Exogenous mesenchymal stem cells affect the function of endogenous
lung stem cells (club cells) in phosgene-induced lung injury
Kaili Ye a, b, c
, Daikun He a, b, c
, Yiru Shao a, b, c
, Ning Xu a, b, c
, Chaoyuan Jin a, b, c
Lin Zhang a, b, c
, Jie Shen a, b, c, *
a Department of Intensive Care Unit, Center of Emergency and Intensive Care Unit, Jinshan Hospital, Fudan University, Shanghai, China
b Department of Intensive Care Unit, Medical Research Center of Chemical Injury, Jinshan Hospital, Fudan University, Shanghai, China
c Department of Intensive Care Unit, Medical Center of Radiation Injury, Jinshan Hospital, Fudan University, Shanghai, China
article info
Article history:
Received 7 April 2019
Accepted 27 April 2019
Available online xxx
Keywords:
Mesenchymal stem cell
Club cell
Club cell secretory protein
Notch signaling pathway
Lung injury
abstract
Exogenous mesenchymal stem cells (MSCs) affect lung cells via cytokines as well as vesicles and activate
the Notch signaling pathway thus affecting the proliferation of endogenous stem cells to repair damaged
tissue. Club cells are endogenous lung stem cells whose proliferation is also closely related to the Notch
signaling pathway. The club cell secretory protein (CCSP) has anti-inflammatory and anti-oxidative
properties. This study aimed to investigate whether exogenous MSCs affect the function of club cells
in an injured lung and whether these effects are related to the Notch signaling pathway. CCSP levels in
bronchoalveolar lavage fluid (BALF) and serum were evaluated using enzyme-linked immunosorbent
assay (ELISA) and the average fluorescence intensity (AFI) of CCSP in club cells was determined using
flow cytometry. Immunohistochemistry and immunofluorescence were used to visualize club cells and
proliferative club cells. The expression of important Notch signaling pathway components including
Notch1~4, c-myc, Hey1 and Hes1 were also assessed. LY3039478 (LY), a specific inhibitor of the Notch
signaling pathway, was applied. After MSCs intervention, CCSP levels decreased, and club cell AFI
increased, indicating that the secretion of club cells had weakened. The expression of Notch1, Notch2, cmyc, Hey1, Hes1 increased, accompanied by an increase in the number of proliferative club cells.
Furthermore, MSCs enhanced the proliferation of club cells, while LY suppressed this phenomenon. In
summary, MSCs reduced the secretion of club cells. And MSCs enhanced the proliferation of club cells
partly via activating the Notch signaling pathway, which promoted lung injury repair.
© 2019 Elsevier Inc. All rights reserved.
1. Introduction
Phosgene is a lively, colorless, toxic gas. Exposure to phosgene
can cause acute lung injury (ALI) and even acute respiratory
distress syndrome (ARDS). Phosgene inhalation can cause damage
of lung epithelial/endothelial cells and inflammatory response [1].
The mortality rate of phosgene-induced lung injury, which has no
current treatment, is high.
Researchers have discovered a variety of endogenous lung stem
cells. Endogenous stem cells play an important role in endogenous
repair of lung injury. Club cells are a type of endogenous stem cells
found in the lungs, mainly in the terminal and respiratory bronchioles. Under normal circumstances, in humans, club cells account
for 11% and 22% of the terminal and respiratory bronchiole
epithelium, respectively, and for 15% and 44% of the number of
proliferating cells in these regions [2]. In mice, the distal airway
epithelium is mainly composed of cells expressing CCSP, and the
number of CCSP-positive cells may exceed 80%. Additionally, club
cells are cuboid cells that contain secretory vesicles. Club cell
secretory protein (CCSP) is a specific secretory protein present in
the cytoplasm and cell membrane of club cells and is the most
abundant protein in airway surface fluid. Therefore, CCSP can be
used as a marker to identify club cells [3].
Club cells have anti-inflammatory effects and have been shown
to be regenerated in lung injury, repairing damaged lung tissue
[4]. A study showed that club cells improved inflammation in
silica-induced lung injury [5]. Other studies found that club cells
* Corresponding author. Center of Emergency & Intensive Care Unit, Medical
Center of Chemical Injury and Medical Research Centre for Chemical Injury,
Emergency and Critical Care, Jinshan Hospital, Fudan University, 1508 Longhang
Road, Shanghai, 201508, PR China.
E-mail address: [email protected] (J. Shen).
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications
journal homepage: www.elsevier.com/locate/ybbrc
https://doi.org/10.1016/j.bbrc.2019.04.182
0006-291X/© 2019 Elsevier Inc. All rights reserved.
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
Please cite this article as: K. Ye et al., Exogenous mesenchymal stem cells affect the function of endogenous lung stem cells (club cells) in
phosgene-induced lung injury, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.04.182
played a key role in host defense, airway barrier, protein secretion,
and substance metabolism [6]. CCSP has anti-inflammatory and
anti-oxidant effects [3] while serum CCSP levels are a sensitive
indicator of lung injury [7,8]. According to pathological features of
ALI/ARDS, the control of inflammation and repair of lung epithelial/endothelial cells are crucial for treatment. Club cells were
chosen as our research focus for two reasons. On the one hand,
CCSP secreted by club cells plays a role in regulating local
inflammation [3], and serum CCSP can be used as an indicator for
monitoring lung injury repair. On the other hand, club cells, as a
type of stem cells, can self-proliferate in damaged lung tissue and
restore the integrity of the airway epithelium. In addition, club
cells can differentiate into ciliated cells and alveolar epithelial
cells [4,9].
MSCs-based therapy is a novel treatment for lung injury as the
protective effects of MSCs on damaged lung tissue have been preliminarily studied. Differentiation, immune regulation, and paracrine signaling of MSCs all have positive effects on lung tissue repair
[10]. Our previous study found that bone marrow-derived MSCs
improved lung inflammation and lung permeability in a phosgeneinduced lung injury model [11]. Other studies demonstrated that
exogenous MSCs affected lung cells (including intravascular endothelial cells, T lymphocytes, B lymphocytes and macrophages) via
cell contact or secretory vesicles. In an animal model of bronchopulmonary dysplasia, MSCs promoted the proliferation of bronchoalveolar stem cells (a type of lung endogenous stem cell that can
differentiate into club cells) and contributed to the reconstruction
of lung epithelial structure [12]. In addition to this study, few previous studies have investigated whether exogenous MSCs have an
effect on lung endogenous stem cells.
The Notch signaling pathway plays a key role in cell differentiation, proliferation and apoptosis. In mammals, the Notch signaling
pathway contains four cognate receptors (Notch1, Notch2, Notch3
and Notch4) that bind to ligands to produce different biological
effects. After binding of the extracellular domain of Notch receptors
to ligands, cleavage of the Notch receptor is induced, releasing the
Notch intracellular domain (NICD). A literature review found that
the expression of constituent proteins and downstream genes in
the Notch signaling pathway in damaged lung tissues varied
[13,14]. In a lung injury model induced by smoke inhalation, MSCs
promoted angiogenesis and Notch1 expression in lung tissue [15].
The proliferation and differentiation of MSCs [16] and club cells [17]
are closely related to the Notch signaling pathway. In summary, the
internal and external repair processes of lung tissue are inextricably
linked to the Notch signaling pathway. Based on these findings, we
believe that MSCs are highly likely to affect club cells via the Notch
signaling pathway in the injured lung tissue.
This study explored whether exogenous MSCs affected club cells
in a phosgene-induced lung injury model, and whether an effect on
proliferation was achieved through the Notch signaling pathway.
2. Materials and methods
2.1. Animals
Animal procedures followed the guidelines for laboratory animal management and use of China. And the experimental protocol
was approved by the Ethics Committee of Jinshan Hospital affiliated
to Fudan University. A total of 108 SPF grade male SD rats weighing
180e220 g each, provided by the Experimental Animal Center of
Naval Medical University, Shanghai, China were used. The rats were
housed at a constant temperature and with independent ventilation systems (12:12 h light and dark cycle). The rats were allowed to
move freely and eat normally during the experiment. The rats were
anesthetized through intraperitoneal injection of 20% carbamate
solution (dose 1.0 g/kg), and were sacrificed at 6, 24 and 48 h after
phosgene exposure.
2.2. Phosgene-induced lung injury
Phosgene was prepared as described previously [18]. The final
concentration of phosgene in the poisoning chamber was 8.33 g/
m3
. Animals were exposed to phosgene for 5 min and then transferred to a room with fresh air.
2.3. Mesenchymal stem cells
Bone marrow-derived mesenchymal stem cells of male SD rats
(KALANG Technology Co., Ltd., Shanghai, China) were cultured in a
37 C incubator (5% CO2). After 24 h, MSCs were completely
adherent. The culture solution was changed and culture was
continued. When MSCs substantially covered the surface of the
flask, trypsin was added to digest intercellular proteins. MSCs were
then transferred into a centrifuge tube. After centrifugation, the
supernatant was discarded. The MSCs were resuspended by adding
a certain amount of PBS. The number of cells was adjusted to
1 106 per 50 mL PBS. The resuspended MSCs were immediately
instilled into the lungs via the trachea.
2.4. Specific inhibitor of the Notch signaling pathway
LY3039478 (LY; Selleck, Houston, USA), a potent Notch signaling
pathway-specific inhibitor of intracellular Notch receptor domain
cleavage, was used. LY was dissolved in a solvent of DMSO, polyethylene glycol, and double distilled water (volume ratio of
2:20:78) and a solution at a concentration of 4 mg/mL was prepared. For the groups treated with the inhibitor, LY solution was
intraperitoneally injected 2 h before phosgene exposure at a dose of
10 mg/kg.
2.5. Experimental grouping
Rats were randomly divided into the following six groups (n ¼ 6
per group at each time point): (1) control group (Air), rats always
exposed to fresh air; (2) phosgene exposure group (PH), rats
exposed to phosgene for 5 min, then transferred into fresh air
immediately; (3) phosgene þ PBS group (PH þ PBS) rats exposed to
phosgene for 5 min, then transferred to fresh air immediately, and
50 mL PBS (without MSCs) slowly instilled into the trachea after
anesthesia; (4) phosgene þ MSCs group (PH þ MSCs) rats exposed
to phosgene for 5 min, then transferred into fresh air immediately,
and 50 mL PBS (containing 1 106 MSCs) slowly instilled into the
trachea after anesthesia; (5) phosgene þ MSCs þ solvent group
(PH þ MSCs þ SO) rats intraperitoneally pre-injected with a certain
volume of the mixed solvent (i.e. mixed solution of DMSO, polyethylene glycol, double distilled water), according to the weight of
each rat 2 h before phosgene exposure. The rats were exposed to
phosgene for 5 min, then transferred into fresh air immediately,
and 50 mL PBS (containing 1 106 MSCs) was slowly instilled into
the trachea after anesthesia; (6) phosgene þ MSCs þ inhibitor
group (PH þ MSCs þ LY) rats intraperitoneally pre-injected with a
certain volume of LY solution (10 mg/kg) 2 h before phosgene
exposure. The rats were exposed to phosgene for 5 min, then
transferred into fresh air immediately, and 50 mL PBS (containing
1 106 MSCs) into the trachea after anesthesia.
2.6. Immunohistochemistry
The middle lobes of the rats were fixed with 4% paraformaldehyde, embedded in paraffin and cut into 4e5 mm slices.
2 K. Ye et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
Please cite this article as: K. Ye et al., Exogenous mesenchymal stem cells affect the function of endogenous lung stem cells (club cells) in
phosgene-induced lung injury, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.04.182
The tissue was then dewaxed and hydrated. And endogenous
peroxidase was blocked. The antigen was then repaired, exposed
and non-specific binding sites were blocked. After adding the primary antibody (CCSP, 1:400, Santa Cruz, Heidelberg, Germany), the
tissue was placed in a wet box at 4 C, overnight. A secondary
antibody was added and re-staining of nuclei was executed with
hematoxylin stain. The tissue was finally observed under an optical
microscope (Olympus, Tokyo, Japan).
2.7. Immunofluorescence
After the tissue slices were dewaxed and hydrated, the antigen
was repaired and exposed. Primary antibodies (CCSP, 1:500, Abcam,
Cambridge, UK; Ki-67, 1:300, Bioss, Beijing, China) were employed
and the tissue was incubated in a wet box at 4 C, overnight. The
slides were washed and the secondary antibodies (Servicebio,
Wuhan, China) were added and the tissue was incubated for 1 h at
20 C. DAPI was used to dye the nuclei. Lung tissue was observed
under a fluorescence microscope (Nikon, Tokyo, Japan). The specific
method was as follows: under 400-fold magnification, on each
slice, six tracheal cross sections of a similar size and shape
(diameter range: 100e200 mm) were selected from each slide. The
number of CCSP and Ki-67 double positive cells and the total
number of nuclei of tracheal epithelial cells were counted. For each
section, the ratio of CCSP and Ki-67 double positive cells were
calculated by dividing the average number of CCSP and Ki-67
double positive cells by the average number of nuclei.
2.8. Enzyme linked immunosorbent assay
The CCSP levels in BALF and serum were tested. After anesthetization, 6 mL of blood was collected from each rat. The left lung of
the rat was ligated and after tracheal intubation, 3 mL of 4 C PBS
was used to repeatedly lavage the left lobe three times. More than
80% of the lavage fluid was recovered. BALF and blood were
centrifuged at 1500 rpm for 10 min at 4 C, and the supernatant was
retained. CCSP levels in BALF and serum were determined using a
rat CCSP ELISA kit (KALANG Technology Co., Ltd., Shanghai, China),
according to the manufacturer’s recommended protocol.
Fig. 1. CCSP levels and the average fluorescence intensity (AFI) of CCSP in club cells. CCSP levels in BALF (A) and serum (B) and CCSP AFI in club cells (C) in the lower lobe were
shown. Results were expressed as mean ± SD (n ¼ 6). Statistical differences were indicated as follows: * p < 0.05, ** p < 0.01, *** p < 0.001, compared with the Air group; # p < 0.05, ##
p < 0.01, compared with the PH group; & p < 0.05, && p < 0.01, compared with the PH þ PBS group.
K. Ye et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx 3
Please cite this article as: K. Ye et al., Exogenous mesenchymal stem cells affect the function of endogenous lung stem cells (club cells) in
phosgene-induced lung injury, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.04.182
2.9. Real-time reverse transcription-polymerase chain reaction
An appropriate amount of tissue was finely ground. An RNA
extraction solution was created using chloroform, isopropanol and
75% ethanol. The concentration and purity of RNA was detected and
the RNA and primers were used to perform reverse transcription
and obtain cDNA, which was used to prepare a reaction system for
amplification. Using the DDCt method normalized with GAPDH,
relative expression levels of genes were determined. The primer
sequences used were listed in the supplemental table.
2.10. Flow cytometry
The AFI of club cells was measured in this experiment. The
entire left inferior lobe was collected from treated rats and 2 mL of
PBS was added. The tissue was then ground thoroughly to prepare a
single-cell suspension. Diluted primary antibody (CCSP, 1:100,
Santa Cruz, Heidelberg, Germany) was added and the sample was
incubated overnight at 4 C. After washing, the mixture was incubated with the secondary antibody (Biyuntian, Shanghai, China) at
4 C for 1 h in the dark. The sample was then centrifuged at
1500 rpm for 10 min; the supernatant was removed, and resuspended in PBS after washing. Each sample was tested by flow
cytometer (BD, Franklin Lakes, USA).
2.11. Statistical analysis
All data in this experiment were analyzed using GraphPad
(version 5.0). Analysis of variance (ANOVA) was used to detect
whether the differences were statistically significant, while unpaired t-test was used to compare differences between two groups.
P < 0.05 was considered to be statistically significant.
3. Results
3.1. MSCs affected the secretion of club cells
This study evaluated whether the secretion of club cells was
changed after phosgene exposure and MSCs intervention. The
results showed that there was no significant difference in CCSP
expression among the lung tissue from four treatment group (Air,
PH, PH þ PBS and PH þ MSCs groups) at 6, 24 and 48 h (negative
results are not shown). At 6, 24 and 48 h, compared to the Air
group, BALF and serum CCSP levels of the PH group increased.
However, after the MSCs intervention (PH þ MSCs group) BALF
and serum CCSP levels decreased, compared with that of the PH
group (Fig. 1A). At 6, 24 and 48 h, compared to the PH and
PH þ PBS groups, club cells were dark brown as shown through
immunohistochemistry and the AFI of the club cells was
enhanced as shown through flow cytometry in the PH þ MSCs
group (Figs. 1B and 2). These results indicated that MSCs intervention weakened the secretion of club cells in the lung tissue
exposed to phosgene.
3.2. MSCs affected the proliferation of club cells
Changes in the proliferation of club cells were evaluated after
phosgene exposure and MSCs intervention. We found that
compared to the Air group, club cells in the tracheas were damaged
and fell into the tracheal cavities after phosgene exposure at 6, 24
and 48 h. At 48 h, compared with the PH and PH þ PBS groups, the
number of club cells in the tracheal cavities of the PH þ MSCs group
were significantly decreased (Fig. 2). As shown with immunofluorescence double labelling, at 6, 24 and 48 h, the numbers of proliferative club cells (i.e., CCSP and Ki-67 double positive cells) in the
middle lobe of the PH group rats increased compared with that of
the Air group. Compared to the PH and PH þ PBS groups, the
Fig. 2. Immunohistochemistry of the middle lobe of the lung. Immunohistochemistry results with CCSP as the antibody were shown. Brown detached club cells were visible (red
sun markers) in the tracheal lumens. Scale bar: 50 mm. Magnification: 400x. . (For interpretation of the references to color in this figure legend, the reader is referred to the Web
version of this article.)
4 K. Ye et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
Please cite this article as: K. Ye et al., Exogenous mesenchymal stem cells affect the function of endogenous lung stem cells (club cells) in
phosgene-induced lung injury, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.04.182
number of proliferative club cells in the PH þ MSCs group further
increased (Fig. 3C and D). These results indicated that in phosgeneinduced lung injury, MSCs intervention enhanced the proliferation
of club cells.
3.3. Activation of the Notch signaling pathway
The expression of important components of the Notch signaling
pathway (including Notch1~4, c-myc, Hey1 and Hes1) in lung tissue
of each group was monitored. It was shown that at 6, 24 and 48 h,
compared to the Air group, the expression of Notch1, c-myc, Hey1
and Hes1 increased, the expression of Notch1 decreased and the
expression of Notch3 and Notch4 did not change significantly.
Compared with the PH and PH þ PBS groups, the expression of
Notch1, Notch2, c-myc, Hey1 and Hes1 of lung tissue in the
PH þ MSCs group increased at 6, 24 and 48 h. It suggested that the
Notch signaling pathway in lung tissue was further activated after
MSCs intervention. At 6, 24 and 48 h, the number of proliferative
club cells in the PH þ MSCs group further increased compared to
the PH and PH þ PBS groups (Figs. 3 and 4). These results indicated
that MSCs might promote the proliferation of club cells through the
Notch signaling pathway.
3.4. Application of Notch signaling pathway inhibitor
We further validated whether changes in the proliferation of
club cells were associated with the Notch signaling pathway by
applying LY, a specific Notch signaling pathway-specific inhibitor.
At 6, 24 and 48 h, the expression of Notch1, Notch2, c-myc, Hey1,
and Hes1 in injured lung tissue decreased after the application of
LY, indicating that the effect of LY was positive and as expected
(Fig. 4). Compared to the PH þ MSCs and PH þ MSCs þ SO groups,
the number of proliferative club cells in the PH þ MSCs þ LY group
decreased significantly (Fig. 3). It indicated that the MSCs-induced
proliferation of club cells was weakened by LY. These results indicated that in phosgene-induced lung injury, MSCs at least partially
activated the Notch signaling pathway to further enhance the
proliferation of club cells.
4. Discussion
Our results showed that in phosgene-induced lung injury, the
intervention of exogenous MSCs reduced the secretion and
enhanced the proliferation of club cells. MSCs enhanced the proliferation of club cells by further activating the Notch signaling
Fig. 3. Immunofluorescence of the middle lobe and the number of CCSP and Ki-67 double positive cells. The immunofluorescence results (D) were shown. Antibodies used
were Ki-67 (red channel) and CCSP (green channel). Proliferative cells (red arrow), club cells (green arrow), and proliferative club cells (yellow arrow) were labeled in the figure.
DAPI (blue channel) was used to locate cell nuclei. Scale bar: 50 mm. Magnification: 400x. The analysis of CCSP and Ki-67 double positive cells was shown (E). Results are expressed
as mean ± SD (n ¼ 6). Statistical differences are indicated as follows: ** p < 0.01, *** p < 0.001, compared with the Air group; # p < 0.05, ## p < 0.01, ### p < 0.001, compared with the
PH group; & p < 0.05, && p < 0.01, &&& p < 0.001, compared with the PH þ PBS group; $$$ p < 0.001, compared with the PH þ MSCs group; @@@ p < 0.001, compared with the
PH þ MSCs þ SO group. . (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
K. Ye et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx 5
Please cite this article as: K. Ye et al., Exogenous mesenchymal stem cells affect the function of endogenous lung stem cells (club cells) in
phosgene-induced lung injury, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.04.182
pathway, thus promoting the repair of injured tissue.
In our experiment, MSCs attenuated the secretion of club cells.
This might be due to the anti-inflammatory, anti-oxidative cytokines (similar to CCSP) secreted by MSCs, resulting in a reduced
“demand” for CCSP for the repair of lung injury. Under normal
circumstances, a small quantity of CCSP in the airways enters the
bloodstream due to the presence of the blood-gas barrier in the
lung. In lung damage caused by phosgene, more CCSP entered
blood circulation due to increased secretion of CCSP and destruction of lung barrier function, resulting in elevated CCSP levels in
serum. After MSCs intervention, the decrease in serum CCSP was
due to a reduction of BALF-derived CCSP and MSCs-induced
improvement in lung permeability [11]. Therefore, to some
extent, the decrease in serum CCSP indicated the repairing effect of
MSCs on lung injury.
Studies have shown that CCSP is a good biomarker of epithelial
damage [19]. After exposure to asbestos [20] and ozone [21], CCSP
levels in serum were often associated with lung epithelial damage.
In asthmatic patients, serum CCSP was associated with the
involvement of small airway [22]. CCSP levels can indicate a change
in lung permeability and are associated with treatment outcomes.
In this experiment, serum CCSP changed after phosgene exposure
and MSCs intervention. However, whether CCSP would be a good
biomarker in phosgene-induced lung injury requires further
investigation.
We used lung homogenates (including all lung interstitial cells
and airway epithelial cells) to assess changes in the Notch signaling
pathway. Our results showed that club cells proliferated in
response to the activation of the Notch signaling pathway. However, we were unable to determine whether the changes in the
Notch signaling pathway were restricted to the airway epithelium.
In future studies, isolation and in vitro culture of relevant cells will
help to further evaluate evidence. Co-culture of club cells with
MSCs in vitro will further reveal the effects of MSCs on club cells
with increased accuracy.
Endogenous stem cells are greatly advantageous for directional differentiation into damaged cells in the repair of lung
injury, but endogenous repair is often difficult to regulate.
Moreover, the lung injury microenvironment where endogenous
stem cells are located can be changed and then repair cannot be
guaranteed. Studies have found that MSCs played a role in
exogenous damage repair through paracrine, differentiation and
other methods [10]; control of cell number and cell function is
better. Both internal and external repair have their own
strengths. If these two repair methods are combined, better
treatment results can be obtained.
Previously, a few studies regarding the effects of exogenous
stem cells on endogenous stem cells have been published but the
mechanism involved has never been explored. Our study found
that MSCs instilled in the airways affected endogenous club cells
in multiple ways (including changes in secretion and proliferation). This influence on the proliferation was related to the
changes of the Notch signaling pathway. In the future, key regulators related to the Notch signaling pathway can be overexpressed in MSCs, promoting the function of club cells and further
enhancing the repair of lung injury, and thereby increasing
clinical efficacy. This research examined the link between
endogenous and exogenous repair pathways in the lungs, which
may help clinical innovation and may provide a new direction for
the treatment of acute lung injury.
Conflicts of interest
The authors declare that there are no conflicts of interest.
Fig. 4. Expression of major constituent proteins in the Notch signaling pathway. The expression of Notch1, Notch2, c-myc, Hey1 and Hes1 were shown. Results were expressed
as mean ± SD (n ¼ 6). Statistical differences were indicated as follows: * p < 0.05, ** p < 0.01, *** p < 0.001, compared with the Air group; ## p < 0.01, ### p < 0.001, compared with the
PH group; && p < 0.01, &&& p < 0.001, compared with the PH þ PBS group; $$ p < 0.01, $$$ p < 0.001, compared with the PH þ MSCs group; @@ p < 0.01, @@@ p < 0.001, compared with
the PH þ MSCs þ SO group.
6 K. Ye et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
Please cite this article as: K. Ye et al., Exogenous mesenchymal stem cells affect the function of endogenous lung stem cells (club cells) in
phosgene-induced lung injury, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.04.182
Acknowledgments
This work was supported by the National Natural Science
Foundation of China (No. 81471850), Shanghai Public Health System (2017-1) and the Research project of Shanghai Health and
Family Planning Commission (No. JSZK2015A01).
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.bbrc.2019.04.182.
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K. Ye et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx 7
Please cite this article as: K. Ye et al., Exogenous mesenchymal stem cells affect the function of endogenous lung stem cells (club cells) in
phosgene-induced lung injury, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.04.182