derived stem cells
and preconditioned BM-MSC transplantations as
bleomycin-induced pulmonary fibrosis therapies [
we demonstrated that AD-MSC transplantation can
reduce the pulmonary inflammatory response of rats after
oral tracheal intubation with silica suspension, indicating
the migration and homing of AD-MSCs toward the
lungs through intravenous infusion to affect the
inflammatory response to silica exposure. Downregulated
expression of TNF-α, IL-1β, IL-6 and IL-10 proteins
indicated that AD-MSCs can also protect the lungs from
injury and fibrosis through an anti-inflammatory
process, which was consistent with ample experimental
evidence in MSC-treated pulmonary fibrosis [
Macrophages can stimulate epithelial cells by activating
and releasing inflammatory cytokines TNF-α, IL-1, IL-6
and IL-10. Activated epithelial cells can regulate local
immunity by recruiting locally inhabiting
immunocompetent cells to provide intercellular and intracellular
communication through autocrine and paracrine
signaling pathways . IL-1β and TNF-α mainly activate the
nuclear factor-κB pathway [
], while IL-6 and IL-10
exert their complex actions through multiple pathways
like the Janus kinase 2/signal transducers and activators
of transcription 3 pathway and the p38
mitogenactivated protein kinase/extracellular signal-regulated
kinase pathway [
]. TNF-α, IL-1β and IL-6 are
regarded as proinflammatory cytokines, and IL-10 is
generally considered to be an anti-inflammatory
cytokine. It was also shown that overexpression of IL-10
aggravates silica-induced pulmonary inflammation and
fibrosis in mice, which might relate to the promotion of
Th2-type cytokine reaction and increased expression of
IL-4 and IL-13 [
]. We found a downregulated
expression of IL-10 at day 28 after transplantation, which
might indicate the benefit of low expression of IL-10 in
lung tissue during pulmonary fibrosis prohibition
exerted by AD-MSC transplantation. In this regard, the
effect of AD-MSC transplantation on pulmonary
inflammatory factor release may directly alter the formation of
pulmonary fibrosis through an anti-inflammatory
Apoptosis, inflammatory reaction and their cascade
reactions can signify secondary damage after lung injury
]. Therefore, we tested the anti-apoptotic effect of
AD-MSCs in a rat silicosis model and found that the
apoptosis index of lung cells in AD-MSC-treated rats
was decreased compared with that of the exposure
group and vehicle group. We also found that the
apoptosis of pulmonary cells in rats exposed to silica involved
regulation processing of Bax, Bcl-2 and Caspase-3, thus
probably resulting in the apoptotic death of cells in lung
tissue. The increase in Caspase family protease activity is
associated with apoptosis, in which Caspase-3 plays a
pivotal part in the apoptosis of bleomycin-induced
pulmonary fibrosis . Further, our data showed decreased
apoptosis in the treatment group using TUNEL assay
(Fig. 6). Thus, the results suggested that AD-MSC
transplantation in rats exposed to silica suspension exerted
an anti-apoptotic effect.
Anti-apoptotic protein Bcl-2, pro-apoptotic protein
Bax and Caspase-3 play an important role in the
progression of apoptosis [
]. Bax regulates apoptosis by
forming homologous dimers or heterodimers with Bcl-2
to produce an apoptotic regulatory system. Owing to the
stability of heterodimer Bax–Bcl-2 compared to Bax–
Bax, the ratio of Bcl-2/Bax regulates the occurrence of
apoptosis and determines cell survival. Pulmonary cells
are in a relatively stable proportion of pro-apoptotic
protein Bax and anti-apoptotic protein Bcl-2 in
physiological conditions, maintaining the steady-state balance
of pulmonary structure and function. The Caspase
family mainly exists in the form of zymogen in cells. When
stimulated by the apoptotic signal, initiator Caspases are
activated, and then the executioner Caspases. They
induce apoptosis by decomposing substrate proteins. It
has been shown that Caspase-3 may play an important
role in apoptosis and has the effect of removing
inhibition to mediate feedback amplification [
we examined the expression of Bax, Bcl-2 and Caspase-3
in apoptosis by western blot assay in order to investigate
the mechanism of apoptosis suppression after AD-MSC
transplantation in rats with oral tracheal intubation with
silica suspension. The results showed that the ratio of
Bcl-2/Bax in the treatment group was significantly
increased compared with the exposure group and vehicle
group (Fig. 7). Although the expression levels of Bax and
Bcl-2 proteins showed no significant difference among
groups after exposure, we found significant
downregulation of Caspase-3 in the treatment group. Therefore, we
hypothesize that the high ratio of Bcl-2/Bax may be
directly related to the release of Caspase-3, which
eventually leads to apoptosis of lung tissue after exposure to
silica. AD-MSC transplantation played an anti-apoptotic
role in suppressing the downregulation of Bcl-2 protein
in rats after oral tracheal intubation with silica
suspension. Accordingly, the inhibition of Caspase-3 on
account of the decrease in the ratio of Bax/Bcl-2 can result
in declined apoptosis in the treatment group. These
findings further confirmed that AD-MSCs appear to
exert pulmonary protection and reduce the process of
apoptosis by inhibiting the expression of proteins related
to the mitochondrial apoptotic pathway in the secondary
damage of experimental silicosis in rats. Thus, the data
from this study indicated that AD-MSCs can mitigate
silica-induced lung fibrosis in rats. We also realized that
this is preliminary work from the point of view of
translation study and more work is required to establish the
intervening and even therapeutic effects of AD-MSCs in
silica-induced lung fibrosis. A study is underway to
explore the effects of AD-MSC on an established silicosis
model and compare their efficacy with BM-MSCs.
Transplanted AD-MSCs can significantly reduce the
pulmonary inflammatory response induced by silica
suspension in rats. AD-MSCs can also inhibit mitochondrial
apoptosis-related protein expression, thereby inhibiting
the process of silicosis. Although the apoptotic pathways
require verification, we provide ample evidence for the
further study of AD-MSCs in the treatment of silicosis.
Our data suggest that AD-MSCs may have a beneficial
effect on pulmonary fibrosis and may represent the basis
of a new treatment for patients with silicosis. The
successful development of stem cells in silicosis therapy will
require a better understanding of host–graft interaction,
the microenvironment and intrinsic characteristics, and
subsequent greater functional improvement. Ongoing
behavioral and biochemical assessment of long-term
positive effects of AD-MSCs will provide further insight
regarding the therapeutic potential of AD-MSCs for
AD-MSC: Adipose-derived mesenchymal stem cell; ANOVA: One-way analysis
of variance; BM-MSC: Bone marrow-derived mesenchymal stem cell;
DMEM: Dulbecco’s modified Eagle’s medium; H&E: Hematoxylin and eosin;
HGF: Hepatocyte growth factors; MSC: Mesenchymal stem cell; OD: Optical
density; PBS: Phosphate buffered solution; SD: Sprague Dawley;
TUNEL: Terminal deoxynucleotide transferase (TdT)-mediated dUTP nick end
The authors would like to express their gratitude to Dr Qiang Jia, Dr
Gongchang Yu and Miss Yan Jiang for their assistance in performing the
This study was supported by grants from the National Natural Science
Foundation of China (NSFC) (No. 81602893), Natural Science Foundation of
Shandong Province (NSFSP) (No. ZR2015YL049), Medical and Health
Technology Development Plan Project of Shandong Province (No.
2016WS0540), Key Research and Development Plan of Shandong Province
(No. 2017GSF18186) and Innovation Project of Shandong Academy of
Availability of data and materials
The data that support the findings of this study are available from the
corresponding author upon request.
SyC performed the experiments, analyzed and interpreted data, and drafted
the manuscript. GqC, EgZ, YY, YjG and XyS performed the analysis and
interpretation of data. CP participated in designing the experiment and
preparing and writing the manuscript. MFL provided technical support for
the analysis and critical revision of the manuscript. ZjD and HS designed the
study and revised the manuscript. Authors read and approved the final
The animal procedures in this study were carried out in accordance with
National Institutes of Health (NIH) Guidelines for the Care and Use of
Laboratory Animals, with approval from the animal ethics committee of
Shandong Academy of Medical Sciences.
Consent for publication
All authors of this manuscript agreed to publication.
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
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