Human amnion-derived mesenchymal stem cells alleviate lung injury induced by white smoke inhalation in rats
Cui et al. Stem Cell Research & Therapy
Human amnion-derived mesenchymal stem cells alleviate lung injury induced by white smoke inhalation in rats
Pei Cui 0 1 5
Haiming Xin 0 4
Yongming Yao 3
Shichu Xiao 2
Feng Zhu 2
Zhenyu Gong 4
Zhiping Tang 1 5
Qiu Zhan 1 5
Wei Qin 4
Yanhua Lai 1 5
Xiaohui Li 1 5
Yalin Tong 1 4 5
Zhaofan Xia 2
0 Equal contributors
1 Research Laboratory of Burns and Trauma, the 181st Hospital of Chinese PLA , Guilin 541002 , People's Republic of China
2 Department of Burn surgery, Changhai Hospital, Naval Military Medical University , Shanghai 200433 , China
3 Trauma Research Center, First Hospital Affiliated to the Chinese PLA General Hospital , Beijing 100048 , People's Republic of China
4 Department of Burns, Plastic and Wound repair surgery, the 181st Hospital of Chinese PLA , Guilin 541002 , People's Republic of China
5 Research Laboratory of Burns and Trauma, the 181st Hospital of Chinese PLA , Guilin 541002 , People's Republic of China
Background: White smoke inhalation (WSI) is an uncommon but potentially deadly cause of acute lung injury and acute respiratory distress syndrome for which no effective pharmaceutical treatment has been developed. This study aimed to determine the protective effects of human amnion-derived mesenchymal stem cells (hAMSCs) against WSI-induced lung injury in rats. Methods: hAMSCs were injected into rats via the tail vein 4 h after WSI. At 1, 3, 7, 14, and 28 days after cell injection, hAMSCs labeled with PKH26 in lung, heart, liver, and kidney tissues were observed by fluorescence microscopy. The lung injury score was determined by hematoxylin and eosin staining. Lung fibrosis was assessed by Masson's trichrome staining. The computed tomography (CT) score was assessed by CT scanning. The wet/dry weight ratio was calculated. The levels of interleukin (IL)-1β, IL-6, and IL-10 were determined by enzyme-linked immunosorbent assays. The expression of surfactant protein (SP)-A, SP-C, and SP-D was measured by Western blotting. Results: The injected hAMSCs were primarily distributed in the lung tissues in WSI-induced rats. Compared with the model and phosphate-buffered saline (PBS) group, hAMSC treatment led to reduced lung injury, lung fibrosis, CT score, and inflammation levels in WSI-induced mice. hAMSC treatment also resulted in increased cell retention in the lung, partial pressure of oxygen (PaO2), and PaO2/fraction of inspired oxygen (FiO2) levels, and pulmonary SP-A, SP-C, and SP-D expression compared with that in the model and PBS group. Conclusions: hAMSCs are a potential cell-based therapy for WSI-induced lung injury.
White smoke inhalation; Human amnion-derived mesenchymal stem cells; Lung injury; Cell-based therapy
Smoke pots and smoke bombs, which are widely used in
fire drills or retreats, can produce white smoke.
Compared to the smoke generated by the combustion of
tobacco or wood dust, white smoke not only contains
carbon tetrachloride, carbon monoxide, and carbon
dioxide, but is also mixed with other highly corrosive,
irritating, and toxic substances that include
tetrachloroethylene, zinc chloride, hexachloroethane, and oxides
(zinc oxide, aluminum oxide, iron oxide, etc.), which
increase the damage caused by white smoke inhalation
]. Accidental exposure to high concentrations
of white smoke, particularly in a confined space, can lead
to inhalation injury, acute respiratory distress syndrome,
pulmonary fibrosis, and even death [
]. Many studies
have reported that lung injury can be caused by a single
or several components in white smoke, and confirmed
that zinc chloride and hexachloroethane are important
substances as inducers of lung injury [
no clinical treatments are available. Therefore, it is
necessary to develop effective therapeutic methods to treat
lung injury induced by white smoke.
Mesenchymal stem cells (MSCs) are multipotent cells
that can differentiate into a variety of lineages. MSCs
have the potential to repair injured tissues by secreting
growth factors and anti-inflammatory molecules [
MSC therapy may be a promising therapeutic strategy
for treatment of white smoke-induced acute respiratory
distress syndrome. It has been reported that MSCs may
recover lung fibroblast function from cigarette
smokeinduced damage [
]. Human amnion-derived MSCs
(hAMSCs) can be collected from the amnion, which is
generally discarded as medical waste. Thus, there are
fewer ethical issues limiting hAMSC collection .
Previous studies demonstrated that hAMSCs have
antiinflammatory effects in dextran sulfate sodium-induced
severe colitis and carbon tetrachloride-induced liver
] and that hAMSCs alleviate lung injury
induced by lipopolysaccharide and ischemia and
However, the therapeutic efficacy of hAMSCs against
lung injury induced by WSI has not been examined. In
this study, we investigated whether tail vein injection of
hAMSCs could improve WSI-induced lung injury in
Dissociation and culture of primary hAMSCs
All pregnant women or their relatives provided written
informed consent for sample collection. The study was
conducted according to principles of the Helsinki
Declaration of 1975 as revised in 1983 and was approved by
the Ethics Committee of No. 181 Hospital of the
People’s Liberation Army. Primary hAMSCs were
dissociated from fresh placenta specimens of full-term
pregnancies delivered by cesarean section. Briefly, the
amniotic membrane was shaken with the same volume
of 0.25% trypsin-0.02% EDTA-disodium salt solution at
200 rpm for 50 min at 37 °C, and the supernatant was
discarded. This step was repeated twice. Next, the
amniotic membrane was cut into pieces and shaken with the
same volume of 0.2 mg/mL type IV collagenase
containing 0.075 mg/mL DNase I (Worthington, Lakewood, NJ,
USA) for 2 h. The solution was filtered through a
200mesh sieve (bore diameter = 0.074 mm) and centrifuged
at 1000 g at 4 °C for 5 min. The cells were washed twice
with phosphate-buffered saline (PBS) and resuspended
in Dulbecco’s modified Eagle’s medium (DMEM)
supplemented with 10% fetal bovine serum (FBS; Gibco, Grand
Island, NY, USA). Then, 5 mL of the cell suspension cells
(1 × 106 cells/mL) in a 25-cm2 flask were maintained at
37 °C in a humidified atmosphere of 95% air and 5% CO2.
After 48 h, the culture medium and nonadherent cells
were discarded and fresh medium was added. Cell growth
was observed under an inverted phase contrast microscope
(Olympus, Tokyo, Japan). When growth reached 80%
confluence, cells were digested and cultured in DMEM
supplemented with 10% FBS. Third-generation hAMSCs
were digested and collected, and the hAMSC surface
markers were measured using a Human MSC Analysis Kit
(BD Biosciences, San Diego, CA, USA) and a FACSCalibur
flow cytometer (BD Biosciences) equipped with Cell Quest
software (BD Biosciences).
Establishment of the WSI model and treatment
All rats were purchased from the animal center of Guilin
Medical College. Rat protocols were approved by the
ethics committee of the No. 181 Hospital of the People’s
Liberation Army and disposal methods followed animal
ethical standards. Rats were allowed free access to water
and food and were housed at 20 to 25 °C in a room that
was continuously ventilated with 12 h of illumination
and 12 h of darkness. The experimental steps to
establish the WSI model are presented in Fig. 1. The rats
were placed in a high-permeability rat cage with an
isolation network to physically separate the rats and to
filter the impact of white smoke. The high-permeability
rat cage was placed in the center of a 2-m2 homemade
laboratory apparatus used for smoke injury research
(Patent No.: 201720002689.0). A smoke pot was located
60 cm below the rat cage. The smoke pot was ignited,
burned, and gradually produced smoke. The WSI time
was calculated from the beginning of smoke generation.
After WSI for 5 min, the rats were fed under normal
conditions. At 4 h post-WSI, rats were randomly divided
into the model, PBS, and hAMSC treatment groups.
Rats in the model group were not injected with
hAMSCs. Rats in the PBS group each received a tail vein
injection of 200 μL PBS. Each rat in the hAMSC
treatment group received a tail vein injection of 200 μL PBS
containing 1 × 106 hAMSCs. Normal rats that were not
treated (n = 6) comprised the control group.
weight). The wet/dry weight ratio was calculated from
the initial and final values.
Computed tomography (CT) evaluation
At 1, 3, 7, 14, and 28 days post-treatment, rats were
fixed to a foam board in the supine position under
general anesthesia induced using chloral hydrate. The rat
breast was scanned with a 64-slice GE LightSpeed VCT
(GE Healthcare, Little Chalfont, UK) at an 80 kV tube
voltage, 150 mA tube current, and medium bowtie filter
(0.625 mm half-value layer). CT scans were scored
according to the radiologist’s score method as previously
]. Briefly, each image slice was divided into
four quadrants (i.e., right and left ventral, and right and
left dorsal) and each quadrant was assigned a numeric
score of 0 (normal), 1 (interstitial markings), 2
(groundglass opacification), or 3 (consolidation). The worst
possible score was given to each quadrant, that is the
presence of a small area of consolidation in a quadrant
yielded a score of 3 even if the remainder of the
quadrant was not consolidated. This score was added across
all quadrants and slices to determine the total
radiologist’s score for the entire scan. A thoracic radiologist
blinded to the group assignments and sampling time
point evaluated each scan score according to the total
radiologist’s score. The CT baseline value was 52.83 ± 2.
14. Theoretically, the CT score is 0. However, individual
image artifacts produced a score between 0 and 52.83.
Arterial blood gas levels
One milliliter of noncoagulated blood was collected from
the abdominal aorta of rats. Each sample was analyzed to
determine the partial pressure of oxygen (PaO2), partial
pressure of carbon dioxide (PaCO2), and the ratio of PaO2
to the fraction of inspired oxygen (PaO2/FiO2) using a
blood gas analyzer (Radiometer Medical, Copenhagen,
Assessment of hAMSC localization
hAMSCs were labeled with PKH26 red fluorescent dye
before tail vein injection. PKH26 labeling has no effect on
the hAMSC phenotype or the behavior of the cells in vitro
or in vivo, and is a safe and effective way to label hAMSCs
]. After treatment, the rats were sacrificed and the
lung, heart, liver, and kidney tissues were collected and
frozen at −80 °C. The frozen tissues were continuously cut
into 4-μm slices that were observed by fluorescence
microscopy (magnification × 100, Olympus, Tokyo, Japan).
Wet/dry weight ratio
After anesthetic overdose and exsanguination (by
severing the inferior vena cava and abdomen aorta), lung
lobes were weighed (wet weight), placed in an oven, and
weighed daily until the weight was unchanged (dry
Assessment of pathological changes
Pathological changes in lung tissues were visualized by
hematoxylin and eosin (H&E) staining. The
diaphragmatic leaves of the right lung of rats from each group
were fixed with 4% formalin, washed, dehydrated,
embedded in paraffin, and cut into 4-μm sections. H&E
staining was performed using a kit according to the
manufacturers’ instructions (Solarbio, Beijing, China).
Semi-quantitative scoring of bleeding, edema, and
inflammation of lung tissues was performed by optical
microscopy examination (magnification × 200, Olympus).
The degree of lung injury was graded as described
]. The pathological score was graded as follows:
a score of 0 indicated no alveolitis; a score of 1+
indicated mild alveolitis with thickening of the alveolar
septum by a mononuclear cell infiltrate, with
involvement limited to focal, pleural-based lesions occupying <
20% of the lung and with good preservation of the
alveolar architecture; a score of 2+ indicated moderate
alveolitis with more widespread alveolitis involving 20 to 50%
of the lung, although still predominantly pleural based;
finally, a score of 3+ indicated severe alveolitis with
diffuse alveolitis involving more than 50% of the lung, with
occasional consolidation of air spaces by the
intraalveolar mononuclear cells and some hemorrhagic areas
within the interstitium and/or alveolus. Tissue sections
were also analyzed by Masson’s trichrome staining. Ten
random fields on a section from each rat were
photographed and blue-stained areas were calculated from the
entire lung cross-sectional area (%, × 20) with a digital
image analyzer (WinROOF; Mitani Co., Fukui, Japan) to
evaluate the degree of lung fibrosis. The degree of lung
fibrosis was graded as follows: a score of 0 indicated no
evidence of fibrosis; a score of 1+ indicated mild lung
fibrosis with focal regions of fibrosis involving < 20% of
the lung, with the fibrosis involving the pleura and the
interstitium of the subpleural parenchyma with some
distortion of alveolar architecture; a score of 2+
indicated moderate fibrosis, which was evident as more
extensive fibrosis involving 20 to 50% of the lung and
fibrotic regions that mostly extend inward from the
pleura and still focal; finally, a score of 3+ indicated
severe fibrosis, which was evident as widespread fibrosis
involving more than 50% of the lung and the presence of
confluent lesions with extensive derangement of
parenchymal architecture, including cystic air spaces lined by
Measurement of inflammatory cytokines
The right lung was separated and the right main bronchus
was ligated. Next, 1 mL of ice saline was injected from the
end of the trachea and washed by lavage three times. A 2.
6-mL sample of bronchoalveolar lavage fluid was collected
and centrifuged at 350 g for 10 min at 4 °C. Transforming
growth factor (TGF)-β1, tumor necrosis factor (TNF)-α,
interleukin (IL)-6, and IL-10 levels in the supernatant of
bronchoalveolar lavage fluid were measured with
enzymelinked immunosorbent assay (ELISA) kits (Multi Science,
Hangzhou, Zhejiang, China).
Western blot analysis was conducted to analyze protein
expression in the lung tissue. Briefly, protein lysis buffer
was added to minced lung tissue and the tissue was
homogenized. The samples were centrifuged at 10,000 g at
4 °C. Protein concentration in the supernatant was
quantified using a BCA protein quantification kit (Tiangen,
Beijing, China). Aliquots containing 50 μg total proteins
were used for 8% sodium dodecyl sulfate-polyacrylamide
gel electrophoresis and the resolved proteins were
transferred to a nitrocellulose membrane (BioTrace Medical,
Menlo Park, CA, USA). The membranes were incubated
with 1:1000 dilutions of antibodies against pulmonary
surfactant protein (SP)-A, SP-C, SP-D, and
glyceraldehyde-3phosphate dehydrogenase (GAPDH) (all from Cell
Signaling Technology, Danvers, MA, USA) at 4 °C on a rotating
shaker overnight. The membranes were subsequently
washed with Tris buffered saline-Tween (TBST) three
times for 5 min each time, followed by incubation with
horseradish peroxidase-labeled goat anti-rabbit IgG (H +
L; Beijing ZSGB Bio, Beijing, China, 1:5000 dilution) for
1 h at 25 °C on a horizontal shaker. The membranes were
washed with TBST twice for 5 min each time, followed by
chemiluminescence detection. The protein band areas
were quantified using Image Lab 6.0 software (Bio-Rad,
Hercules, CA, USA). The GAPDH antibody was used as
an internal reference.
Statistical analyses were performed using SPSS v19.0
software (SPSS, Inc., Chicago, IL, USA). All data are expressed
as the mean ± standard deviation (SD). The data were
analyzed by one-way analysis of variance followed by
posthoc tests of the least significant difference for multiple
pairwise comparisons. P < 0.05 was considered statistically
hAMSC morphology and verification of phenotype
hAMSCs displayed MSC characteristics including
adhesion and a spindle-shaped and flat morphology
(Fig. 2a). hAMSCs were negative for CD34 and
positive for CD105, CD90, and CD73 mesenchymal markers
Distribution of hAMSCs in WSI-induced rats
hAMSCs labeled with PKH26 in the lung, heart, liver,
and kidney tissues were observed using fluorescence
microscopy (Fig. 3). hAMSCs were clearly observed in
the lung tissues but were gradually reduced at 1, 3, 7,
14, and 28 days after treatment with hAMSCs.
Additionally, nearly no hAMSCs were detected in the
heart, liver, and kidney tissues. The results suggest
that hAMSCs mainly migrated to the lung tissue in
CT score increased by WSI is reduced by hAMSC treatment
CT scans of WSI-induced rats after treatment revealed
varying increases of lung marking, ground-glass
opacities, and lung consolidation in the model and PBS
groups at 1, 3, 7, 14, and 28 days after treatment (Fig. 4).
In the hAMSC treatment group, the CT score was
increased at 3 and 7 days after treatment followed by
reductions at 14 and 28 days after treatment. The
pathological score was significantly reduced at 28 days after
hAMSC treatment compared with that at 1 day after
hAMSC treatment (P < 0.05). Compared with the model
group and PBS group, the CT score in the hAMSC
group was significantly decreased at 28 days
posthAMSC treatment (P < 0.001). However, the CT score
did not decrease to the normal value after hAMSC
treatment at 28 days.
Wet/dry weight ratio in WSI-induced rats is reduced by hAMSC treatment
At 1, 3, 7, 14, and 28 days after treatment, the wet/dry
weight ratio was measured (Fig. 5). The wet/dry weight
ratio gradually decreased after hAMSC treatment and was
significantly reduced at 28 days after hAMSC treatment
compared with that at 1 day after hAMSC treatment.
Additionally, the wet/dry weight ratio was significantly
decreased at 28 days after hAMSC treatment compared with
that in the model group and PBS group at the same time
point (P < 0.05). However, the wet/dry weight ratio did not
decrease to the normal value after hAMSC treatment at
Pathological score increased by WSI is reduced by hAMSC treatment
Histopathological features of lung tissue were evaluated
by H&E staining. The lung tissue of WSI-induced rats in
the model, PBS, and hAMSC treatment groups exhibited
histopathological features of lung injury including
congestion of airway mucosa, edema, and bleeding,
neutrophil infiltration, alveolar wall thickening, and alveolar
wall collapse (Fig. 6). In the hAMSC treatment group,
the pathological score was increased at 3 days after
treatment followed by reductions at 7, 14, and 28 days after
treatment. The pathological score was significantly
reduced after hAMSC treatment at 14 and 28 days
compared with after hAMSC treatment at 1 day (P < 0.05).
Additionally, the pathological scores after hAMSC
treatment at 14 and 28 days were significantly decreased
compared with those in the model group and PBS group
at the same time point (P < 0.01). However, the
pathological score did not decrease to the normal value after
hAMSC treatment at 28 days.
Lung fibrosis grade increased by WSI is reduced by hAMSC treatment
Lung fibrosis was measured at 14, 21, and 28 days after
treatment by Masson trichrome staining (Fig. 7). In the
hAMSC treatment group, lung fibrosis was reduced at
14, 21, and 28 days after treatment. Lung fibrosis in the
hAMSC treatment group at 14 and 28 days was
significantly decreased compared with that in the model group
and PBS group at the same time point (P < 0.001).
However, the lung fibrosis grade did not decrease to the
normal value after hAMSC treatment at 28 days.
hAMSC treatment alleviates the changes in arterial blood
gases induced by WSI
Figure 8 depicts the levels of PaO2, PaCO2, and PaO2/FiO2
in WSI-induced rats after treatment. The levels of PaO2 and
PaO2/FiO2 in the hAMSC treatment group gradually
increased, while the level of PaCO2 gradually decreased at 1, 3,
7, 14, and 28 days after treatment. The levels of PaO2 and
PaO2/FiO2 were significantly enhanced after hAMSC
treatment at 28 days compared with that after hAMSC treatment
at 1 day (P < 0.01), while the levels of PaO2 and PaO2/FiO2
were significantly reduced after hAMSC treatment at 14 and
28 days compared with that after hAMSC treatment at
1 day (P < 0.05). The levels of PaO2 and PaO2/FiO2 were
significantly increased (P < 0.001), while the level of PaCO2 was
significantly inhibited (P < 0.01), at 7, 14, and 28 days after
hAMSC treatment compared with that in the model group
and PBS group at the same time points (P < 0.01). However,
the levels of PaO2, PaCO2, and PaO2/FiO2 did not recover
to the normal values after hAMSC treatment at 28 days.
hAMSC treatment increases the expression of SP-A, SP-C,
and SP-D decreased by WSI
The expressions of SP-A, SP-C, and SP-D were
measured by Western blotting (Fig. 9). The expressions of
SP-A, SP-C, and SP-D gradually increased at 1, 3, 7, 14,
and 28 days after hAMSC treatment. The expressions of
SP-A, SP-C, and SP-D were significantly increased at 7,
14, and 28 days after hAMSC treatment compared with
that after hAMSC treatment at 1 day (P < 0.01). The
expressions of SP-A, SP-C, and SP-D were increased after
hAMSC treatment compared with that in the model
group and PBS group at the same time point. However,
the SP-A, SP-C, and SP-D expressions did not increase
to the normal values after hAMSC treatment at 28 days.
hAMSC treatment reduced the levels of inflammatory
cytokines increased by WSI
The levels of inflammatory cytokines were measured by
ELISA (Fig. 10). The levels of IL-6, TNF-α, and TGF-β1
were gradually decreased, while the level of IL-10 was
gradually increased at 1, 3, 7, 14, and 28 days after
hAMSC treatment. The levels of IL-6 and TGF-β1 were
significantly reduced at 7, 14, and 28 days after hAMSC
treatment compared with those after hAMSC treatment
at 1 day (P < 0.01), while the level of IL-10 was
significantly enhanced after hAMSC treatment at 3, 7, 14, and
28 days compared with that at 1 day after hAMSC
treatment (P < 0.05). However, the level of TNF-α was not
significantly reduced at 3, 7, 14, and 28 days after
hAMSC treatment compared with that at 1 day after
hAMSC treatment. The levels of IL-6, TNF-α, and
TGFβ1 were decreased, while the level of IL-10 was
increased (P < 0.01) after hAMSC treatment at 3, 7, 14,
and 28 days compared with those in the model group
and PBS group at the same time point, but the level of
TNF-α was not significantly reduced. However, the levels
of inflammatory cytokines did not recover to the normal
values after hAMSC treatment at 28 days.
Clinical studies revealed that patients severely affected
by exposure to white smoke display significant
pathological characteristics that include pulmonary edema,
bleeding, inflammatory infiltration, diffuse alveolar
damage, and pulmonary fibrosis [
]. CT scanning results
have shown similar characteristics as pulmonary vascular
lesions in adult respiratory distress syndrome caused by
inhalation of zinc chloride smoke [
]. In this study, we
found that rats induced by WSI exhibited congestion of
airway mucosa, edema and bleeding, neutrophil
infiltration, alveolar edema and diffuse bleeding, alveolar wall
thickening, and alveolar wall collapse. Lung fibrosis, lung
marking, ground-glass opacities, and lung consolidation
were increased to different degrees after WSI in rats,
similar to the pathological characteristics of WSI
patients. hAMSCs can proliferate in lung tissues and
improve lung injury induced by lipopolysaccharide and
ischemia and reperfusion [
]. Similar to the findings
of previous studies, we found that hAMSCs introduced
by tail vein injection become primarily distributed in the
lung tissues in WSI-induced rats, and that hAMSC
treatment reduced lung injury and lung fibrosis, and
decreased the CT score increased by WSI according to the
results of CT scanning, H&E staining, and Masson
trichrome staining. The wet/dry weight ratio and degree
of pulmonary edema were increased after smoke
inhalation in an ovine model . In this study, we found
that the wet/dry weight ratio was gradually decreased
after hAMSC treatment compared with that in the WSI
model, similar to the findings from H&E-stained lungs,
suggesting that the degree of pulmonary edema was
decreased after hAMSC treatment. These results suggest
that hAMSC treatment improves lung injury induced by
WSI, however, lung injury did not recover to the normal
values after hAMSC treatment at 28 days. Additionally,
we found that edema is no longer evident at 14 and
28 days according to H&E results in the model and PBS
groups; rather, an increase in fibrosis is observed at these
time points in the model and PBS groups, with possible
reasons being that white smoke could persist in the
airways for a long time and inflame lung tissue due to the
tiny particles in the smoke, resulting in infiltrated
collagen fibers in most areas of the lung. Although edema
was no longer apparent in the model and PBS groups, it
was still present in the lung tissue at these time points
and the reasons for this still need further verification.
As pattern-recognition molecules of the collectin
family of C-type lectins, SP-A, SP-C, and SP-D play
important roles in mediating pulmonary immune defense [
The changes in SP-A, SP-C, and SP-D caused by
cigarette smoking contribute to the development of lung
]. Previous studies also showed that smoke
inhalation injury frequently induces severe hypoxemia
and increases the risk of acute respiratory distress
]. Our results showed that hAMSC treatment
increased SP-A, SP-C, and SP-D expressions, increased
PaO2 and PaO2/FiO2 levels, and decreased PaCO2 levels
compared with those in the WSI model. These results
suggest that hAMSC treatment can improve the
respiratory and immune functions inhibited by WSI. However,
the respiratory and immune functions did not recover to
the normal values after hAMSC treatment at 28 days.
Furthermore, we found that PaO2 and the PaO2/FiO2
ratio were significantly increased at all time points starting
from day 3 in the hAMSC-treated group compared with
the PBS and model groups, but the levels of lung injury
were similar in all three groups at day 3 and day 7.
Possible reasons are as follows: PaO2 and the PaO2/FiO2
ratio do not correspond exactly to the degree of lung
injury since the body automatically begins respiratory
compensation under acute hypoxia and, therefore, PaO2
and the PaO2/FiO2 ratio do not decrease. However,
when the body is heavily damaged, and the
compensatory respiration range is exceeded, PaO2 and the PaO2/
FiO2 ratio cannot increase. In our study, PaO2 and the
PaO2/FiO2 ratio decreased significantly on day 3 in the
PBS and model groups compared with the
hAMSCtreated group because, in the PBS and model groups,
each body was heavily damaged, and the compensatory
respiration range was exceeded, resulting in decrease in
PaO2 and PaO2/FiO2 ratio. With the recovery of the
body, the body entered the compensatory respiration
range, and PaO2 and the PaO2/FiO2 ratio increased on
day 7 in the PBS and model groups. However, PaO2 and
the PaO2/FiO2 ratio exhibited no significant change with
hAMSC treatment. Additionally, pathological results
could reflect the state of lung injury. We observed that
on days 3 and 7, the degrees of lung injury in all three
groups were similar because the therapeutic effects of
hAMSCs on lung injury take some time to manifest.
Inflammatory cytokines, such as TGF-β1, TNF-α, IL-1β,
and IL-10, act as tracheobronchial markers of lung injury
in smoke inhalation victims [
]. TGF-β1, TNF-α, and
IL1β inhibit lung injury repair and promote lung cell
apoptosis and fibrosis [
], the levels of which are increased
by cigarette smoke . Reduced pulmonary inflammation
contributes to remodeling of lung injury [
]. IL-10, an
anti-inflammatory cytokine, reportedly inhibits cigarette
smoke-induced pulmonary neutrophilic inflammation and
TNF-α expression in mice . In this study, we found
that hAMSC treatment decreased the levels of IL-6,
TNFα, and TGF-β1, but increased the level of IL-10 compared
with those in the WSI model. These results suggest that
hAMSC treatment improves the levels of inflammatory
cytokines changed by WSI. However, the levels of
inflammatory cytokines did not recover to the normal values
after hAMSC treatment at 28 days.
In conclusion, hAMSC treatment can improve lung
injury and respiratory and immune functions and inhibit
the levels of inflammatory cytokines induced by WSI.
These results implicate hAMSCs as a potential
cellbased therapy for WSI-induced lung injury. However,
the mechanism underlying hAMSC treatment for
WSIinduced lung injury requires further investigation.
CT: Computed tomography; DMEM: Dulbecco’s modified Eagle’s medium;
ELISA: Enzyme-linked immunosorbent assay; FBS: Fetal bovine serum;
FiO2: Fraction of inspired oxygen; GAPDH: Glyceraldehyde-3-phosphate
dehydrogenase; H&E: Hematoxylin and eosin; hAMSC: Human
amnionderived mesenchymal stem cell; IL: Interleukin; MSC: Mesenchymal stem cell;
PaCO2: Partial pressure of carbon dioxide; PaO2: Partial pressure of oxygen;
PBS: Phosphate-buffered saline; SD: Standard deviation; SP: Surfactant
protein; TBST: Tris buffered saline-Tween; TGF: Transforming growth factor;
TNF: Tumor necrosis factor; WSI: White smoke inhalation
This work was supported by grants from the Academician Workstation
Program, the Lijiang Scholars Program, Guilin Science and Technology
Project (No. 20170109–35), and Guangxi Science and Technology Project
Availability of data and materials
The datasets generated and/or analyzed during the current study are
available from the corresponding author on reasonable request.
PC, HX, YY, SX, FZ, YT, and ZX designed and planned the study. PC, HX, ZG,
ZT, QZ, WQ, YL, and XL collected the data, and PC and HX analyzed the data.
PC was a major contributor in writing the manuscript. HX amended the
manuscript. YT and ZX provided the Funds. All authors read and approved
the final manuscript.
Rat protocols were approved by the ethics committee of the No. 181
Hospital of the People’s Liberation Army and disposal methods followed
animal ethics standards.
Consent for publication
All authors have approved the submission for 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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