Inhalation of MSC-EVs is a noninvasive strategy for ameliorating acute lung injury

Abstract

Mesenchymal stem cell-derived small extracellular vesicles (MSC-EVs) are promising nanotherapeutic agent for pneumonia (bacterial origin, COVID-19), but the optimal administration route and potential mechanisms of action remain poorly understood. This study compared the administration of MSC-EVs via inhalation and tail vein injection for the treatment of acute lung injury (ALI) and determined the host-derived mechanisms that may contribute to the therapeutic effects of MSC-EVs in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells (macrophage cell line) and animal models. Luminex liquid chip and hematoxylin and eosin (HE) staining revealed that, compared with the vehicle control, inhaled MSC-EVs outperformed those injected via the tail vein, by reducing the expression of pro-inflammatory cytokines, increasing the expression of anti-inflammatory cytokine, and decreasing pathological scores in ALI. MSC-EV administration promoted the polarization of macrophages towards a M2 phenotype in vitro and in vivo (via inhalation). RNA sequencing revealed that immune and redox mediators, including TLR4, Arg1, and HO-1, were associated with the activity MSC-EVs against ALI mice. Western blotting and immunofluorescence revealed that correlative inflammatory and oxidative mediators were involved in the therapeutic effects of MSC-EVs in LPS-stimulated cells and mice. Moreover, variable expression of Nrf2 was observed following treatment with MSC-EVs in cell and animal models, and knockdown of Nrf2 attenuated the anti-inflammatory and antioxidant activities of MSC-EVs in LPS-stimulated macrophages. Together, these data suggest that inhalation of MSC-EVs as a noninvasive strategy for attenuation of ALI, and the adaptive regulation of Nrf2 may contribute to their anti-inflammatory and anti-oxidant activity in mice.

Full text

Journal of Controlled Release 345 (2022) 214–230
Available online 18 March 2022
0168-3659/© 2022 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
Inhalation of MSC-EVs is a noninvasive strategy for ameliorating acute
lung injury
Ruijing Zhao
a
,
1
, Lina Wang
a
,
1
, Tian Wang
a
,
b
, Panpan Xian
a
, Hongkang Wang
a
,
Qianfa Long
a
,
c
,
*
a
Mini-invasive Neurosurgery and Translational Medical Center, Xi’an Central Hospital, Xi’an Jiaotong University. No. 161, West 5
th
Road, Xincheng District, Xi’an
710003, China
b
Shaanxi Lon-EV Biotechnology Limited Company, No.9 Jiazi, Renyi village, Beilin District, Xi’an 710054, China
c
College of Medicine, Yan’an University, Yongxiang Road, Baota District, Yan’an 716000, China
ARTICLE INFO
Keywords:
Small extracellular vesicles
Inhalation
Acute lung injury
Immunomodulation
Redox system
ABSTRACT
Mesenchymal stem cell-derived small extracellular vesicles (MSC-EVs) are promising nanotherapeutic agent for
pneumonia (bacterial origin, COVID-19), but the optimal administration route and potential mechanisms of
action remain poorly understood. This study compared the administration of MSC-EVs via inhalation and tail
vein injection for the treatment of acute lung injury (ALI) and determined the host-derived mechanisms that may
contribute to the therapeutic effects of MSC-EVs in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells
(macrophage cell line) and animal models. Luminex liquid chip and hematoxylin and eosin (HE) staining
revealed that, compared with the vehicle control, inhaled MSC-EVs outperformed those injected via the tail vein,
by reducing the expression of pro-inammatory cytokines, increasing the expression of anti-inammatory
cytokine, and decreasing pathological scores in ALI. MSC-EV administration promoted the polarization of
macrophages towards a M2 phenotype in vitro and in vivo (via inhalation). RNA sequencing revealed that immune
and redox mediators, including TLR4, Arg1, and HO-1, were associated with the activity MSC-EVs against ALI
mice. Western blotting and immunouorescence revealed that correlative inammatory and oxidative mediators
were involved in the therapeutic effects of MSC-EVs in LPS-stimulated cells and mice. Moreover, variable
expression of Nrf2 was observed following treatment with MSC-EVs in cell and animal models, and knockdown of
Nrf2 attenuated the anti-inammatory and antioxidant activities of MSC-EVs in LPS-stimulated macrophages.
Together, these data suggest that inhalation of MSC-EVs as a noninvasive strategy for attenuation of ALI, and the
adaptive regulation of Nrf2 may contribute to their anti-inammatory and anti-oxidant activity in mice.
1. Introduction
Acute lung injury (ALI) is characterized by impaired pulmonary gas
exchange, bilateral inltrates, and noncardiogenic edema, and can be
induced by direct injury and systemic stimuli, such as mechanical
trauma, bacteria, and viruses (e.g., SARS-CoV-2), resulting in human and
economic burden [1,2]. If pulmonary disease is not effectively managed
during the early stage, acute respiratory distress syndrome (ARDS) can
develop, and is associated with high mortality [3]. ALI is associated with
severe acute inammation, as well as the complications of infections,
such as increased permeability of blood vessels and the death of
pulmonary epithelial and endothelial cells [2]. Stem cell therapy has
demonstrated great potential in the treatment of lung injury, including
that induced by COVID-19, owing to its immunomodulatory and tissue
repair properties [4,5]. However, the cellular candidates, optimal
management, and therapeutic mechanisms are not well understood.
Increasing evidence, including data from our previous studies, sug-
gests that small extracellular vesicles (EVs) exhibit more potential than
their parental cells (e.g. MSCs) as therapeutics against inammatory
diseases, owing to characteristics such as blood-air barrier permeability,
freeze / thaw resistance, and targeting to injured cells [6,7]. Recent
studies have shown that EV-based therapies hold potential for the
* Corresponding author at: Mini-invasive Neurosurgery and Translational Medical Center, Xi’an Central Hospital, Xi’an Jiaotong University, No. 161, West 5th
Road, Xincheng District, Xi’an 710003, China.
E-mail address: lonva@live.cn (Q. Long).
1
These authors contributed this work equally.
Contents lists available at ScienceDirect
Journal of Controlled Release
journal homepage: www.elsevier.com/locate/jconrel
https://doi.org/10.1016/j.jconrel.2022.03.025
Received 18 November 2021; Received in revised form 21 February 2022; Accepted 14 March 2022

Journal of Controlled Release 345 (2022) 214–230
215
treatment of lung injury, such as that induced by COVID-19, as they can
target multiple pathways and enhance tissue regeneration [8]. In addi-
tion, MSC-EVs can attenuate ALI via mitochondrial or miRNA transfer
and modulate macrophage polarization because they contain multiple
functional cargoes, including proteins, lipids, RNA, and metabolites
[6,9,10]. Besides to determine the therapeutic agents carried by EVs, it
also is important to elucidate the therapeutic mechanisms, including
immunomodulation, antioxidation, and tissue regeneration in target
cells or injured tissues during ALI.
Inammatory stimuli can evoke the excessive production of reactive
oxygen species (ROS) in pulmonary tissue, followed by the development
of ALI. Oxidative stress is an early contributor to ALI, and can cause
macrophage activation, cellular inltration, and enhanced pulmonary
cytokine production [11,12]. Thus, crosstalk between oxidation and
inammation is important for regulating the initiation and progression
of ALI. Specically, the nuclear factor kappa beta (NF-κB) pathway is
activated by a variety of stimuli in ALI, and activation of NF-κB down-
stream of Toll-like receptor 4 (TLR4) and transcription factors such as
signal transducer and activator of transcription 3 (STAT3) mediates
macrophage plasticity and inammation [13]. Also, the anti-
inammatory or antioxidative activity in ALI depends on the regula-
tion of nuclear factor erythroid 2-related factor 2 (Nrf2, a key mediator
in oxidative stress) [12,14]. Together, these mediators may orchestrate
inammation and oxidation during lung injury. Previously, we showed
that MSC-EVs exert signicant biological activities in models of
inammation and oxidative stress [15,16], which suggests that EV
therapy may have potential to regulate the crosstalk between oxidation
and inammation in ALI.
In the present study, inhalation and tail vein injection were used as
administration methods to examine the potential activity of MSC-EVs in
mice with ALI. This was followed by RNA sequencing (RNA-Seq), mo-
lecular pattern tests, and Nrf2 knockdown to elucidate the therapeutic
mechanism of MSC-EVs in lipopolysaccharide (LPS)-stimulated cells or
mice. The results showed that administration of MSC-EVs via inhalation
has potential against acute lung inammation and oxidation, high-
lighting the clinical value of MSC-EV inhalation in ALI, even in that
induced by COVID-19.
2. Materials and methods (Fig. S1)
2.1. Cell preparation
For preparation of allogeneic MSCs, informed consent was obtained
before cell collection, and 3 donors (age 27–29) were selected from full-
term puerpera in good health. All procedures were approved by the
Ethical Committee of the Xi’an Central Hospital, Xi’an Jiaotong Uni-
versity, as well as in accordance with the Guidelines of the National
Institutes of Health. MSCs were obtained from Wharton’s jelly in the
umbilical cord and characterized by ow cytometry, the gating strategy
was employed by using Fluorescence Minus One control, as in our pre-
vious reports [15,17]. Primary antibodies including rabbit polyclonal
anti-CD105, CD90, CD73, CD45, CD34 and CD11b (1:100 dilution)
(Bioss, Wuhan, CHN), and secondary antibody Alexa Fluor 488 goat
anti-rabbit IgG (1:500) (Invitrogen, A-21206, CA, USA) were used to
detect the surface antigens of fth-passage MSCs. At least three cell
culture samples were examined on an FACS Calibur instrument (Becton
Dickinson) and the data were analyzed using Cell Quest software (Bec-
ton Dickinson). Multi-potency of MSCs was detected by StemPro®
Osteogenesis (Gibco, A1007201, MD, USA), Chondrogenesis (Gibco,
A1007101, MD, USA) and Adipogenesis (Gibco, A1007001, MD, USA)
differentiation Kits (37 ◦C, 5% CO2) according to the instructions. RAW
264.7 cells, a murine macrophage cell line, were purchased from the Cell
Bank of Type Culture Collection of Chinese Academy of Sciences
(Shanghai, China) and cultured in Dulbecco’s modied Eagle medium
(DMEM) / F12 +10% fetal bovine serum (FBS) at 37 ◦C, 5% CO
2
.
2.2. Isolation, characterization and labelling of MSC-EVs
EVs were isolated from the supernatants of fth-passage MSCs, as
previously described [15]. Briey, the ratio of live and dead MSCs was
detected by using an automatic cell counter (Bodboge, Shenzhen, CHN),
the batch of which contained more than 99% live cells. The MSCs were
cultured in
α
MEM containing 10% EV-depleted FBS for 24 h, and then
the supernatants were harvested and processed via a series of centrifu-
gation steps (300 ×g for 10 min, 2000 ×g for 10 min, and 10,000 ×g for
30 min; ST16R, Thermo Fisher, USA). Subsequently, the EVs were
collected via ultracentrifugation at 100,000 ×g for 70 min (XPN-100,
Beckman Coulter, USA). The nanoparticles were then characterized by
western blotting based on the positive markers TSG101 and CD9, as well
as negative marker calnexin, and examined via transmission electron
microscopy (TEM) and nanoparticle tracking analysis (NTA) to evaluate
morphology and size distribution, respectively. In addition, C5
Maleimide-Alexa 594 (CM-A954) (Invitrogen, A10256, California, USA)
was used to label MSC-EVs as our previous reports [17].
2.3. Macrophage activation and intervention
RAW 264.7 cells were pretreated with 10
μ
g / mL MSC-EVs
(Fig. S2A) for 12 h to ensure uptake, and then activated with 100 ng /
mL lipopolysaccharide (LPS; L2880, Sigma-Aldrich, CA, USA) for 12 h,
as previously reported [18]. ML385 (15
μ
M; HY-100523, Medchem
Express, New Jersey, USA), a pharmacological inhibitor of Nrf2, was
used to downregulate Nrf2 expression in RAW 264.7 cells.
2.4. Animal procedures
97 adult male (8–10-weeks old) C57BL/6 mice were purchased from
the Experimental Animal Center of Xi’an Jiaotong University, they were
housed in groups and were allowed a period to acclimatize to the lab-
oratory environment before the start of the study. All animal procedures
were performed in accordance with the ARRIVE guidelines and
approved by the Ethics Review Board of Xi’an Central Hospital, Xi’an
Jiaotong University. Animals were housed under a controlled environ-
ment with a 12 / 12 h light / dark cycle with food and water provided.
Mice were intraperitoneally anesthetized using 4.0% chloralhydrate
(10 mL / kg) and administered LPS (10 mg / kg, diluted with saline)
intratracheally. A sham operation was performed in a similar manner
using saline solution (Sham group, n =5). After LPS induction for 3 h,
50
μ
g MSC-EVs (diluted in 50
μ
L saline, Fig. S2B and C) and 50
μ
L saline
(vehicle) were administered via inhalation using an atomizer (YSKD Bio-
Tec, Beijing, China) as the ALI-inh +EVs (n =20) and ALI-inh +Veh (n
=15) groups, or administered by tail vein injection as the ALI-iv +EVs
(n =15) and ALI-iv +Veh (n =15) groups, respectively. The mice were
sacriced at random via isourane at 24 h, 4 days (d), and 14 d after EV
administration, and lung tissues and blood samples were collected for
further analyses (Fig. S1). Also, to track the MSC-EVs in vitro, ALI mice
received MSC-EVs via inhalation (negative control, n =3), or CM-A594
labeled MSC-EVs via inhalation (n =3) and tail vein injection (n =3) for
24 h, the lung tissues were then processed as above procedures. Addi-
tionally, 8 mice were excluded due to failed injection via tail vein, and
10 mice were died in the present experiments.
2.5. Luminex liquid chip
Following treatment with MSC-EVs for 24 h, whole blood was
collected from mouse orbits and centrifuged (10,000 rpm) for 10 min.
Supernatants from each group were assayed using a Luminex liquid chip
(Luminex 200, USA) for interleukin (IL)-1β, macrophage chemo-
attractant protein-1 (MCP-1), IL-1
α
, tumor necrosis factor (TNF)
α
, IL-12,
and IL-10 (MHSTCMAG-70 K, Mouse High Sensitivity T Cell Magnetic
Bead Panel, USA), according to the manufacturer’s instructions.
R. Zhao et al.

Journal of Controlled Release 345 (2022) 214–230
216
Fig. 1. Characterization of MSC-EVs.
MSC-EVs were collected using a series of centrifugation steps. (A) western blotting showed that, compared with MSCs, the nanovesicles strongly express the
membrane protein TSG 101 and CD 9, and do not express calnexin; (B) transmission electron microscope (TEM) revealed that MSC-EVs form a classical “rim of a cup”
shape; (C) nanoparticle tracking analysis (NTA) shows the mean size distribution of the collected EVs; (D) Representative images show the location of C5 Maleimide-
Alexa 594 (CM-A954) labeled MSC-EVs (red) in the lung tissues of ALI model (bar =20
μ
m), the square area appears with higher magnication in the image on the
lower left (bar =10
μ
m). (For interpretation of the references to colour in this gure legend, the reader is referred to the web version of this article.)
R. Zhao et al.

Journal of Controlled Release 345 (2022) 214–230
217
2.6. Histological study
Mice were randomly selected from each group (n =5–6) and used to
measure cell inltration and alveolar collapse in ALI. Sections were
deparafnized, stained with hematoxylin and eosin (HE), subjected to
light microscopy (Leica, Germany) and analyzed using a numerical scale
[19]. Image-Pro Plus was used to determine the lesion area in the
pathological sections, and the pathological score was analyzed as pre-
viously described [20,21].
For immunostaining, lung tissues from each group were harvested at
random, processed as previously described [16], and then treated for 30
min in PBS solution containing 0.1% Triton-X 100 and 10% serum,
selected based on the species in which the chosen secondary antibody
was raised. Primary antibodies against 8-OHdG (oxidative marker)
(1:200; AB5830, Millipore, MA, USA), CD86 (M1 marker) (1:200;
19,599, CST, MA, USA), CD206 (M2 marker) (1:200; ab64693, Abcam,
CA, USA) and ED1 (macrophage marker) (1:200; ab53444, Abcam, CA,
USA). After overnight incubation (4 ◦C), the samples were incubated
with an appropriate secondary antibody solution for 2 h at room tem-
perature, and the cell nuclei were probed using 4′,6-diamidino-2-phe-
nylindole (DAPI; D9542, Sigma-Aldrich, CA, USA). Images were
captured using a microscope (THUNDER Imager Tissue 3D, Leica, Ger-
many). Mean uorescence intensity (MFI) was analyzed using the
Image-Pro Plus software.
2.7. RNA sequencing (RNA-Seq)
To evaluate variation in the immune and redox systems following
MSC-EV treatment for 4 d in ALI, total RNA was extracted from Sham (n
=4), ALI-inh +EVs (n =3), and ALI-inh +Veh (n =3) mice using the
RNAiso Plus Total RNA extraction reagent (9109, Takara, Japan). The
RNA integrity number was determined to conrm RNA integrity using
an Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA,
US). Total RNA was further puried using the RNAClean XP Kit
(A63987, Beckman Coulter, Inc. Kraemer Boulevard Brea, CA, USA) and
RNase-Free DNase Set (79,254, QIAGEN, GmBH, Germany). Then, RNA
libraries were generated using 1
μ
g of total RNA based on the VAHTS
Universal V6 RNA-seq Library Prep Kit for Illumina® (NR604–02,
Vazyme, Nanjing, China), according to the manufacturer’s instructions.
The quality of the cDNA library was assessed using a DNA chip on an
Agilent 4200 Bioanalyzer. Finally, the library was sorted using the
Illumina HiSep X Ten platform. RNA sequencing and analyses were
performed by the Biotechnology Corporation (BC200469–1, Shanghai,
China). Differential gene analysis was performed using edgeR [22], and
the differentially expressed mRNAs were screened according to the ab-
solute value of differential multiple fold change (FC) >2 and P <0.05.
2.8. Western blotting
Western blotting was performed to evaluate changes in protein
expression in each group (n =5 per group), as previously described [17].
Proteins were extracted from cells or tissue samples using radio-
immunoprecipitation assay (RIPA) lysis buffer (P0013B, Beyotime,
Beijing, China) and quantied via bicinchoninic acid assay. Normalized
protein samples were subjected to sodium dodecyl-sulfate poly-
acrylamide gel electrophoresis (SDS-PAGE) and transferred to poly-
vinylidene uoride membranes (Millipore, MA, USA). Membranes were
blocked using 5% skim milk at room temperature for 1.5 h and then
incubated with primary antibodies against CD9 (1:800; ab92726,
Abcam, CA, USA), TSG101 (1:800; ab125011, Abcam, CA, USA), Nrf2
(1:1000; PA5–27882, Invitrogen, IL, USA), HO-1 (1:1000; 10,701–1-AP,
Proteintech, IL, USA), Keap1 (1:1000; 10,503–2-AP, Proteintech, IL,
USA), CD86 (1:1000; 19,599, CST, CA, USA), inducible nitric oxide
synthase (iNOS; 1:1000; 18,985–1-AP, Proteintech, IL, USA), Arg1
(1:10,000; 16,001–1-AP, Proteintech, IL, USA), NF-κB p65 (1:1000;
6956S, CST, MA, USA), TLR4 (1:1000; 66,350–1-lg, Proteintech, IL,
USA), TNF-
α
(1:1000; ab183218, Abcam, CA, USA), IL-1β (1:1000;
31202S, CST, MA, USA), high mobility group box-1 protein (HMGB1;
1:1000; 10,829–1-AP, Proteintech, IL, USA), and β-actin (1:100,000;
AC026, Abclonal, Wuhan, China) overnight at 4 ◦C, followed by washing
with TBST three times. The membranes were then incubated with
horseradish peroxidase-conjugated secondary antibodies (Thermo
Fisher Scientic, NY, USA) at room temperature for 1.5 h. Labeled
proteins were detected using a Bio-Rad imaging system (Bio-Rad, Her-
cules, CA, USA) and quantied using the Image Lab software package
(Bio-Rad, CA, USA) based on β-actin protein expression.
2.9. Real time quantitative PCR (qPCR)
Total RNA was extracted from cell samples (n =5 per group) using
TRIzol reagent (15596026, Life Technologies, New York, USA) and
processed as previously reported [17]. All primer sequences were
designed and optimized by TaKaRa (TaKaRa, Dalian, China), as follows:
Nfe2f2: Forward 5′-TTGGCAGAGACATTCCCATTTG-3′, Reverse 5′-
AAACTTGCTCCATGTCCTGCTCTA-3′; Keap1: Forward 5′-AGCA-
GATCGGCTGCACTGAA-3′, Reverse 5′-AGCTGGCAGTGTGACAGGTTG-
3; IL-6: Forward 5′-CCACTTCACAAGTCGGAGGCTTA-3′, Reverse 5′-
TGCAAGTGCATCATCGTTGTTC-3′, Tnf
α
: Forward 5′-ACTC-
CAGGCGGTGCCTATGT-3′, Reverse 5′-GTGAGGGTCTGGGCCATAGAA-
3′; β-actin: Forward 5′-CATCCGTAAAGACCTCTATGCCAAC-3′, Reverse
5′-ATGGAGCCACCGATCCACA-3′. Reverse transcription was performed
using the Prime Script RT reagent kit with a gDNA eraser (RR047A,
Takara, Japan). qPCR was performed using TB Green Premix Ex Taq II
(RR820A, Takara, Japan) on a CFX Connect real-time PCR detection
system (Bio-Rad, CA, USA). The data were analyzed using Bio-Rad CFX
2.1 in triplicate.
2.10. Statistical analysis
All observers were blinded to the experimental group assignment
and data were examined by the Kolmogorov-Smirnov (K
–
S) test to
determine distribution (P ≥0.05 was employed in the following anal-
ysis). Data are expressed as the mean ±standard error of the mean
(SEM). Multiple comparisons were analyzed using one- or two-way an-
alyses of variance (ANOVA) via the least signicant difference (LSD) test
using SPSS 22.0.0; GraphPad Prism 8 (GraphPad Prism, USA) was used
to construct the histograms. Statistical signicance was set at P <0.05.
3. Results
3.1. Identication of MSCs and their derived EVs
Flow cytometry results showed that MSCs strongly expressed stromal
markers CD105 (99.5%), CD90 (99.1%), and CD73 (80.7%), and weakly
expressed hematopoietic markers CD45 (2.6%) and CD34 (2.9%). Also,
analyses of multipotency manifested that MSCs differentiated into os-
teoblasts, chondroblasts, and adipocytes following induction, using
differentiation kits, respectively. Thus, these results indicated that the
characteristics of these cells met the specic criteria of MSCs. Nano-
vesicles isolated from the culture medium of MSCs via a series of ultra-
centrifugation steps were positive for the markers TSG 101 and CD 9,
which occur on the surface of MSCs, and negative for calnexin (Fig. 1A).
The TEM and NTA results revealed that the vesicles presented a classical
“rim of a cup” and granule shape (Fig. 1B), and the mean diameter was
123.3 ±40.8 nm (number and concentration; Fig. 1C). Together, these
results indicated that the isolated vesicles met the typical criteria for
small EVs, as previously reported [15,23].
R. Zhao et al.

Journal of Controlled Release 345 (2022) 214–230
218
(caption on next page)
R. Zhao et al.

Journal of Controlled Release 345 (2022) 214–230
219
3.2. MSC-EVs administered via inhalation performed better than those
administered via tail vein injection regarding anti-inammation and tissue
repair in ALI mice
Following the treatment of ALI mice with MSC-EVs for 24 h, we
found more CM-A594 (red uorescence) labeled MSC-EVs were located
in the lung tissue via inhalation compared with tail vein injection
(Fig. 1D). Also, the expression of pro- and anti-inammatory cytokines
in serum was determine using a Luminex liquid chip. Our results showed
that both inhalation (ALI-inh +EVs) and tail vein injection (ALI-iv +
EVs) of MSC-EVs reduced (P <0.05) the levels of the pro-inammatory
cytokines IL-1β (Fig. 2A), MCP-1 (Fig. 2B), IL-1
α
(Fig. 2C), TNF
α
(Fig. 2D), and IL-12 (Fig. 2E), and increased (P <0.05) the level of the
anti-inammatory cytokine IL-10 (Fig. 2F) compared with the vehicle
group (ALI-inh +Veh or ALI-iv +Veh). Notably, compared with the ALI-
iv +EV group, lower levels (P <0.05) of pro-inammatory cytokines
(Fig. 2A–E) were observed following the inhalation of MSC-EVs (ALI-inh
+EVs) by ALI mice, while no signicant difference (P >0.05) in the
expression of anti-inammatory cytokine was found between the ALI-iv
+EVs and ALI-inh +EV groups. Moreover, to investigate the tissue
repair effects of MSC-EVs in LPS-induced lung injury, HE staining was
performed to evaluate pathological changes at different time points. As
shown in Fig. 2G, changes in lung tissues compared with the sham group
were apparent, and included alveolar wall thickening, interalveolar
septum congestion, and inammatory inltration after LPS stimulation
(ALI-iv +Veh and ALI-inh +Veh) on day 4 and 14. These changes were
further identied by the increased pathological scores (Fig. 2H). Mark-
edly, MSC-EV treatments (ALI-iv +EVs and ALI-inh +EVs) reversed the
changes observed in the vehicle control group (Fig. 2G); however, a
signicant difference (P <0.01) in pathological scores was only
observed between EV therapy (ALI-inh +EVs) and saline treatment
(ALI-inh +Veh) on day 4. Notably, lower pathological scores (P <
0.001) were observed in the ALI-inh +EVs group compared with the
ALI-iv +Veh group at 4 d (Fig. 2H). Collectively, these data indicated
that MSC-EVs ameliorated the inammatory response and pathological
alterations at an early stage, and that MSC-EVs administered via inha-
lation performed better than those administered via tail vein injection
against LPS-induced inammation and tissue injury in mice.
3.3. MSC-EV administration promoted the polarization of macrophages
from the M1 to the M2 phenotype in LPS-stimulated cell and animal
models
To investigate macrophage activation, M1 and M2 phenotypic
changes in each group were evaluated by western blotting and immu-
nouorescence. For the in vitro study, Western blotting (Fig. 3A) showed
that, compared with the control group, LPS stimulation (Fig. 3B)
resulted in an increase (P <0.01) in CD 86 expression in the RAW 264.7
cells, while no signicant difference (P >0.05) was found between the
LPS and control group (Fig. 3C) for Arg1 expression. Following MSC-EV
administration (LPS +EVs), there was a signicant decrease (P <0.01)
in the expression of the CD86 (Fig. 3B), and an increase (P <0.01) in the
expression of the Arg1 (Fig. 3C) compared with the LPS group. Double
immunostaining for ED1 and CD86 (Fig. 3D) also manifested the vari-
ations of CD86 (Fig. 3D) as above results. For the in vivo study, protein
assay (Fig. 3E) revealed that LPS administration induced an increase (P
<0.001) of iNOS expression in ALI-inh +Veh group only at 4 d (Fig. 3F),
but no signicant change (P >0.05) for the Arg1 expression was found
between Sham and ALI-inh +Veh group (Fig. 3G). Notably, after MSC-
EV therapy, a reduction (P <0.01) of iNOS expression and an increase
(P <0.01) of Arg1 expression was shown in the ALI-inh +EVs group
compared with the ALI-inh +Veh group at 4 d, while these variations
were not signicant (P >0.05) at 14 d. Immuno-staining with ED1 and
CD86 / CD206 (another M2 marker) was further used to detect the al-
terations of M1 (Fig. 3H; Fig. S3A) and M2 (Fig. S3B) phenotypic pat-
terns in each group at 4 d and 14 d, statistical analyses revealed that
MSC-EV treatment (ALI-inh +EVs) only decreased (Fig. 3I, P <0.01)
and increased (Fig. S3C, P <0.01) the MFI of CD86 and CD206,
respectively, compared to that observed in the ALI-inh +Veh group at 4
d. Together, these results suggested that MSC-EV administration pro-
moted the polarization of macrophages from the M1 to the M2 pheno-
type in LPS-stimulated cell and animal models (at 4 d).
3.4. RNA-Seq predicted the immune- and redox-regulatory roles of MSC-
EVs in a mouse model of ALI
Following the treatment of ALI mice with MSC-EVs for 4 d, tran-
scriptional group sequencing was performed to evaluate variations in
gene expression among the sham, ALI-inh +Veh, and ALI-inh +EVs
groups (Fig. 4A). Differential gene expression was analyzed by edgeR
according to the fragments per kilobase of exon model per million
mapped reads (FPKM) [22], which were selected for detailed functional
genomics based on fold change (FC) >2 and P value <0.05 (Fig. 4B;
Fig. S4A). Compared with the sham group, mice in the ALI model group
presented 866 upregulated mRNAs (Table S1, top 50 genes) and 272
downregulated mRNAs (Table S2, top 50 genes). A total of 154 upre-
gulated mRNAs (Table 1) and 68 downregulated mRNAs (Table 2) were
selected between the ALI-inh +Veh and ALI-inh +EV groups. As it’s
shown, the selected differential genes included a large number of genes
related to immune and redox regulation, including Tlr4, arg1, and
Hmox1 (HO-1) (Fig. 4B; Tables 1 and 2). Moreover, Kyoto Encyclopedia
of Genes and Genomes (KEGG) enrichment analysis revealed that,
compared with the sham group, more genes in immune and signal
transduction pathways were upregulated in the ALI-inh +Veh group
(Fig. S4B; Tables S1 and S2), and between the ALI-inh +Veh and ALI-
inh +EV groups (Fig. 4C; Tables 1 and 2). The top 30 pathway en-
richments revealed that, compared to the sham group, multiple immune
system including TLR signaling pathway, phagosome, cytokine-cytokine
receptor interaction participated the regulation in response to LPS
stimulation (ALI-inh +Veh) (Fig. S4C). After MSC-EVs treatment (ALI-
inh +EVs), the enriched signaling such as TLR, NOD-like receptor,
cytosolic DNA-sensing pathway was observed in comparison to the ALI-
inh +Veh group (Fig. 4D). In addition, GO enrichment analysis of
differentially expressed genes related to biological processes, cellular
components, and molecular functions (Fig. S5D and E) showed that,
compared with the sham group, the top 30 GO enrichments (Fig. S4D)
and GO classication (Fig. S4E) were involved in immune system pro-
cess and oxidase activity in the ALI-inh +Veh group. After MSC-EV
administration (ALI-inh +EVs), the top 30 GO enrichments (Fig. 4E)
showed positive regulation of inammatory (e.g., IL-10, interferon
α
production) and immune response (e.g., response to interferon β and γ),
and a series of genes (e.g., Tlr4, arg1, and Hmox1) were shown in the
participation of immune regulation and antioxidant activity in com-
parison to ALI-inh +Veh group (Fig. 4F; Tables 1 and 2). Together, these
Fig. 2. Anti-inammatory roles and tissue repair mechanisms of MSC-EVs administered to mice via tail vein injection and inhalation.
(A–G) Histograms show the concentration of pro-inammatory cytokines IL-1β (A), MCP-1 (B), IL-1
α
(C), TNF
α
(D), IL-12 (E) and the anti-inammatory cytokine IL-
10 (F) following the treatment of ALI mice with vehicle or MSC-EVs via tail vein or inhalation for 24 h; (G) representative images showing morphological changes in
LPS-induced lung injury treated with tail vein injection of MSC-EVs or MSC-EVs inhalation for 24 h, 4 d and 14 d, bar =200
μ
m; (H) histograms showing the
pathological scores at different time points for the sham-, vehicle-, and MSC-EV-treated (tail vein injection and inhalation) groups. ALI: acute lung injury, Veh:
vehicle, inh: inhalation, iv: tail vein injection, EVs: extracellular vesicles, IL-1β: interleukin-1β, MCP-1: macrophage chemoattractant protein-1, TNF
α
: tumor necrosis
factor
α
, ns: no signicance. All data are presented as the mean ±SEM and the normal distribution is conrmed by Kolmogorov-Smirnov (K
–
S) test, multiple
comparisons were analyzed using two-way ANOVA with Tukey correction.
ns
P >0.05, *P <0.05, **P <0.01, ***P <0.001, ****P <0.0001.
R. Zhao et al.

Journal of Controlled Release 345 (2022) 214–230
220
Fig. 3. The inuence of MSC-EVs on macrophage polarization in LPS-stimulated cell and animal models.
(A) Western blots against M1 (CD86) and M2 (Arg1) markers in LPS-stimulated RAW 264.7 cells; (B
–
C) protein analysis shows the expression of CD86 (B) and Arg1
(C) among the control, LPS, and LPS +EVs groups; (D) representative images display the double immuno-staining with ED1 (macrophage marker, green) and CD86
(M1 marker, red) in the experimental groups. Left image in each group, bar =100
μ
m; right image in each group, bar =25
μ
m; (E) western blots of M1 (iNOS) and
M2 (Arg1) markers in an ALI model; (F–G) Protein analysis shows the expression of iNOS (F) and Arg1 (G) in each group (sham, ALI-inh +Veh, and ALI-inh +EVs) at
different time points; (H) representative images show double immuno-staining with ED1 (green) and CD86 (red) in the sham, ALI-inh +Veh, and ALI-inh +EVs
group at 4 d. Bottom image in each group, bar =50
μ
m; top image in each group, bar =20
μ
m. ALI: acute lung injury, Veh: vehicle, inh: inhalation, EVs: extracellular
vesicles, Arg1: arginase-1, iNOS: inducible nitric oxide synthase, ns: no signicance. All data are presented as the mean ±SEM and the normal distribution is
conrmed by Kolmogorov-Smirnov (K
–
S) test, multiple comparisons analyzed using one-way ANOVA by the least signicant difference (LSD) test.
ns
P >0.05, **P
<0.01, ***P <0.001, ****P <0.0001. (For interpretation of the references to colour in this gure legend, the reader is referred to the web version of this article.)
R. Zhao et al.

Journal of Controlled Release 345 (2022) 214–230
221
(caption on next page)
R. Zhao et al.

Journal of Controlled Release 345 (2022) 214–230
222
RNA-Seq data suggested that MSC-EVs exert a regulatory role in the
immune and redox systems in the ALI mouse model.
3.5. Inammatory and oxidative mediators participated in the therapeutic
effects of MSC-EVs in vitro and in vivo
To investigate the regulatory role of MSC-EVs in LPS-stimulated cells
and animal models, typical inammatory and oxidative signals were
examined by western blotting and immunouorescence. In the cell study
(Fig. 5A), the addition of LPS to RAW264.7 cells signicantly increased
(P <0.001) the protein expression of the inammatory mediators TLR4
(Fig. 5B), TNF
α
(Fig. 5F) and IL-1β (Fig. 5G), and the oxidative regula-
tors Nrf2 (Fig. 5C), HO-1 (Fig. 5D), and HMGB1 (Fig. 5E) comparison
with the control group. Double immunostaining with 8-OHDG (an
oxidative marker) and ED1 (Fig. 5H) increased the number of 8-OHDG-
positive cells in the LPS group compared with the control group.
Notably, the expression of these inammatory and oxidative mediators
(Fig. 5B–H) was reversed (P <0.01) following the addition of MSC-EVs
(LPS +EVs) compared with the LPS group. In the animal study, western
blotting (Fig. 5I) revealed that, compared with the sham group, the
protein expression of TLR4 (Fig. 5J), NF-κB p65 (Fig. 5K), HO-1
(Fig. 5L), Nrf2 (Fig. 5M), and Keap1 (Fig. 5N), was increased (P <
0.05) in the vehicle group (ALI-inh +Veh) at 4 d. Except for Nrf2
(Fig. 5M), there was no signicant difference (P >0.05) in the expres-
sion of these mediators between the sham and vehicle control groups
(Fig. 5J-N) at 14 d. After MSC-EV treatment, the expression of TLR4
(Fig. 5J), NF-κB p65 (Fig. 5K), and Keap1 (Fig. 5N) decreased (P <0.05),
while that of Nrf2 (Fig. 5K) and HO-1 (Fig. S6C) increased (P <0.05)
compared with the vehicle group at 4 d. Interestingly, following treat-
ment with MSC-EVs for 14 d, there was no signicant difference (P >
0.05) in the expression of TLR4 (Fig. 5J), NF-κB p65 (Fig. 5K), and HO-1
(Fig. 5L), but a marked decrease (P <0.001) in the expression of Nrf2
(Fig. 5M)and an increase (P <0.05) in the expression of Keap1 (Fig. 5N)
were shown in the ALI-inh +EVs compared with the ALI-inh +Veh
group. In addition, immunostaining analyses conrmed the anti-
oxidation of MSC-EVs typied by the reduction of 8-OHdG expression
between ALI-inh +Veh and ALI-inh +EV group at 4 d (Fig. 5O).
Together, these results indicated a role for these inammatory and
oxidative mediators in the response to MSC-EV therapy.
3.6. Nrf2 knockdown attenuated the anti-inammatory and antioxidant
activity of MSC-EVs in LPS-stimulated macrophages
Nfe2f2 knocked down (Nrf2
−
) was employed to clarify the thera-
peutic mechanism of MSC-EVs in LPS-stimulated cell injury. qPCR
analysis conrmed the successful knockdown of Nfe2f2 in RAW264.7
cells (Fig. 6A, Nrf2
−
versus NC). Except for the increased (P <0.0001)
expression of Keap1 (Fig. 6C), there were no signicant differences (P >
0.05) in Hmox1 (Fig. 6B), IL-6 (Fig. 6D), or Tnf
α
(Fig. 6E) expression
between the NC and Nrf2
−
groups. Following LPS stimulation of
RAW264.7, the mRNA expression of Nfe2f2 (Fig. 6A), Hmox1 (Fig. 6B),
IL-6 (Fig. 6D), and Tnf
α
(Fig. 6E) increased (P <0.05), while the mRNA
expression of Keap1 (Fig. 6C) decreased (P <0.0001) in the Nfr2
−
+LPS
group. Following the addition of MSC-EVs to LPS-stimulated Nrf2
−
RAW264.7 cells (Nfr2
−
+LPS +EVs), the expression of Hmox1 (Fig. 6B)
and IL-6 (Fig. 6D) was upregulated (P <0.0001) compared with that in
the Nfr2
−
+LPS group, while there was no change (P >0.05) in the
expression of Nfe2f2 (Fig. 6A), Keap1 (Fig. 6C), and Tnf
α
(Fig. 6E) be-
tween the Nfr2
−
+LPS +EVs and Nfr2
−
+LPS groups. Collectively,
these results suggested that Nrf2 knockdown attenuate the anti-
inammatory and antioxidant activity of MSC-EVs in LPS-stimulated
RAW264.7 cells.
4. Discussion
Many measures are used to regulate the inammatory response in
lung injury, and regenerative medicine approaches, including EV-based
therapies, have shown promise in ALI induced by COVID-19 and other
stimuli [8]; however, the optimal delivery methods and therapeutic
mechanisms are poorly understood. Here, we report that MSC-EVs exert
robust anti-inammatory and tissue regenerative effects in LPS-
stimulated ALI models during the acute stage. Notably, inhalation of
MSC-EVs demonstrated greater feasibility for the treatment of ALI
compared to tail vein injection. In addition, RNA-Seq predicted that the
immune and redox systems are involved in the response to EV-based
therapy, and a series of classical inammatory (e.g., TLR4, NF-κB,
TNF-
α
, etc.) and oxidative (e.g., Nrf2, HO-1, Keap1) mediators partici-
pate in the response to MSC-EV treatment in vitro and in vivo. Further-
more, regulation of redox signaling in the anti-inammatory response
was claried by Nrf2 knockdown in LPS-stimulated macrophages.
In the present study, we identied that inhalation of EVs resulted in
stronger immune regulation and tissue regeneration compared to tail
vein injection in the acute lung injury. Multiple data suggest that
injecting MSC-EVs via the tail vein may offer a novel therapeutic
approach for ALI owing to their anti-inammatory, immunomodulatory,
mitochondrial regeneration, and tissue repair properties [24–26].
However, here, we evaluated a non-invasive route of administration
(inhalation) for the treatment of ALI mice. The inability to pass through
the pulmonary barrier is a major problem for the treatment of ALI and
ARDS. Nanomedicine, including EV-based therapy, provides a strategy
to cross the air-blood barrier, alveolar epithelial-endothelial barrier, and
other biological barriers [27,28]. In the present experiment, the vesicle
tracking results indicated inhalation of MSC-EVs show better perfor-
mance than those tail vein injection to reach the lung tissue in mice
model. Together, the present data suggest that equal doses of MSC-EVs
favor the repair of acute lung injury via inhalation rather than tail
vein injection. Of course, other problems (blood stimulation, circulation,
metabolization, exhalation, et al.) should be considered to inuence the
bioavailability of EVs, but the detailed mechanism needs to be studied in
the near future. Considering the signicance of macrophage polarization
in ALI, we investigated the regulatory role of MSC-EVs in LPS-stimulated
cells and mice (via inhalation). M1 and M2 macrophages are mainly
involved in pro- and anti-inammatory responses, respectively; thus,
modulating macrophage activation may be an effective method for the
treatment of ALI [29]. Here, EV therapy signicantly reduced the M1
phenotype and increased the M2 phenotype in cell and animal models (4
d), indicating the anti-inammation during the acute stage of lung
injury.
MSC-EVs participate in various biological processes, such as immune
responses, inammation, and metabolism [25,30,31]; however, their
regulatory role in ALI is poorly understood, especially when adminis-
tered via inhalation. KEGG classication and GO analysis revealed the
Fig. 4. Differential gene expression in sham-, vehicle-, and MSC-EV-treated mice.
(A) Heatmap showing the upregulated (red) and downregulated (green) genes among the sham, vehicle and MSC-EVs inhalation groups; (B) scattergram showing the
fold change (FC) expression of genes including Arg1, Hmox1, and TLR4 between the ALI +EVs and ALI +Veh group; (C) KEGG classication of the differential genes
in cellular processes, environmental information processing, genetic information processing, metabolism, and organismal systems in ALI +EVs compared with the
ALI +Veh group; (D) top 30 enriched pathways for the differential genes associated with multiple immunomodulatory signaling pathways including Toll-like re-
ceptor signal pathway, NOD-like receptor signaling pathway, and arginine biosynthesis; (E) top 30 GO enrichments showing the participation of differential genes
with regard to the response to interferon-β, positive regulation of IL-10 production, etc. (F) GO classication for the differential genes in ALI +EVs compared with
ALI +Veh group. ALI: acute lung injury, Veh: vehicle, EVs: extracellular vesicles, Arg1: arginase-1, KEGG: Kyoto Encyclopedia of Genes and Genomes, GO: gene
ontology. (For interpretation of the references to colour in this gure legend, the reader is referred to the web version of this article.)
R. Zhao et al.

Journal of Controlled Release 345 (2022) 214–230
223
Table 1
Up-regulated mRNA in MSC-EVs treated group after LPS stimulation.
Gene ID Gene name Description log2FC P value
ENSMUSG00000104148 Pcdha2 Protocadherin_alpha_2 Inf 1.22E-11
ENSMUSG00000064220 Hist2h2aa1 Histone_cluster_2_H2aa1 Inf 0.000121889
ENSMUSG00000022991 Lalba Lactalbumin_alpha Inf 0.000171073
ENSMUSG00000031710 Ucp1 Uncoupling_protein_1_mitochondrial_proton_carrier_ 6.86722263 6.54E-05
ENSMUSG00000076710 Ighv1–49 Immunoglobulin_heavy_variable_1_49 5.835396899 0.000111972
ENSMUSG00000097439 Gm16754 Predicted_gene_16754 4.466267159 0.000410792
ENSMUSG00000022650 Retnlb Resistin_like_beta 4.37568772 0.000272911
ENSMUSG00000047562 Mmp10 Matrix_metallopeptidase_10 3.910984322 2.88E-05
ENSMUSG00000107480 RP24-483 J20.2 – 3.8037255 1.02E-05
ENSMUSG00000019987 Arg1 Arginase_liver 3.700563817 7.52E-10
ENSMUSG00000034730 Adgrb1 Adhesion_G_protein_coupled_receptor_B1 3.408511803 3.01E-05
ENSMUSG00000013353 4931406B18Rik RIKEN_cDNA_4931406B18_gene 3.328635559 0.000516203
ENSMUSG00000034416 Pkd1l2 Polycystic_kidney_disease_1_like_2 3.313869157 0.00060597
ENSMUSG00000009350 Mpo Myeloperoxidase 3.043574325 0.000319336
ENSMUSG00000031551 Ido1 Indoleamine_2_3_dioxygenase_1 2.948211336 9.21E-11
ENSMUSG00000108596 Itgam Integrin_alpha_M 2.618862438 3.04E-10
ENSMUSG00000060183 Cxcl11 Chemokine_C_X_C_motif_ligand_11 2.601155993 7.27E-06
ENSMUSG00000022126 Irg1 Immunoresponsive_gene_1 2.553995318 1.37E-05
ENSMUSG00000078616 Trim30c Tripartite_motif_containing_30C 2.517860728 2.51E-06
ENSMUSG00000034850 Tmem127 Transmembrane_protein_127 2.468083025 1.15E-06
ENSMUSG00000020826 Nos2 Nitric_oxide_synthase_2_inducible 2.44290498 1.50E-11
ENSMUSG00000000204 Slfn4 Schlafen_4 2.329811414 1.14E-09
ENSMUSG00000029417 Cxcl9 Chemokine_C_X_C_motif_ligand_9 2.327917277 5.08E-20
ENSMUSG00000049848 Ceacam19 Carcinoembryonic_antigen_related_cell_adhesion_molecule_19 2.297789371 0.000114298
ENSMUSG00000105096 Gbp10 Guanylate_binding_protein_10 2.2671004 2.88E-08
ENSMUSG00000093765 Gm20658 Predicted_gene_20658 2.160673046 0.00014692
ENSMUSG00000063234 Gpr84 G_protein_coupled_receptor_84 2.076539012 0.000375818
ENSMUSG00000034855 Cxcl10 Chemokine_C_X_C_motif_ligand_10 2.044830255 1.33E-13
ENSMUSG00000029005 Draxin Dorsal_inhibitory_axon_guidance_protein 2.021367616 2.08E-07
ENSMUSG00000022584 Ly6c2 Lymphocyte_antigen_6_complex_locus_C2 2.014189764 3.85E-10
ENSMUSG00000049734 Trex1 Three_prime_repair_exonuclease_1 1.992921807 3.00E-08
ENSMUSG00000045932 It2 Interferon_induced_protein_with_tetratricopeptide_repeats_2 1.96243906 2.50E-17
ENSMUSG00000020641 Rsad2 Radical_S_adenosyl_methionine_domain_containing_2 1.930987416 3.20E-11
ENSMUSG00000035105 Egln3 egl_9_family_hypoxia_inducible_factor_3 1.896723668 5.83E-05
ENSMUSG00000035455 Fignl1 Fidgetin_like_1 1.89248234 2.72E-05
ENSMUSG00000040017 Saa4 Serum_amyloid_A_4 1.845810356 9.02E-06
ENSMUSG00000040026 Saa3 Serum_amyloid_A_3 1.837934029 9.77E-06
ENSMUSG00000078922 Tgtp1 T_cell_specic_GTPase_1 1.837543849 1.27E-10
ENSMUSG00000033213 AA467197 Expressed_sequence_AA467197 1.810187306 6.99E-10
ENSMUSG00000006403 Adamts4 a_disintegrin_like_and_metallopeptidase_reprolysin_type_with_thrombospondin_type_1_motif_4 1.792384309 8.39E-07
ENSMUSG00000022534 Mefv Mediterranean_fever 1.769250644 2.82E-07
ENSMUSG00000040328 Olfr56 Olfactory_receptor_56 1.76163496 0.000634648
ENSMUSG00000032661 Oas3 2_5_oligoadenylate_synthetase_3 1.748028193 6.00E-11
ENSMUSG00000030077 Chl1 Cell_adhesion_molecule_with_homology_to_L1CAM 1.747729979 4.47E-06
ENSMUSG00000035692 Isg15 ISG15_ubiquitin_like_modier 1.742828866 2.97E-12
ENSMUSG00000046031 Fam26f Family_with_sequence_similarity_26_member_F 1.735164013 3.55E-06
ENSMUSG00000105504 Gbp5 Guanylate_binding_protein_5 1.714142214 3.83E-11
ENSMUSG00000055254 Ntrk2 Neurotrophic_tyrosine_kinase_receptor_type_2 1.699850337 2.98E-05
ENSMUSG00000028270 Gbp2 Guanylate_binding_protein_2 1.674098289 5.62E-10
ENSMUSG00000024675 Ms4a4c Membrane_spanning_4_domains_subfamily_A_member_4C 1.666753629 5.07E-08
ENSMUSG00000063388 BC023105 cDNA_sequence_BC023105 1.658539575 0.00024741
ENSMUSG00000041827 Oasl1 2_5_oligoadenylate_synthetase_like_1 1.645877017 1.49E-06
ENSMUSG00000070524 Fcrlb Fc_receptor_like_B 1.635942988 0.000711091
ENSMUSG00000023913 Pla2g7 Phospholipase_A2_group_VII_platelet_activating_factor_acetylhydrolase_plasma_ 1.621254861 3.73E-06
(continued on next page)
R. Zhao et al.

Journal of Controlled Release 345 (2022) 214–230
224
Table 1 (continued )
Gene ID Gene name Description log2FC P value
ENSMUSG00000053318 Slamf8 SLAM_family_member_8 1.620494587 9.79E-09
ENSMUSG00000022180 Slc7a8 Solute_carrier_family_7_cationic_amino_acid_transporter_y +_system_member_8 1.618648091 4.14E-08
ENSMUSG00000050914 Ankrd37 Ankyrin_repeat_domain_37 1.608620006 0.000128941
ENSMUSG00000038037 Socs1 Suppressor_of_cytokine_signaling_1 1.58786231 3.46E-05
ENSMUSG00000026573 Xcl1 Chemokine_C_motif_ligand_1 1.517552985 0.000850449
ENSMUSG00000073489 I204 Interferon_activated_gene_204 1.516520382 6.90E-09
ENSMUSG00000070427 Il18bp Interleukin_18_binding_protein 1.515103993 1.59E-06
ENSMUSG00000026981 Il1rn Interleukin_1_receptor_antagonist 1.483212372 0.000321565
ENSMUSG00000032796 Lama1 Laminin_alpha_1 1.47491355 9.82E-05
ENSMUSG00000054072 Iigp1 Interferon_inducible_GTPase_1 1.474246966 9.80E-10
ENSMUSG00000004098 Col5a3 Collagen_type_V_alpha_3 1.469003218 2.41E-06
ENSMUSG00000027514 Zbp1 Z_DNA_binding_protein_1 1.46887593 1.76E-08
ENSMUSG00000055629 B4galnt4 Beta_1_4_N_acetyl_galactosaminyl_transferase_4 1.461993313 0.000601701
ENSMUSG00000034570 Inpp5j Inositol_polyphosphate_5_phosphatase_J 1.459717021 0.000118582
ENSMUSG00000025161 Slc16a3 Solute_carrier_family_16_monocarboxylic_acid_transporters_member_3 1.45451477 1.56E-07
ENSMUSG00000078763 Slfn1 Schlafen_1 1.45199247 4.12E-08
ENSMUSG00000079362 Gbp6 Guanylate_binding_protein_6 1.447185026 1.49E-08
ENSMUSG00000034459 It1 Interferon_induced_protein_with_tetratricopeptide_repeats_1 1.436491638 1.25E-10
ENSMUSG00000015947 Fcgr1 Fc_receptor_IgG_high_afnity_I 1.430731494 6.27E-08
ENSMUSG00000092021 Gbp11 Guanylate_binding_protein_11 1.430067541 0.000379839
ENSMUSG00000028364 Tnc Tenascin_C 1.426011894 3.96E-09
ENSMUSG00000026536 Mnda Myeloid_cell_nuclear_differentiation_antigen 1.421172485 8.45E-09
ENSMUSG00000025877 Hk3 Hexokinase_3 1.4192114 7.42E-07
ENSMUSG00000005413 Hmox1 Heme_oxygenase_1 1.413823378 7.11E-06
ENSMUSG00000073412 Lst1 Leukocyte_specic_transcript_1 1.40486746 4.72E-06
ENSMUSG00000030144 Clec4d C_type_lectin_domain_family_4_member_d 1.391084178 0.000274792
ENSMUSG00000069830 Nlrp1a NLR_family_pyrin_domain_containing_1A 1.382252984 0.000736524
ENSMUSG00000068606 Gm4841 Predicted_gene_4841 1.379025328 0.000802212
ENSMUSG00000054676 1600014C10Rik RIKEN_cDNA_1600014C10_gene 1.369578726 3.99E-05
ENSMUSG00000024397 Aif1 Allograft_inammatory_factor_1 1.365184414 3.79E-07
ENSMUSG00000029915 Clec5a C_type_lectin_domain_family_5_member_a 1.363656394 1.18E-05
ENSMUSG00000047945 Marcksl1 MARCKS_like_1 1.333855435 4.07E-09
ENSMUSG00000046311 Zfp62 Zinc_nger_protein_62 1.333701932 3.36E-06
ENSMUSG00000035493 Tgfbi Transforming_growth_factor_beta_induced 1.330790917 4.18E-09
ENSMUSG00000030111 A2m Alpha_2_macroglobulin 1.320221268 0.00035884
ENSMUSG00000031444 F10 Coagulation_factor_X 1.30297311 6.00E-05
ENSMUSG00000039196 Orm1 Orosomucoid_1 1.289327724 0.000509261
ENSMUSG00000000386 Mx1 MX_dynamin_like_GTPase_1 1.288554965 0.000493531
ENSMUSG00000076441 Ass1 Argininosuccinate_synthetase_1 1.279581268 2.92E-09
ENSMUSG00000101628 Gm28177 Predicted_gene_28177 1.270142365 1.79E-05
ENSMUSG00000025044 Msr1 Macrophage_scavenger_receptor_1 1.255203972 4.67E-08
ENSMUSG00000025498 Irf7 Interferon_regulatory_factor_7 1.254086563 4.65E-07
ENSMUSG00000026358 Rgs1 Regulator_of_G_protein_signaling_1 1.250097436 0.000104446
ENSMUSG00000022586 Ly6i Lymphocyte_antigen_6_complex_locus_I 1.236056925 3.49E-05
ENSMUSG00000024679 Ms4a6d Membrane_spanning_4_domains_subfamily_A_member_6D 1.233546344 2.41E-07
ENSMUSG00000104713 Gbp6 Guanylate_binding_protein_6 1.225081125 3.76E-07
ENSMUSG00000107068 Gm42742 Predicted_gene_42742 1.215945814 0.000301727
ENSMUSG00000053101 Gpr141 G_protein_coupled_receptor_141 1.205958625 1.30E-05
ENSMUSG00000020178 Adora2a Adenosine_A2a_receptor 1.186724602 6.91E-05
ENSMUSG00000078771 Evi2a Ecotropic_viral_integration_site_2a 1.183439777 0.000477638
ENSMUSG00000078853 Igtp Interferon_gamma_induced_GTPase 1.178870807 3.79E-06
ENSMUSG00000035208 Slfn8 Schlafen_8 1.171719163 3.99E-06
ENSMUSG00000090231 Cfb Complement_factor_B 1.166265986 0.000368957
ENSMUSG00000046687 Gm5424 Predicted_gene_5424 1.164873512 1.48E-08
ENSMUSG00000048572 Tmem252 Transmembrane_protein_252 1.156883183 1.04E-05
(continued on next page)
R. Zhao et al.

Journal of Controlled Release 345 (2022) 214–230
225
Table 1 (continued )
Gene ID Gene name Description log2FC P value
ENSMUSG00000057191 AB124611 cDNA_sequence_AB124611 1.14590096 3.12E-06
ENSMUSG00000035042 Ccl5 Chemokine_C_C_motif_ligand_5 1.14367436 0.00010231
ENSMUSG00000028268 Gbp3 Guanylate_binding_protein_3 1.139757589 2.00E-06
ENSMUSG00000039519 Cyp7b1 Cytochrome_P450_family_7_subfamily_b_polypeptide_1 1.134939043 8.32E-05
ENSMUSG00000052776 Oas1a 2_5_oligoadenylate_synthetase_1A 1.130406892 3.24E-05
ENSMUSG00000079419 Ms4a6c Membrane_spanning_4_domains_subfamily_A_member_6C 1.129644066 2.71E-07
ENSMUSG00000032691 Nlrp3 NLR_family_pyrin_domain_containing_3 1.125677688 0.000738196
ENSMUSG00000027078 Ube2l6 Ubiquitin_conjugating_enzyme_E2L_6 1.119305745 1.43E-06
ENSMUSG00000002489 Tiam1 T_cell_lymphoma_invasion_and_metastasis_1 1.11137199 6.10E-06
ENSMUSG00000054203 I205 Interferon_activated_gene_205 1.107756182 1.78E-06
ENSMUSG00000075010 AW112010 Expressed_sequence_AW112010 1.107592835 3.79E-07
ENSMUSG00000035004 Igsf6 Immunoglobulin_superfamily_member_6 1.107117957 0.00044031
ENSMUSG00000027199 Gatm Glycine_amidinotransferase_L_arginine_glycine_amidinotransferase 1.104685894 2.28E-05
ENSMUSG00000045763 Basp1 Brain_abundant_membrane_attached_signal_protein_1 1.10356929 5.99E-06
ENSMUSG00000033307 Mif Macrophage_migration_inhibitory_factor 1.100832435 1.32E-05
ENSMUSG00000046879 Irgm1 Immunity_related_GTPase_family_M_member_1 1.090995372 5.87E-07
ENSMUSG00000024053 Emilin2 Elastin_microbril_interfacer_2 1.077530157 9.74E-06
ENSMUSG00000079227 Ccr5 Chemokine_C_C_motif_receptor_5 1.076069098 1.93E-05
ENSMUSG00000028261 Ndufaf4 NADH_dehydrogenase_ubiquinone_1_alpha_subcomplex_assembly_factor_4 1.072339851 0.000490484
ENSMUSG00000049037 Clec4a1 C_type_lectin_domain_family_4_member_a1 1.071710107 9.44E-06
ENSMUSG00000040552 C3ar1 Complement_component_3a_receptor_1 1.070053271 0.000311051
ENSMUSG00000001751 Naglu Alpha_N_acetylglucosaminidase_Sanlippo_disease_IIIB_ 1.06714193 4.93E-07
ENSMUSG00000022148 Fyb FYN_binding_protein 1.065392178 2.17E-06
ENSMUSG00000078921 Tgtp2 T_cell_specic_GTPase_2 1.059232875 4.78E-05
ENSMUSG00000021451 Sema4d Sema_domain_immunoglobulin_domain_Ig_transmembrane_domain_TM_and_short_cytoplasmic_domain_semaphorin_4D 1.050239088 1.78E-05
ENSMUSG00000045322 Tlr9 Toll_like_receptor_9 1.040472335 0.000169875
ENSMUSG00000027611 Procr Protein_C_receptor_endothelial 1.040207417 0.000174922
ENSMUSG00000074622 Mafb v_maf_musculoaponeurotic_brosarcoma_oncogene_family_protein_B_avian_ 1.036703403 3.17E-05
ENSMUSG00000056529 Ptafr Platelet_activating_factor_receptor 1.033051521 3.66E-05
ENSMUSG00000029322 Plac8 Placenta_specic_8 1.031873267 7.76E-06
ENSMUSG00000036067 Slc2a6 Solute_carrier_family_2_facilitated_glucose_transporter_member_6 1.022443149 0.000298085
ENSMUSG00000073555 Gm4951 Predicted_gene_4951 1.022280133 0.000212646
ENSMUSG00000024953 Prdx5 Peroxiredoxin_5 1.020978944 3.54E-05
ENSMUSG00000034438 Gbp8 Guanylate_binding_protein_8 1.019622037 0.000199613
ENSMUSG00000023456 Tpi1 Triosephosphate_isomerase_1 1.018370208 3.24E-06
ENSMUSG00000048234 Rnf149 Ring_nger_protein_149 1.015818168 4.49E-05
ENSMUSG00000049988 Lrrc25 Leucine_rich_repeat_containing_25 1.014928247 0.000306512
ENSMUSG00000016496 Cd274 CD274_antigen 1.012980063 6.32E-07
ENSMUSG00000078920 I47 Interferon_gamma_inducible_protein_47 1.011529935 6.91E-06
ENSMUSG00000040907 Atp1a3 ATPase_Na +_K +_transporting_alpha_3_polypeptide 1.009991272 0.000342156
ENSMUSG00000056749 Nl3 Nuclear_factor_interleukin_3_regulated 1.009610524 0.000485923
ENSMUSG00000074896 It3 Interferon_induced_protein_with_tetratricopeptide_repeats_3 1.006877761 2.99E-05
ENSMUSG00000058715 Fcer1g Fc_receptor_IgE_high_afnity_I_gamma_polypeptide 1.003506911 5.17E-06
ENSMUSG00000030187 Klra2 Killer_cell_lectin_like_receptor_subfamily_A_member_2 1.003402201 3.50E-05
R. Zhao et al.

Journal of Controlled Release 345 (2022) 214–230
226
genetic signatures in ALI mice, as previously reported [32,33]. A large
number of genes were associated with immune and oxidative systems,
indicating the involvement of inammatory and oxidative signaling
pathways in the development of ALI. The mediators, including TLR and
interferon-β, have been identied as targets of inammation or
oxidative stress in ALI [34–36]. suggesting a regulatory role for MSC-EV
inhalation in ALI mice. In addition, analysis of differential gene (e.g.
Tlr4, Hmox1, Arg1) expression revealed that MSC-EVs exert anti-
inammatory and antioxidant activity in animal models of ALI. Based
on the results of RNA-Seq, we investigated variations in typical
Table 2
Down regulated mRNA in ALI +EVs in comparison to ALI +Veh group.
Gene ID Gene name Description log2FC Pvalue
ENSMUSG00000076668 Ighv7–4 Immunoglobulin_heavy_variable_7_4 −8.117303019 0.000265116
ENSMUSG00000108815 RP23-325D10.3 – −6.851466107 3.64E-06
ENSMUSG00000098387 Pet117 PET117_homolog_S._cerevisiae_ −6.038722257 1.78E-16
ENSMUSG00000001670 Tat Tyrosine_aminotransferase −5.49915803 9.19E-05
ENSMUSG00000076709 Ighv1–47 Immunoglobulin_heavy_variable_1_47 −4.823690524 0.000229927
ENSMUSG00000044022 Pcdhb21 Protocadherin_beta_21 −4.634783678 4.08E-10
ENSMUSG00000076512 Igkv9–123 Immunoglobulin_kappa_variable_9_123 −4.492389142 0.000124595
ENSMUSG00000076543 Igkv4–74 Immunoglobulin_kappa_variable_4_74 −4.134330517 0.00039497
ENSMUSG00000102888 Ighv1–11 Immunoglobulin_heavy_variable_V1_11 −4.126434633 0.000478945
ENSMUSG00000075707 Dio3 Deiodinase_iodothyronine_type_III −4.052277342 0.00087219
ENSMUSG00000076688 Ighv15–2 Immunoglobulin_heavy_variable_V15_2 −3.894926855 0.000218882
ENSMUSG00000045545 Krt14 Keratin_14 −3.851994931 0.000136651
ENSMUSG00000097740 E030044B06Rik RIKEN_cDNA_E030044B06_gene −3.731804347 0.000356703
ENSMUSG00000023829 Slc22a1 Solute_carrier_family_22_organic_cation_transporter_member_1 −3.632740439 0.000642579
ENSMUSG00000094356 Igkv8–28 Immunoglobulin_kappa_variable_8_28 −3.427650329 0.000188234
ENSMUSG00000037386 Rims2 Regulating_synaptic_membrane_exocytosis_2 −3.253392741 1.34E-06
ENSMUSG00000094102 Ighv9–2 Immunoglobulin_heavy_variable_V9_2 −3.23137377 0.000119952
ENSMUSG00000029390 Tmed2 Transmembrane_emp24_domain_trafcking_protein_2 −2.733434674 5.23E-05
ENSMUSG00000063681 Crb1 Crumbs_family_member_1_photoreceptor_morphogenesis_associated −2.704541117 9.43E-06
ENSMUSG00000057836 Xlr3a X_linked_lymphocyte_regulated_3A −2.501305246 0.000596072
ENSMUSG00000045114 Prrt2 Proline_rich_transmembrane_protein_2 −2.326412258 1.68E-06
ENSMUSG00000079509 Zfx Zinc_nger_protein_X_linked −2.303324887 9.32E-26
ENSMUSG00000006642 Tcf23 Transcription_factor_23 −2.26239927 5.77E-05
ENSMUSG00000031283 Chrdl1 Chordin_like_1 −2.148855195 8.20E-06
ENSMUSG00000041794 Myrip Myosin_VIIA_and_Rab_interacting_protein −2.114322712 6.56E-06
ENSMUSG00000043110 Lrrn4 Leucine_rich_repeat_neuronal_4 −1.888511993 0.000393275
ENSMUSG00000041644 Slc5a12 Solute_carrier_family_5_sodium_glucose_cotransporter_member_12 −1.826318195 0.000224631
ENSMUSG00000021750 Fam107a Family_with_sequence_similarity_107_member_A −1.823108929 5.28E-05
ENSMUSG00000042985 Upk3b Uroplakin_3B −1.798378698 2.53E-05
ENSMUSG00000026393 Nek7 NIMA_never_in_mitosis_gene_a_related_expressed_kinase_7 −1.680050647 1.92E-05
ENSMUSG00000104184 Gm37818 Predicted_gene_37818 −1.673644962 0.00013507
ENSMUSG00000039005 Tlr4 Toll_like_receptor_4 ¡1.5989624 0.000756567
ENSMUSG00000026840 Lamc3 Laminin_gamma_3 −1.586545741 6.72E-05
ENSMUSG00000008845 Cd163 CD163_antigen −1.546582522 0.000648699
ENSMUSG00000019278 Dpep1 Dipeptidase_1_renal_ −1.539800197 1.18E-05
ENSMUSG00000107134 Gm42528 Predicted_gene_42528 −1.470544436 0.000150557
ENSMUSG00000030711 Sult1a1 Sulfotransferase_family_1A_phenol_preferring_member_1 −1.447703409 0.000151239
ENSMUSG00000051022 Hs3st1 Heparan_sulfate_glucosamine_3_O_sulfotransferase_1 −1.40778605 0.00012126
ENSMUSG00000105843 Gm42644 Predicted_gene_42644 −1.407172113 0.000358996
ENSMUSG00000028862 Map3k6 Mitogen_activated_protein_kinase_kinase_kinase_6 −1.339143035 0.000657209
ENSMUSG00000020627 Klhl29 Kelch_like_29 −1.335534092 0.000155582
ENSMUSG00000055368 Slc6a2 Solute_carrier_family_6_neurotransmitter_transporter_noradrenalin_member_2 −1.317610017 3.69E-06
ENSMUSG00000026691 Fmo3 Flavin_containing_monooxygenase_3 −1.316198039 1.29E-05
ENSMUSG00000031613 Hpgd Hydroxyprostaglandin_dehydrogenase_15_NAD_ −1.312392763 8.45E-05
ENSMUSG00000025936 Gm4956 Predicted_gene_4956 −1.308869505 0.000117148
ENSMUSG00000028871 Rspo1 R_spondin_1 −1.289586922 0.000675482
ENSMUSG00000039405 Prss23 Protease_serine_23 −1.262270763 0.000216445
ENSMUSG00000023046 Igfbp6 Insulin_like_growth_factor_binding_protein_6 −1.262158859 3.73E-06
ENSMUSG00000025934 Gsta3 Glutathione_S_transferase_alpha_3 −1.256077173 2.20E-05
ENSMUSG00000026712 Mrc1 Mannose_receptor_C_type_1 −1.185509168 8.80E-07
ENSMUSG00000030737 Slco2b1 Solute_carrier_organic_anion_transporter_family_member_2b1 −1.148775679 4.08E-05
ENSMUSG00000000805 Car4 Carbonic_anhydrase_4 −1.128075738 0.00010447
ENSMUSG00000074971 Fibin Fin_bud_initiation_factor_homolog_zebrash_ −1.122123859 1.32E-05
ENSMUSG00000102697 Pcdhac2 Protocadherin_alpha_subfamily_C_2 −1.094902407 0.000636972
ENSMUSG00000015354 Pcolce2 Procollagen_C_endopeptidase_enhancer_2 −1.076367024 2.37E-05
ENSMUSG00000061740 Cyp2d22 Cytochrome_P450_family_2_subfamily_d_polypeptide_22 −1.07410918 0.000217591
ENSMUSG00000104475 D630036G22Rik RIKEN_cDNA_D630036G22_gene −1.071753106 0.000735149
ENSMUSG00000054986 Sec14l3 SEC14_like_lipid_binding_3 −1.060632826 3.13E-08
ENSMUSG00000041351 Rap1gap Rap1_GTPase_activating_protein −1.052040267 0.000104382
ENSMUSG00000040170 Fmo2 Flavin_containing_monooxygenase_2 −1.047504112 3.38E-05
ENSMUSG00000097336 Fendrr Foxf1_adjacent_non_coding_developmental_regulatory_RNA −1.039874631 3.41E-05
ENSMUSG00000041361 Myzap Myocardial_zonula_adherens_protein −1.030769269 8.59E-05
ENSMUSG00000044349 Snhg11 Small_nucleolar_RNA_host_gene_11 −1.025072763 9.28E-06
ENSMUSG00000030306 Tmtc1 Transmembrane_and_tetratricopeptide_repeat_containing_1 −1.018148647 3.60E-05
ENSMUSG00000095538 Gm21983 Predicted_gene_21983 -Ifn 1.22E-06
ENSMUSG00000107705 Umad1 UMAP1_MVP12_associated_UMA_domain_containing_1 -Ifn 0.000120596
ENSMUSG00000076646 Ighv2–6-8 Immunoglobulin_heavy_variable_2_6_8 -Ifn 0.000346067
R. Zhao et al.

Journal of Controlled Release 345 (2022) 214–230
227
(caption on next page)
R. Zhao et al.

Journal of Controlled Release 345 (2022) 214–230
228
inammatory and oxidative signals in vitro and in vivo. TLRs are involved
in both innate and acquired immunity, and TLR4 plays a crucial role in
the regulation of inammation and signal transduction, and triggers
signaling pathways, including the activation of proinammatory factors
IL-1β, IL-6, and NF-κB p65 [35,37]. Following MSC-EV treatment, the
decreased expression of TLR4 and its downstream signals (including IL-
1β, IL-6, and NF-κB p65) indicated that inhalation of MSC-EVs was able
to alleviate inammation in response to LPS stimulation. Additionally,
there was no signicant difference in these inammatory signals be-
tween sham and vehicle control at 14 d, which may be ascribed to the
self-healing in ALI mice [38].
Notably, Nrf2 mediates oxidative stress by regulating the transcrip-
tion of a wide array of genes, such as HO-1 [39], and protects against ALI
by regulating TLR4 and other inammatory signaling pathways [36]. In
particular, under physiological conditions, Nrf2 is maintained at very
low levels by the negative regulator Keap1; conversely, under oxidative
stress, Nrf2 dissociates from Keap1 following phosphorylation by
various kinases and defends the lung against damage [40,41]. In the
present study, oxidative stress in response to LPS stimulation (in vitro
and in vivo) was demonstrated by the increased expression of 8-OHdG
(DNA damage marker), Nrf2, HO-1, and Keap1. Interestingly, after
MSC-EV administration, the expression of these markers changed in
both cells and animal models at different stages of lung injury. The
activation of Nrf2 and its target genes has demonstrated protection
against ALI [36,39]. In animal models, increased Nrf2 expression
(accompanied by increased HO-1 expression, and decreased Keap1
expression) in ALI-inh +EVs 4 d (versus ALI-inh +Veh 4 d), and the anti-
inammatory and tissue repair response of MSC-EVs at this time point,
indicate that the effects of EV-therapy against ALI are associated with
the early activation of Nrf2 in mice. Following the inhalation of MSC-
EVs for 14 d, there were no signicant changes in inammatory and
partial oxidative signals (NF-κB p65, TLR4, and HO-1) between the MSC-
EV group and the vehicle control. However, the expression of Nrf2
decreased, while that of Keap1 increased compared with that in the ALI-
Fig. 5. Involvement of inammatory and oxidative mediators in MSC-EV-treated cell and animal models.
(A) Western blots of the inammatory mediators TLR4, TNF
α
, and IL-1β, and the oxidative mediators Nrf2, HO-1, and HMGB1 in LPS-stimulated RAW 264.7 cells;
(B–G) protein analysis showing the expression of TLR4 (B), Nrf2 (C), HO-1 (D), HMGB1 (E), TNF
α
(F), and IL-1β (G) in the experimental groups; (H) representative
images display double immuno-staining with ED1 (macrophage marker, green) and 8-OHdG (oxidative stress marker, red) in each group. Left image of each group,
bar =100
μ
m; right image of each group, bar =25
μ
m; (I) western blots of typical inammatory and oxidative mediators in the animal model at different time points;
(J–K) histograms showing the protein expression of TLR4 (J), NF-κB p65 (K), HO-1 (L), Nrf2 (M), and Keap1 (N) in the experimental groups; (H) representative
images displaying double immuno-staining with ED1 (macrophage marker, green) and 8-OHdG (oxidative marker, red) in the sham, ALI-inh +Veh, and ALI-inh +
EVs groups at 4 d. Bottom image of each group, bar =50
μ
m; top image of each group, bar =20
μ
m. ALI: acute lung injury, Veh: vehicle, inh: inhalation, EVs:
extracellular vesicles, TLR4: Toll-like receptor 4, Nrf2: nuclear factor erythrocyte 2-related factor 2, HO-1: heme oxygenase-1, HMGB1: high mobility group box-1
protein, TNF
α
: tumor necrosis factor
α
, and IL-1β: Interleukin 1β, ns: no signicance. All data are presented as the mean ±SEM and normal distribution is conrmed
by the K
–
S test, multiple comparisons were analyzed using one-way ANOVA with the LSD test.
ns
P >0.05, *P <0.05, **P <0.01, ***P <0.001, ****P <0.0001.
(For interpretation of the references to colour in this gure legend, the reader is referred to the web version of this article.)
Fig. 6. Effect of Nrf2 knockdown on the anti-inammatory and antioxidant activity of MSC-EVs in LPS-stimulated RAW 264.7 cells.
(A) Histogram showing the RNA level of Nrf2 following knockdown in RAW 264.7 cells (Nrf2
−
). LPS-stimulated Nrf2
−
RAW 264.7 cells received PBS (Nrf2
−
+LPS) or
MSC-EVs (Nrf2
−
+LPS +EVs); (B–E) RNA analysis displaying variation in the expression of HO-1 (B), Keap1 (C), IL-6 (D), and TNF
α
(E) in the experimental groups.
NC: negative control, LPS: lipopolysaccharide, EVs: extracellular vesicles, Nrf2: nuclear factor erythrocyte 2-related factor 2, Nrf2
−
: Nrf2 knockdown, HO-1: heme
oxygenase-1, Keap1: Kelch-like epichlorohydrin-associated protein 1, IL: interleukin, TNF
α
: tumor necrosis factor
α
, ns: no signicance. All data are presented as the
mean ±SEM and normal distribution is conrmed by the K
–
S test, multiple comparisons were analyzed using one-way ANOVA by the least LSD test.
ns
P >0.05, *P
<0.05, ****P <0.0001.
R. Zhao et al.

Journal of Controlled Release 345 (2022) 214–230
229
inh +Veh 14-day group. Furthermore, after MSC-EV administration,
reduced oxidative signaling (8-OHdG, Nrf2, and HO-1) together with
down-regulated inammatory mediators were observed in LPS-
stimulated macrophages. To elucidate the potential mechanism of
Nrf2 in ALI after EV therapy, Nrf2 was knocked down to determine
variations in typical oxidative and inammatory mediators in LPS-
stimulated macrophages. Interestingly, LPS stimulation resulted in the
oxidative and inammatory responses typied by the increased
expression of Nrf2, HO-1, TNF
α
and decreased expression of Keap1 in
LPS-stimulated Nrf2
−
RAW 264.7 cells (versus the NC group); however,
no differences were observed in Nrf2, Keap1, and TNF
α
expression
following treatment with MSC-EVs (Nrf2
−
+LPS vs. Nrf2
−
+LPS +EVs).
A series of studies indicated that activation of Nrf2 is favor to protect the
lung from oxidative and inammatory injury [39], but persistent or
hyper activation of Nrf2 pathway will result in the redox imbalance to
induce tissue injury [42,43]. Therefore, the above results implied that
Nrf2 expression is variable in LPS-stimulated cells and animal models,
even at different stages of ALI, suggesting that adaptive regulation of
Nrf2 is favorable for the treatment of ALI mice with MSC-EVs. Changes
in the expression of HO-1, IL-6, and other mediators in MSC-EV-treated
ALI mice should be explored further in the future.
5. Conclusions
In summary, our results showed that the inhalation of MSC-EVs
presented better performance than those administered via tail vein in-
jection for the treatment of ALI, as well as exhibited robust anti-
inammatory and antioxidative activity in LPS-stimulated cells and
animal models. Moreover, in vitro and in vivo studies revealed that Nrf2
may play an important role in the treatment of ALI, and its regulation
might restore lung inammation and oxidative damage. From a clinical
perspective, the benecial effects of inhaled MSC-EVs in mice suggest
that EV-based nano-therapeutics that regulate inammatory and
oxidative mediators during macrophage activation and ALI may atten-
uate COVID-19 patients.
Credit author statement
Ruijing Zhao: Conceptualization, Methodology, Writing- Original
draft preparation. Lina Wang: Acquisition, Methodology. Tian Wang:
Methodology, Software. Panpan Xian: Visualization, Interpretation.
Hongkang Wang: Data curation, Investigation. Qianfa Long: Supervi-
sion, Writing- Reviewing and Editing.
Declaration of Competing Interest
The authors declare that they have no conict of interest.
Acknowledgements
The present study was mainly supported by the Clinical Emergency
Study of MSC-EVs in the Treatment of COVID-19 (20200001YX001(1)),
partially supported by the National Natural Science Foundation of China
(82171353), Science and Technology Innovation of Shaanxi Province
(2018ZDXM-SF-046), and Major Projects of Xi’an Medical Research
(201805104YX12SF38-1). We would like to thank Editage (www.ed
itage.com) for English language editing.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.jconrel.2022.03.025.
References
[1] M. Khoury, J. Cuenca, F.F. Cruz, F.E. Figueroa, P.R.M. Rocco, D.J. Weiss, Current
status of cell-based therapies for respiratory virus infections: applicability to
COVID-19, Eur. Respir. J. 55 (2020) 2000858.
[2] A.P. Wheeler, G.R. Bernard, Acute lung injury and the acute respiratory distress
syndrome: a clinical review, Lancet 369 (2007) 1553–1564.
[3] Acute respiratory distress syndrome, Nat. Rev. Dis. Primers 5 (2019) 19.
[4] X. Cao, COVID-19: immunopathology and its implications for therapy, Nat. Rev.
Immunol. 20 (2020) 269–270.
[5] J.G. Wilson, K.D. Liu, H. Zhuo, L. Caballero, M. McMillan, X. Fang, K. Cosgrove,
R. Vojnik, C.S. Calfee, J.W. Lee, A.J. Rogers, J. Levitt, J. Wiener-Kronish, E.
K. Bajwa, A. Leavitt, et al., Mesenchymal stem (stromal) cells for treatment of
ARDS: a phase 1 clinical trial, Lancet Respir. Med. 3 (2015) 24–32.
[6] R. Kalluri, V.S. LeBleu, The biology, function, and biomedical applications of
exosomes, Science 367 (2020) eaau6977.
[7] G. van Niel, G. D’Angelo, G. Raposo, Shedding light on the cell biology of
extracellular vesicles, Nat. Rev. Mol. Cell Biol. 19 (2018) 213–228.
[8] K. Khalaj, R.L. Figueira, L. Antounians, G. Lauriti, A. Zani, Systematic review of
extracellular vesicle-based treatments for lung injury: are EVs a potential therapy
for COVID-19? J. Extracell. Vesicles 9 (2020) 1795365.
[9] J. Wang, R. Huang, Q. Xu, G. Zheng, G. Qiu, M. Ge, Q. Shu, J. Xu, Mesenchymal
stem cell-derived extracellular vesicles alleviate acute lung injury via transfer of
miR-27a-3p, Crit. Care Med. 48 (2020) e599–e610.
[10] T.J. Morrison, M.V. Jackson, E.K. Cunningham, A. Kissenpfennig, D.F. McAuley, C.
M. O’Kane, A.D. Krasnodembskaya, Mesenchymal stromal cells modulate
macrophages in clinically relevant lung injury models by extracellular vesicle
mitochondrial transfer, Am. J. Respir. Crit. Care Med. 196 (2017) 1275–1286.
[11] M.H. Sohn, M.J. Kang, H. Matsuura, V. Bhandari, N.Y. Chen, C.G. Lee, J.A. Elias,
The chitinase-like proteins breast regression protein-39 and YKL-40 regulate
hyperoxia-induced acute lung injury, Am. J. Respir. Crit. Care Med. 182 (2010)
918–928.
[12] H. Amatullah, T. Maron-Gutierrez, Y. Shan, S. Gupta, J.N. Tsoporis, A.K. Varkouhi,
A.P. Teixeira Monteiro, X. He, J. Yin, J.C. Marshall, P.R.M. Rocco, H. Zhang, W.
M. Kuebler, C.C. Dos Santos, Protective function of DJ-1/PARK7 in
lipopolysaccharide and ventilator-induced acute lung injury, Redox Biol. 38
(2021), 101796.
[13] M. Mittal, C. Tiruppathi, S. Nepal, Y.Y. Zhao, D. Grzych, D. Soni, D.J. Prockop, A.
B. Malik, TNF
α
-stimulated gene-6 (TSG6) activates macrophage phenotype
transition to prevent inammatory lung injury, Proc. Natl. Acad. Sci. U. S. A. 113
(2016) E8151–e8158.
[14] Y. Qin, L. Cao, L. Hu, Sirtuin 6 mitigated LPS-induced human umbilical vein
endothelial cells inammatory responses through modulating nuclear factor
erythroid 2-related factor 2, J. Cell. Biochem. (2019), https://doi.org/10.1002/
jcb.28407. Online ahead of print.
[15] T. Wang, Z. Jian, A. Baskys, J. Yang, J. Li, H. Guo, Y. Hei, P. Xian, Z. He, Z. Li, N. Li,
Q. Long, MSC-derived exosomes protect against oxidative stress-induced skin
injury via adaptive regulation of the NRF2 defense system, Biomaterials 257
(2020), 120264.
[16] Q. Long, D. Upadhya, B. Hattiangady, D.K. Kim, S.Y. An, B. Shuai, D.J. Prockop, A.
K. Shetty, Intranasal MSC-derived A1-exosomes ease inammation, and prevent
abnormal neurogenesis and memory dysfunction after status epilepticus, Proc.
Natl. Acad. Sci. U. S. A. 114 (2017) E3536–E3545.
[17] P. Xian, Y. Hei, R. Wang, T. Wang, J. Yang, J. Li, Z. Di, Z. Liu, A. Baskys, W. Liu,
S. Wu, Q. Long, Mesenchymal stem cell-derived exosomes as a nanotherapeutic
agent for amelioration of inammation-induced astrocyte alterations in mice,
Theranostics 9 (2019) 5956–5975.
[18] L. Wang, J. Jing, H. Yan, J. Tang, G. Jia, G. Liu, X. Chen, G. Tian, J. Cai, H. Shang,
H. Zhao, Selenium pretreatment alleviated LPS-induced immunological stress via
upregulation of several Selenoprotein encoding genes in murine RAW264.7 cells,
Biol. Trace Elem. Res. 186 (2018) 505–513.
[19] T. Ashcroft, J.M. Simpson, V. Timbrell, Simple method of estimating severity of
pulmonary brosis on a numerical scale, J. Clin. Pathol. 41 (1988) 467–470.
[20] S.V. Szapiel, N.A. Elson, J.D. Fulmer, G.W. Hunninghake, R.G. Crystal, Bleomycin-
induced interstitial pulmonary disease in the nude, athymic mouse, Am. Rev.
Respir. Dis. 120 (1979) 893–899.
[21] K.S. Lee, S.R. Kim, H.S. Park, S.J. Park, K.H. Min, K.Y. Lee, Y.H. Choe, S.H. Hong,
H.J. Han, Y.R. Lee, J.S. Kim, D. Atlas, Y.C. Lee, A novel thiol compound, N-
acetylcysteine amide, attenuates allergic airway disease by regulating activation of
NF-kappaB and hypoxia-inducible factor-1alpha, Exp. Mol. Med. 39 (2007)
756–768.
[22] M.D. Robinson, D.J. McCarthy, G.K. Smyth, edgeR: a Bioconductor package for
differential expression analysis of digital gene expression data, Bioinformatics 26
(2010) 139–140.
[23] C. Thery, K.W. Witwer, E. Aikawa, M.J. Alcaraz, J.D. Anderson,
R. Andriantsitohaina, A. Antoniou, T. Arab, F. Archer, G.K. Atkin-Smith, D.C. Ayre,
J.M. Bach, D. Bachurski, H. Baharvand, L. Balaj, et al., Minimal information for
studies of extracellular vesicles 2018 (MISEV2018): a position statement of the
International Society for Extracellular Vesicles and update of the MISEV2014
guidelines, J. Extracell. Vesicles 7 (2018) 1535750.
[24] E. Bari, I. Ferrarotti, M.L. Torre, A.G. Corsico, S. Perteghella, Mesenchymal stem/
stromal cell secretome for lung regeneration: the long way through
"pharmaceuticalization" for the best formulation, J. Control. Release 309 (2019)
11–24.
[25] M. Zhao, S. Liu, C. Wang, Y. Wang, M. Wan, F. Liu, M. Gong, Y. Yuan, Y. Chen,
J. Cheng, Y. Lu, J. Liu, Mesenchymal stem cell-derived extracellular vesicles
R. Zhao et al.

Journal of Controlled Release 345 (2022) 214–230
230
attenuate mitochondrial damage and inammation by stabilizing mitochondrial
DNA, ACS Nano 15 (2021) 1519–1538.
[26] J. Dutra Silva, Y. Su, C.S. Calfee, K.L. Delucchi, D. Weiss, D.F. McAuley, C. O’Kane,
A.D. Krasnodembskaya, Mesenchymal stromal cell extracellular vesicles rescue
mitochondrial dysfunction and improve barrier integrity in clinically relevant
models of ARDS, Eur. Respir. J. 58 (2021) 2002978.
[27] J.D. Silva, A.D. Krasnodembskaya, Investigation of the MSC paracrine effects on
alveolar-capillary barrier integrity in the in vitro models of ARDS, Methods Mol.
Biol. 2269 (2021) 63–81.
[28] Q. Qiao, X. Liu, T. Yang, K. Cui, L. Kong, C. Yang, Z. Zhang, Nanomedicine for
acute respiratory distress syndrome: the latest application, targeting strategy, and
rational design, Acta Pharm. Sin. B 11 (2021) 3060–3091.
[29] X. Huang, H. Xiu, S. Zhang, G. Zhang, The role of macrophages in the pathogenesis
of ALI/ARDS, Mediat. Inamm. 2018 (2018) 1264913.
[30] H. Kim, M.J. Lee, E.H. Bae, J.S. Ryu, G. Kaur, H.J. Kim, J.Y. Kim, H. Barreda, S.
Y. Jung, J.M. Choi, T. Shigemoto-Kuroda, J.Y. Oh, R.H. Lee, Comprehensive
molecular proles of functionally effective MSC-derived extracellular vesicles in
immunomodulation, Mol. Ther. 28 (2020) 1628–1644.
[31] R. Huang, C. Qin, J. Wang, Y. Hu, G. Zheng, G. Qiu, M. Ge, H. Tao, Q. Shu, J. Xu,
Differential effects of extracellular vesicles from aging and young mesenchymal
stem cells in acute lung injury, Aging (Albany NY) 11 (2019) 7996–8014.
[32] C. Liu, Z. Yin, T. Feng, M. Zhang, Z. Zhou, Y. Zhou, An integrated network
pharmacology and RNA-Seq approach for exploring the preventive effect of
Lonicerae japonicae os on LPS-induced acute lung injury, J. Ethnopharmacol. 264
(2021), 113364.
[33] H. Zhu, S. Wang, C. Shan, X. Li, B. Tan, Q. Chen, Y. Yang, H. Yu, A. Yang,
Mechanism of protective effect of xuan-bai-cheng-qi decoction on LPS-induced
acute lung injury based on an integrated network pharmacology and RNA-
sequencing approach, Respir. Res. 22 (2021) 188.
[34] P.M. Cobelens, I.A. Tiebosch, R.M. Dijkhuizen, P.H. van der Meide, R. Zwartbol, C.
J. Heijnen, J. Kesecioglu, W.M. van den Bergh, Interferon-β attenuates lung
inammation following experimental subarachnoid hemorrhage, Crit. Care 14
(2010) R157.
[35] Y. Imai, K. Kuba, G.G. Neely, R. Yaghubian-Malhami, T. Perkmann, G. van Loo,
M. Ermolaeva, R. Veldhuizen, Y.H. Leung, H. Wang, H. Liu, Y. Sun, M. Pasparakis,
M. Kopf, C. Mech, et al., Identication of oxidative stress and toll-like receptor 4
signaling as a key pathway of acute lung injury, Cell 133 (2008) 235–249.
[36] J. Yan, J. Li, L. Zhang, Y. Sun, J. Jiang, Y. Huang, H. Xu, H. Jiang, R. Hu, Nrf2
protects against acute lung injury and inammation by modulating TLR4 and Akt
signaling, Free Radic. Biol. Med. 121 (2018) 78–85.
[37] H. Sun, J. Zhang, F. Chen, X. Chen, Z. Zhou, H. Wang, Activation of RAW264.7
macrophages by the polysaccharide from the roots of Actinidia eriantha and its
molecular mechanisms, Carbohydr. Polym. 121 (2015) 388–402.
[38] F.R. D’Alessio, Mouse models of acute lung injury and ARDS, Methods Mol. Biol.
2018 (1809) 341–350.
[39] Q. Liu, Y. Gao, X. Ci, Role of Nrf2 and its activators in respiratory diseases,
Oxidative Med. Cell. Longev. 2019 (2019) 7090534.
[40] C. Tonelli, I.I.C. Chio, D.A. Tuveson, Transcriptional regulation by Nrf2, Antioxid.
Redox Signal. 29 (2018) 1727–1745.
[41] H.Y. Cho, S.P. Reddy, S.R. Kleeberger, Nrf2 defends the lung from oxidative stress,
Antioxid. Redox Signal. 8 (2006) 76–87.
[42] P. Hiebert, M.S. Wietecha, M. Cangkrama, E. Haertel, E. Mavrogonatou,
M. Stumpe, H. Steenbock, S. Grossi, H.D. Beer, P. Angel, J. Brinckmann, D. Kletsas,
J. Dengjel, S. Werner, Nrf2-mediated broblast reprogramming drives cellular
senescence by targeting the Matrisome, Dev. Cell 46 (2018) 145–161.e110.
[43] R.F. Fan, K.K. Tang, Z.Y. Wang, L. Wang, Persistent activation of Nrf2 promotes a
vicious cycle of oxidative stress and autophagy inhibition in cadmium-induced
kidney injury, Toxicology 464 (2021), 152999.
R. Zhao et al.