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Citation: Kim, J.-S.; Lee, H.-J.; Yoon,
E.-J.; Lee, H.; Ji, Y.; Kim, Y.; Park, S.-J.;
Kim, J.; Bae, S. Protective Effect of
Iris germanica L. Rhizome-Derived
Exosome against Oxidative-Stress
-Induced Cellular Senescence in
Human Epidermal Keratinocytes.
Appl. Sci. 2023,13, 11681. https://
doi.org/10.3390/app132111681
Academic Editor: Snezana
Agatonovic-Kustrin
Received: 30 September 2023
Revised: 19 October 2023
Accepted: 23 October 2023
Published: 25 October 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
applied
sciences
Article
Protective Effect of Iris germanica L. Rhizome-Derived Exosome
against Oxidative-Stress-Induced Cellular Senescence in
Human Epidermal Keratinocytes
Ji-Seon Kim 1, Hyun-Jeong Lee 1, Eun-Jeong Yoon 2, Hyunsang Lee 2, Youngeun Ji 3, Youngseok Kim 3,
Si-Jun Park 2, Junoh Kim 3and Seunghee Bae 1, *
1Department of Cosmetics Engineering, Konkuk University, 120 Neungdong-ro, Gwangjin-gu,
Seoul 05029, Republic of Korea; jskimkyapt@konkuk.ac.kr (J.-S.K.); dlguswjd9018@konkuk.ac.kr (H.-J.L.)
2
Advanced Actives of Plants & Life Science Institute, ABio Materials Co., Ltd., Cheonan 31005, Republic of Korea;
ejyoon@a-bio.co.kr (E.-J.Y.); hyunsanglee@a-bio.co.kr (H.L.); coolsijun@a-bio.co.kr (S.-J.P.)
3Shinsegae International Inc., Seoul 06015, Republic of Korea; youngeunji@sikorea.co.kr (Y.J.);
yskim23@sikorea.co.kr (Y.K.); junohkim@sikorea.co.kr (J.K.)
*Correspondence: sbae@konkuk.ac.kr
Abstract:
Plant-derived exosomes can exert therapeutic effects against various dermatological con-
ditions. Several studies have demonstrated that plant-derived exosomes can have positive effects
on the skin, preventing aging, hyperpigmentation, and hair loss. In this study, the protective effects
of
Iris germanica L.
rhizome-derived exosomes (Iris-exosomes) on oxidative-stress-induced cellular
dysfunction were investigated in human epidermal keratinocytes (nHEKs). Iris-exosomes with a
diameter range of 100–300 nm were detected. In the cytotoxicity assay, Iris-exosomes with up to
10
7
particles per milliliter were found to possess no cytotoxicity, and we recovered H
2
O
2
-induced cell
viability loss. In nHEKs, H
2
O
2
-induced ROS levels were significantly reduced using Iris-exosomes
and additionally associated with increases in antioxidant enzyme transcription. The H
2
O
2
-induced
SA-
β
-gal-positive nHEKs were decreased using Iris-exosomes; these effects correlate with the changed
levels of cell cycle arrest marker p21. Furthermore, the H
2
O
2
-induced loss of
in vitro
wound-healing
properties and early detection of keratin 1 and 10—keratinization markers—were restored to control
levels using Iris-exosomes. Altogether, these results indicate the possibility that Iris-exosomes exert
antioxidant and anti-senescence effects in order to protect against oxidative-stress-induced cellular
dysfunction in nHEKs.
Keywords: antioxidant; exosome; Iris germanica L. rhizome; keratinocytes; senescence
1. Introduction
Exosomes are among the extracellular vesicles produced by eukaryotic cells, a category
of cell that includes plant cells [
1
]. Possessing a lipid bilayer structure, exosomes are
between 30 and 200 nm in diameter, with an average diameter of 100 nm [
2
,
3
]. A variety of
investigations have found that exosomes can contain various cellular constituents including
DNA, RNA, lipids, metabolites, and cytosolic and cell-surface proteins, depending on the
origin of the cells [
3
]. The physiological roles of exosomes are based on maintaining
cellular homeostasis by removing unnecessary constituents from cells [
4
]. However, recent
studies have found that exosomes play a large part in intercellular communication and
can alter biological responses, including immune responses and cancer progression [
5
–
8
].
Therefore, these properties can be used in the treatment and diagnosis of various diseases,
including cancer [
3
,
9
]. In addition, recent studies have shown that exosomes have low
immunogenicity and high hemocompatibility and are effective in treating skin problems
such as aging, atopic dermatitis, and wounds [10]. Moreover, exosomes are being studied
for their potential use as ingredients in skin care products. One study found that exosomes
Appl. Sci. 2023,13, 11681. https://doi.org/10.3390/app132111681 https://www.mdpi.com/journal/applsci
Appl. Sci. 2023,13, 11681 2 of 18
secreted by dermal papilla cells helped hair follicle development [
11
]. Another found
that cardiac progenitor cell-derived exosomes protected cardiomyocytes against oxidative-
stress-induced apoptosis [
12
]. Moreover, treatment with blueberry-derived exosomes
protected human endothelial cells from tumor necrosis factor-
α
(TNF-
α
)-induced reactive
oxygen species (ROS) production and viability loss [
13
]. Recent studies have indicated the
functional therapeutic roles of plant-derived exosomes originating from cabbage, ginseng
(Panax ginseng), and green tea (Camellia sinensis) [
14
–
19
]. According to research, exosomes
can be absorbed effectively into keratinocytes owing to their biocompatibility. Additionally,
comparative gene expression analysis has shown that exosome treatment contributes to
maintaining skin condition better than several plant extract treatment groups in genes
related to skin aging, regeneration, skin barrier, and moisturization. These findings indicate
that plant-derived exosomes affect human cells, including skin cells, differently than
the extracts, offering a new perspective on their potential application as cosmeceutical
ingredients [18].
Oxygen molecules with unpaired electrons, known as free radicals, are produced by
extrinsic factors, including UV light, pollution, stress, and smoking, as well as endogenous
factors inside the body. Antioxidant defense systems within cells counteract ROS and
maintain a balance between ROS generation and neutralization [
20
]. The inability of
the body to scavenge free radicals, as occurs when an imbalance between ROS and the
antioxidative defense system emerges, results in skin aging and diseases such as psoriasis
and atopic dermatitis [
21
–
23
]. Skin aging and such diseases are closely associated with
keratinocyte functioning. Keratinocytes play an important role in skin regeneration and
barrier functions [
24
–
26
]. When oxidative stress is excessive, keratinocyte differentiation
becomes abnormal [
25
,
27
], resulting in an abnormal skin barrier that causes skin diseases
and accelerates the overall aging of the skin due to excessive moisture evaporation [
28
,
29
].
Therefore, if one is to maintain good skin health, it is crucial to protect keratinocytes from
oxidative stress.
The skin is the largest organ in the body [
30
] and comprises the following three layers:
epidermis, dermis, and subcutaneous fat [
31
]. Multiple layers of keratinocytes are present
in the epidermis, each with a different degree of differentiation [
32
,
33
]. As keratinocytes
differentiate, they migrate toward the surface and become fully differentiated corneo-
cytes [
33
,
34
]. Located in the outermost layer of the skin, keratinocytes are continually
exposed to various kinds of stresses, as well as UV irradiation [
35
,
36
]. UV is one of the
major factors contributing to ROS generation [
37
,
38
], and UV-induced ROS generation
reduces physiological antioxidant levels in the epidermis, causing oxidative stress [
39
]. Ker-
atinocytes are susceptible to oxidative stress, which can cause serious skin diseases [
40
,
41
].
Significant ROS accumulation in keratinocytes is associated with oxidative modifications of
nucleic acids, lipids, proteins, and other intracellular molecules, leading to cellular dysfunc-
tion such as cell viability loss, cellular senescence, and apoptosis [
42
–
46
]. The epidermis
is the first barrier against pathogens, pollutants, toxic chemicals, and UV irradiation—all
factors that contribute to oxidative stress [
47
]. Therefore, the identification of bioactive
molecules that protect keratinocytes from oxidative stress can be a promising therapeutic
and/or cosmeceutical strategy in preventing oxidative-stress-related skin diseases.
Various novel bioactive molecules are produced by plants and used to prepare a
variety of medicines [
48
]. Iris species, members of the Iridaceae family, are valuable tra-
ditional medicines with a variety of applications, such as relieving joint pain and cough
symptoms [
49
–
51
]. Several studies have shown that Iris extracts have anti-inflammatory,
anti-cancer, antioxidant, and antimicrobial activities [
52
–
56
]. In addition,
Iris germanica L.
contains many secondary metabolites with strong antioxidant potential, including flavonoids
and triterpenes [
57
–
59
]. Specifically, methanolic extracts of Iris germanica L. rhizomes possess
antioxidant and anti-inflammatory properties due to their high content of isoflavonoids,
such as tectoridin, iridin, 8-hydroxyirilone, and tectorigenin [
60
]. There are also bioac-
tive molecules in Iris germanica L., including isoflavones such as irigenin, iristectoiri-
genin A, nigricin, irisflorentin, irilone, and irisolidone [
59
]. Furthermore, previous phyto-
Appl. Sci. 2023,13, 11681 3 of 18
chemical studies have demonstrated that Iris germanica L. includes a variety of chemical
compounds [
61
,
62
], including flavonoids, triterpenes, sterols, phenolic compounds, ce-
ramides, and benzoquinone derivatives. Therefore, it is reasonable to assume that exosomes
derived from Iris rhizomes are an optimum source of antiaging agents for action against
intracellular ROS. However, there are limited studies on how exosomes derived from the
rhizome of Iris germanica L. affect the antioxidant activity of keratinocytes.
In this study, we investigate whether Iris-exosomes possess antioxidant properties
and protect against oxidative stress. H
2
O
2
is a common oxidizing agent that accumulates
mostly in the epidermis and can move into and out of cells and tissues [
63
–
65
]; therefore,
we used H2O2in order to induce oxidative stress in keratinocytes.
2. Materials and Methods
2.1. Isolation and Characterization of Iris germanica L. Rhizome-Derived Exosomes (Iris-Exosomes)
Iris germanica L. rhizomes were purchased from Produits Prestiges S.A.
(Montreux, Switzerland). The dry Iris rhizomes (Iris germanica L./100 g) were packed
in a plastic pack with distilled water and sealed well to prevent air infiltration. Then,
ultra-high-pressure treatment was performed for 30 sec at 25
◦
C at a pressure of 200 Mpa.
Iris rhizomes treated with ultrahigh pressure were then extracted with a general juicer
using a low-speed screw at 30 rpm, and the obtained Iris rhizome extracts were filtered
using a mesh net. The Iris rhizome extracts were stored at −80 ◦C until purification could
be performed. For exosome purification, the Iris rhizome extracts were centrifugated
at 10,000
×
gfor 10 min at 4
◦
C to remove large contaminants. The supernatant was
collected, frozen at
−
80
◦
C for 20 h, and then dried in a freeze dryer for 100 h under
vacuum conditions. The lyophilized supernatant was mixed with distilled water (DW)
and an aqueous two-phase system (ATPS). The ATPS was prepared by adding 3.3% PEG
(Sigma-Aldrich, St. Louis, MO, USA)
with a molecular weight of 10,000–35,000 and 1.7%
dextran (Sigma-Aldrich, St. Louis, MO, USA) with a molecular weight of 300,000–650,000.
After mixing the lyophilized supernatant and PEG/dextran solution, centrifugation at
1000
×
gfor 10 min was performed at 4
◦
C. Exosomes were recovered via centrifugation af-
ter the supernatant was removed. To increase the purity, the recovered lower-layer solution
was washed with an additional aqueous two-phase system at the same concentration. The
final exosome-concentrated layer was recovered after repeating the same washing process
three times (Figure 1a). As the exosomes purified from Iris germanica L. rhizome are in
the form of an aqueous solution of nanoparticles with an inherent tendency to sediment,
they were carefully treated with cells using gentle pipetting to prevent the destabilization
of the exosomes.
Appl. Sci. 2023, 13, x FOR PEER REVIEW 3 of 19
also bioactive molecules in Iris germanica L., including isoflavones such as irigenin, iristec-
toirigenin A, nigricin, irisflorentin, irilone, and irisolidone [59]. Furthermore, previous
phytochemical studies have demonstrated that Iris germanica L. includes a variety of chem-
ical compounds [61,62], including flavonoids, triterpenes, sterols, phenolic compounds,
ceramides, and benzoquinone derivatives. Therefore, it is reasonable to assume that exo-
somes derived from Iris rhizomes are an optimum source of antiaging agents for action
against intracellular ROS. However, there are limited studies on how exosomes derived
from the rhizome of Iris germanica L. affect the antioxidant activity of keratinocytes.
In this study, we investigate whether Iris-exosomes possess antioxidant properties
and protect against oxidative stress. H2O2 is a common oxidizing agent that accumulates
mostly in the epidermis and can move into and out of cells and tissues [63–65]; therefore,
we used H2O2 in order to induce oxidative stress in keratinocytes.
2. Materials and Methods
2.1. Isolation and Characterization of Iris germanica L. Rhizome-Derived Exosomes
(Iris-Exosomes)
Iris germanica L. rhizomes were purchased from Produits Prestiges S.A. (Montreux,
Swierland). The dry Iris rhizomes (Iris germanica L./100 g) were packed in a plastic pack
with distilled water and sealed well to prevent air infiltration. Then, ultra-high-pressure
treatment was performed for 30 sec at 25 °C at a pressure of 200 Mpa. Iris rhizomes treated
with ultrahigh pressure were then extracted with a general juicer using a low-speed screw
at 30 rpm, and the obtained Iris rhizome extracts were filtered using a mesh net. The Iris
rhizome extracts were stored at −80 °C until purification could be performed. For exosome
purification, the Iris rhizome extracts were centrifugated at 10,000 × g for 10 min at 4 °C to
remove large contaminants. The supernatant was collected, frozen at −80 °C for 20 h, and
then dried in a freeze dryer for 100 h under vacuum conditions. The lyophilized superna-
tant was mixed with distilled water (DW) and an aqueous two-phase system (ATPS). The
ATPS was prepared by adding 3.3% PEG (Sigma-Aldrich, St. Louis, MO, USA) with a mo-
lecular weight of 10,000–35,000 and 1.7% dextran (Sigma-Aldrich, St. Louis, MO, USA)
with a molecular weight of 300,000–650,000. After mixing the lyophilized supernatant and
PEG/dextran solution, centrifugation at 1000× g for 10 min was performed at 4 °C. Exo-
somes were recovered via centrifugation after the supernatant was removed. To increase
the purity, the recovered lower-layer solution was washed with an additional aqueous
two-phase system at the same concentration. The final exosome-concentrated layer was
recovered after repeating the same washing process three times (Figure 1a). As the exo-
somes purified from Iris germanica L. rhizome are in the form of an aqueous solution of
nanoparticles with an inherent tendency to sediment, they were carefully treated with
cells using gentle pipeing to prevent the destabilization of the exosomes.
Figure 1.
Isolation and characterization of Iris germanica L. rhizome-derived exosome. (
a
) Isolation of
Iris germanica L. rhizome-derived exosome. (
b
) Representative image of Iris germanica L. rhizome-derived
exosome visualized using Cryo-TEM (scale bars, 500 nm and 50 nm). Iris-exosomes are pointed by
black arrows within the image. (
c
) A graph showing the results derived with NTA of Iris-exosomes.
(d)Iris-exosomes were stained with oil red O and visualized using a microscope (scale bar, 100 nm).
Appl. Sci. 2023,13, 11681 4 of 18
2.1.1. Cryo-TEM Analysis
We placed 5
µ
L of the Iris-exosomes sample on a Lacey carbon grid. This was set-
tled on a cryoholder under conditions of a wait time of 30 s and blot time of 5 s in Vit-
robot Mark IV (FEI Company, Hillsboro, OR, USA) using cryogenic transmission electron
microscopy (Cryo-TEM).
2.1.2. Nanoparticle Tracking Analysis (NTA)
Exosomes separated from Iris germanica L. rhizome were analyzed using nanoparticle
tracking analysis (NTA) to verify particle size distributions and particle numbers per unit
volume. ZetaView (Particle Metrix GmbH, Meerbusch, Germany) was used to conduct
nanoparticle tracking analysis (NTA) measurements. After diluting the sample to an
appropriate concentration where the number of detected particles was between 50 and 300,
about 1 mL of the sample was injected into the cell in the device. We then examined the
product at a wavelength of 488 nm in scatter mode.
2.1.3. Oil Red O Staining
In total, 1 mL of oil red O solution (Sigma Aldrich) was added to the Iris-exosomes.
After 10 min, the Iris-exosomes were centrifuged again to remove any remaining oil red
O. Afterward, 1 mL of distilled water was added, and centrifugation was repeated twice
to eliminate any remaining oil red O. Iris-exosomes stained with oil red O were observed
under a microscope.
2.2. Cell Culture and Reagents
Primary normal human epidermal keratinocytes (nHEKs) provided by CELLnTEC
(Bern, Switzerland) were maintained in Keratinocyte Basal Medium-2 (Lonza, Basel, Switzer-
land) supplemented with bovine pituitary extract (BPE), human epidermal growth factor
(hEGF), insulin, hydrocortisone, transferrin, epinephrine, and gentamicin sulfate-amphotericin
(GA-1000). nHEKs were cultured in collagen-coated plates (SPL Life Sciences, Pocheon city,
Republic of Korea) and incubated at 5% CO
2
and 37
◦
C. The cells were washed with HEPES-
buffered saline solution (HSS) (Promocell, Heidelberg, Germany) and subcultured using
trypsin/ethylenediaminetetraacetic acid (EDTA) at a ratio of 0.04%/0.03% (Promocell). Pas-
sages 4–6 of nHEKs were used in this experiment.
Human dermal fibroblast (nHDF) purchased from Promocell (Heidelberg, Germany)
were maintained in Dulbecco’s Modified Eagle’s medium (DMEM) supplemented with
10% fetal bovine serum (FBS), 1% streptomycin (100 mg/mL), and penicillin (100 U/mL) at
37
◦
C and 5% CO
2
. Passages 12–14 of nHDFs were used in this study. N-acetyl-l-cysteine
(NAC) (Sigma-Aldrich, St. Louis, MO, USA) was used as an antioxidant control.
2.3. In Vitro DPPH and ABTS Radical Scavenging Assays
1,1-Diphenyl-2-picrylhydrazyl (DPPH) was used to measure radical scavenging activ-
ity via a modification of the Blois method (Blois, 1958) [
66
]. The DPPH reagent (0.2 mM)
and various concentrations of Iris-exosomes were mixed in a 96-well plate and incubated
for 15 min. The absorbance was measured at 517 nm using a microplate reader.
2,2
0
-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical cations were pre-
pared by reacting 7 mM ABTS solution (MedChemExpress ,Monmouth Junction, NJ, USA)
with potassium peroxydisulfate (Supelco, Bellefonte, PA, USA) to a final concentration of 2.45
mM. The mixture was then incubated overnight in the dark at room temperature. After incu-
bation, the solution was diluted until the absorbance at 734 nm was 0.70 (
±
0.02). L-ascorbic
acid (Sigma-Aldrich) was used as a positive control. Each concentration of Iris-exosomes and
L-ascorbic acid were mixed with ABTS solution in 96 wells. Then, absorbance was measured
at 734 nm using a microplate reader after shaking in the dark for 10 min.
Appl. Sci. 2023,13, 11681 5 of 18
2.4. Cell Viability Assay
To evaluate cell viability, the nHEKs and nHDFs were subjected to a Water-Soluble
Tetrazolium-1 (WST-1) assay. The cells were seeded in 96-well plates and treated with vari-
ous concentrations of Iris-exosomes or H
2
O
2
and incubated at 37
◦
C for 24 h. Subsequently,
EZ-Cytox solution (DoGenBio, Seoul, Republic of Korea) was added to the plate, and the
cells were incubated at 37
◦
C with 5% CO
2
for 30 min. Cell viability was analyzed using an
iMark microplate reader measuring absorbance at 450 nm.
For the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay,
cells were seeded in a 12-well plate and incubated at 37
◦
C for 24 h. Subsequently, the
cells were pretreated with Iris-exosomes or NAC for 4 h, followed by exposure to 200
µ
M
H
2
O
2
for another 24 h. Next, 500
µ
g/mL of MTT solution was added to the cells, which
were incubated at 37
◦
C with 5% CO
2
for 3 h in the dark. The MTT solution was carefully
removed, and 1 mL of DMSO (Sigma-Aldrich) was added to each well. The plates were
then shaken for 5 min to ensure proper dissolution of the formazan crystals. Finally, the
absorbance was measured at 595 nm using a microplate reader.
2.5. Intracellular ROS Measurement Assay
Intracellular ROS levels were evaluated using an H
2
DCFDA assay. nHEKs were seeded
in 6-well plates and incubated for 24 h. After pretreatment with Iris-exosomes for 4 h, DCF-DA
solution (10
µ
M) was added and incubated for 30 min in the dark. The cells were then treated
with 500
µ
M H
2
O
2
to induce intracellular ROS production and incubated for 30 min in the
dark. The intracellular ROS levels were determined using fluorescence microplate reader at
485 nm for excitation and 520 nm for emission.
2.6. Cellular-Senescence-Analysis-Associated SA-β-Galactosidase Activity
nHEKs were treated with 200
µ
M H
2
O
2
for 4 h after the treatment of Iris-exosomes for
4 h. Using a senescence
β
-galactosidase staining kit (Cell Signaling Technology, Danvers,
MA, USA), the cells were fixed for 5 min and incubated in a staining solution at 37
◦
C for
8 h. Cellular senescence levels were assessed by counting senescent cells using a
bright-field microscope.
2.7. Polymerase Chain Reaction (PCR) and Quantitative Reverse Transcription PCR (RT-qPCR)
nHEKs were pretreated with Iris-exosomes for 4 h, followed by treatment with 200
µ
M H
2
O
2
for 4 h. The cells were further incubated for 40 h, and total RNAs were extracted
using RiboEx reagent (GeneAll Biotechnology, Seoul, Korea). cDNA was synthesized using
Moloney murine leukemia virus (M-MLV) reverse transcriptase (Thermo Fisher Scientific,
Waltham, MA, USA). RT-qPCR was conducted using a reaction mixture containing each
primer and the Evagreen qPCR master mix (Solis BioDyne, Tartu, Estonia). Gene expression
was normalized to that of GAPDH. The primer sequences for specific genes used in the anal-
ysis were as follows: p16 forward, 5
0
-TGCCTTTTCACTGTGTTGGA-3
0
and p16 reverse 5
0
-
GCCATTTGCTAGCAGTGTGA-3
0
;p21 forward, 5
0
-GAACTTCGACTTTGTCACCGAGAC-
3
0
and p21 reverse, 5
0
-TGGAGTGGTAGAAATCTGTCATGCT-3
0
;p53 forward, 5
0
-CGGGAT
CCCGAATGTTGTACCTGGAAAACAATG-3
0
and p53 reverse, 5
0
-GCTCTAGAGCT
CACTCCCCCTCCTCTTTGATG-3
0
;p63 forward, 5
0
-CGGGATCCCGAATGTTGTACCTGG
AAAACAATG-3
0
and p63 reverse, 5
0
-GCTCTAGAGCTCACTCCCCCTCCTCTTTGATG-3
0
;
HO-1 forward, 5
0
-GCCCTTCAGCATCCTCAGTTCC-3
0
and HO-1 reverse, 5
0
-AGTGGTCAT
GGCCGTGTCAAC-3
0
;NQO-1 forward, 5
0
-GGGAGACAGCCTCTTACTTGCC-3
0
and NQO-
1reverse, 5
0
-AACACCCAGCCGTCAGCTATTG-3
0
;GSS forward, 5
0
-TAGATGCCCCACG
TGCTTGT-3
0
and GSS reverse, 5
0
-ATCCTCATGGAGAAGATCGA-3
0
;SOD forward, 5
0
-
CCAGTGCAGGGCATCATCA-3
0
and SOD reverse, 5
0
-TTGGCCCACCGTGTTTTCT-3
0
;
GPx1 forward, 5
0
-GCAGCTCGTTCATCTGGGTG-3
0
and GPx1 reverse, 5
0
-ATGTGTGCT
GCTCGGCTAGC-3
0
;Nrf2 forward, 5
0
-ATAGCTGAGCCCAGTATC-3
0
and Nrf2 reverse, 5
0
-
CATGCACGTGAGTGCTCT-3
0
;GAPDH forward, 5
0
-TCCAAAATCAAGTGGGGCGATGC-
Appl. Sci. 2023,13, 11681 6 of 18
3
0
,GAPDH reverse, 5
0
-GCCAGTAGAGGCAGGGATGATGT-3
0
and
β
-actin forward, 5
0
-
CGCTCGGTGAGGATCTTCATG-3
0
,
β
-actin reverse, 5
0
-GGATTCCTATGTGGGCGACGA-3
0
.
2.8. Immunoblot Analysis
nHEK cells were pretreated using the indicated concentrations of Iris-exosomes for 4 h,
after which they were treated with 200
µ
M H
2
O
2
for another 4 h. After 40 h of further incuba-
tion, the cells were harvested, and total proteins were lysed using radioimmunoprecipitation
assay (RIPA) buffer containing protease inhibitors. Total protein quantification was performed
using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Samples containing 20
µ
g of
protein were separated via sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis,
and the proteins were then transferred onto a polyvinylidene fluoride (PVDF) membrane.
These membranes were blocked using 2% skim milk in Tris-buffered saline with Tween
20 (TBST) buffer and incubated with the corresponding primary antibodies overnight at 4
◦
C.
Then, the membranes were incubated with a horseradish peroxidase (HRP)-conjugated sec-
ondary antibody. The Western blot results were analyzed using enhanced chemiluminescence
(ECL) solution (Thermo Fisher Scientific) and visualized using a chemiluminescence detector.
The antibodies used were as follows: ERK (1:1000, CST), p-ERK (Thr202/Tyr204)(1:1000,
CST), p16 (1:1000, CST), Lamin B1 (1:1000, Abcam), p21 (1:1000, CST), Nrf2 (1:1000, CST), p53
(1:200, Santa Cruz), p-p53 (Ser15), (1:1000, CST), p38 (1:1000, CST), p-p38 (T180/Y182), (1:1000,
CST), JNK (1:1000, CST), p-JNK (T183/Y185), (1:1000, CST), Cytokeratin 1 (1:1000, Abcam),
Cytokeratin 10 (1:1000, Abcam), Loricrin (1:1000, Abcam), and
β
-actin (1:1000, Santa Cruz).
Quantification of protein level was conducted with ImageJ software (ImageJ 1.52a, National
Institutes of Health, Bethesda, MD, USA).
2.9. Keratinocyte Differentiation
nHEKs maintained in a KBM-2 medium containing 0.06 mM CaCl
2
solution (Pro-
mocell) were seeded on a 100 mm cell culture dish. The seeding date was adjusted so
that all cells could be harvested simultaneously on day 12. The cells were pretreated with
Iris-exosomes for 4 h before treatment with 200
µ
M H
2
O
2
for 4 h. To induce differentiation,
the cells were incubated in a KBM-2 medium containing 1.2 mM CaCl
2
for 0, 4, 8, and
12 days. Media with 1.2 mM CaCl
2
were changed once every three days. Cell lysates were
used to examine the expression levels of the differentiation markers K1, K10, and LOR
via Western blotting. The following antibodies were used: Cytokeratin 1 (1:1000, Abcam),
Cytokeratin 10 (1:1000, Abcam), Loricrin (1:1000, Abcam), and
β
-actin (1:1000, Santa Cruz).
2.10. Wound-Healing Assay
nHEKs were treated with the indicated concentrations of Iris-exosomes and/or
200 µM
H
2
O
2
for 4 h. Following incubation for 24 h, the confluent cells were scratched with a
pipette tip and then washed using HSS buffer. The wound area was photographed at
24 h intervals while incubating under conditions of 5% CO
2
and 37
◦
C. The gap area was
determined and measured using the ImageJ software.
2.11. Statistical Analysis
All experiments were performed independently at least thrice. The treatment groups
were assessed as statistically significant at p< 0.05 via one-way analysis of variance
(ANOVA). Different group means were compared using Tukey’s test. Data are expressed as
mean ±standard deviation (SD).
3. Results
3.1. Purification and Characterization of Iris germanica L. Rhizome-Derived Exosome
An in-depth description of Iris-exosome isolation is presented in the Materials and
Methods section. Briefly, an aqueous two-phase solution was prepared via a process
that used PEG and dextran to isolate Iris-exosomes from the Iris rhizome (Figure 1a).
By analyzing the total protein in the Iris-exosomes, it was determined that there was
Appl. Sci. 2023,13, 11681 7 of 18
approximately 21.08
µ
g/mL of protein in the solution. The morphological structure of
the exosomes derived from the Iris germanica L. rhizomes was analyzed using cryogenic
transmission electron microscopy (Cryo-TEM). The phospholipid particles exhibited a
spherical bilayer structure, with an average size of 160.7
±
3.60 nm (Figure 1b). Nanoparticle
tracking analysis (NTA) was conducted using ZetaView to determine the particle size
distribution and number per unit volume within the purified exosomes derived from the
Iris germanica L. rhizomes. According to the measurements, the average size of the particles
was 172.1
±
66.4 nm, and the concentration was confirmed to be 1.61
×
10
9
particles/mL
(Figure 1c). Next, oil red O staining was conducted to estimate the lipid levels in the
Iris-exosomes. This confirmed that the Iris-exosome is composed of a phospholipid bilayer
(Figure 1d). The results indicate that exosomes were present in the extracts derived from
the Iris germanica L. rhizomes.
3.2. Iris-Exosome Protects nHEKs from the Loss of Cell Viability Caused by Oxidative Stress
Before analyzing the effects of Iris-exosomes on H
2
O
2
-induced cellular stress in nHEKs,
we assessed the potential cytotoxicity of the nHEKs treated with Iris-exosomes for 24 h. As
shown in Figure 2a, nHEKs treated with a concentration of less than 10
7
(particles/mL)
showed no cytotoxicity. In order to evaluate the effect of H
2
O
2
on nHEKs, cell viability
was determined via 24 h treatment with various concentrations of H
2
O
2
. Consequently,
cell viability was reduced by 80% at 200
µ
M (Figure 2b). Therefore, 10
5
, 10
6
, and 10
7
Iris-exosomes were used in the following experiments along with 200
µ
M H
2
O
2
. The MTT
assay was performed on the nHEKs to determine whether the exosome treatment could
recover the reduction in cell viability caused by H
2
O
2
. The viability of nHEKs pretreated
with Iris-exosomes increased in a concentration-dependent manner compared with that of
cells treated with H
2
O
2
alone (Figure 2c). These results indicate that Iris-exosomes have
protective effects against the H2O2-induced loss of cell viability in nHEKs.
Appl. Sci. 2023, 13, x FOR PEER REVIEW 7 of 19
2.11. Statistical Analysis
All experiments were performed independently at least thrice. The treatment groups
were assessed as statistically significant at p < 0.05 via one-way analysis of variance
(ANOVA). Different group means were compared using Tukey’s test. Data are expressed
as mean ± standard deviation (SD).
3. Results
3.1. Purification and Characterization of Iris germanica L. Rhizome-Derived Exosome
An in-depth description of Iris-exosome isolation is presented in the Materials and
Methods section. Briefly, an aqueous two-phase solution was prepared via a process that
used PEG and dextran to isolate Iris-exosomes from the Iris rhizome (Figure 1a). By ana-
lyzing the total protein in the Iris-exosomes, it was determined that there was approxi-
mately 21.08 µg/mL of protein in the solution. The morphological structure of the exo-
somes derived from the Iris germanica L. rhizomes was analyzed using cryogenic trans-
mission electron microscopy (Cryo-TEM). The phospholipid particles exhibited a spheri-
cal bilayer structure, with an average size of 160.7 ± 3.60 nm (Figure 1b). Nanoparticle
tracking analysis (NTA) was conducted using ZetaView to determine the particle size dis-
tribution and number per unit volume within the purified exosomes derived from the Iris
germanica L. rhizomes. According to the measurements, the average size of the particles
was 172.1 ± 66.4 nm, and the concentration was confirmed to be 1.61 × 10
9
particles/mL
(Figure 1c). Next, oil red O staining was conducted to estimate the lipid levels in the Iris-
exosomes. This confirmed that the Iris-exosome is composed of a phospholipid bilayer
(Figure 1d). The results indicate that exosomes were present in the extracts derived from
the Iris germanica L. rhizomes.
3.2. Iris-Exosome Protects nHEKs from the Loss of Cell Viability Caused by Oxidative Stress
Before analyzing the effects of Iris-exosomes on H
2
O
2
-induced cellular stress in
nHEKs, we assessed the potential cytotoxicity of the nHEKs treated with Iris-exosomes
for 24 h. As shown in Figure 2a, nHEKs treated with a concentration of less than 10
7
(par-
ticles/mL) showed no cytotoxicity. In order to evaluate the effect of H
2
O
2
on nHEKs, cell
viability was determined via 24 h treatment with various concentrations of H
2
O
2
. Conse-
quently, cell viability was reduced by 80% at 200 µM (Figure 2b). Therefore, 10
5
, 10
6
, and
10
7
Iris-exosomes were used in the following experiments along with 200 µM H
2
O
2
. The
MTT assay was performed on the nHEKs to determine whether the exosome treatment
could recover the reduction in cell viability caused by H
2
O
2
. The viability of nHEKs pre-
treated with Iris-exosomes increased in a concentration-dependent manner compared
with that of cells treated with H
2
O
2
alone (Figure 2c). These results indicate that Iris-exo-
somes have protective effects against the H
2
O
2
-induced loss of cell viability in nHEKs.
Figure 2. Protective effect of Iris-exosome on cell viability against H
2
O
2
-induced cytotoxicity in hu-
man keratinocytes. (a) Cell viabilities pretreated with Iris-exosomes for 24 h and examined using
Figure 2.
Protective effect of Iris-exosome on cell viability against H
2
O
2
-induced cytotoxicity in
human keratinocytes. (a) Cell viabilities pretreated with Iris-exosomes for 24 h and examined using
WST-1 assay. (
b
) Cell viability of nHEKs with H
2
O
2
for 24 h. (
c
) nHEKs were treated with 200
µ
M
H
2
O
2
for 24 h after Iris-exosomes for 4 h and cell viability was measured via MTT assay. Values are
expressed as mean
±
SD of three independent experiments. * p< 0.05 and *** p< 0.001 compared
with untreated cells; ### p< 0.001 compared with H2O2-treated cells.
Furthermore, we conducted the same experiment on nHDFs to determine whether the
protective effects observed in nHEKs are also found in other types of primary skin cells.
As shown in Supplementary Figure S1a, nHDFs showed no cytotoxicity and increased
cell viability at concentrations below 10
9
(particles/mL). Furthermore, the viability of the
nHDFs was reduced by 80% at 300
µ
M H
2
O
2
(Supplementary Figure S1b). Therefore,
we treated 10
6
, 10
7
, and 10
8
Iris-exosomes with 300
µ
M H
2
O
2
. nHDFs were treated with
exosomes to determine whether exosome treatment could restore the viability of the cells
post-H
2
O
2
treatment. Compared to the cells treated with H
2
O
2
alone, pretreatment with
Iris-exosomes increased the viability of the nHDFs in a concentration-dependent manner
Appl. Sci. 2023,13, 11681 8 of 18
(Supplementary Figure S1c). Considering these results, it is evident that Iris-exosomes are
protective against the reduction in cell viability caused by H
2
O
2
in not only nHEKs but
also nHDFs.
3.3. Iris-Exosome Reduces Intracellular ROS Levels in nHEKs
Our previous results led us to investigate the antioxidant potential of Iris-exosomes.
The DPPH and ABTS assays were initially conducted to determine whether Iris-exosomes
possess a radical scavenging effect. The Iris-exosomes showed concentration-dependent
scavenging activity against DPPH and ABTS radicals but revealed a less potent overall
effect than L-ascorbic acid (Figure 3a,b). To determine whether exosomes can scavenge
intracellular ROS in nHEKs, a DCF-DA assay was conducted in which nHEKs were pre-
treated using Iris-exosomes for 4 h and then exposed to 500
µ
M H
2
O
2
for 30 min. As shown
in Figure 3c,d, the increased DCF fluorescence intensity caused by the H
2
O
2
treatment
decreased in the Iris-exosome-treated cells in a concentration-dependent manner. To con-
firm whether these results could be explained by the expression of antioxidant-related
genes in the cells, several antioxidant-related genes were measured after Iris-exosome
treatment. HO-1,CAT,GSS,GPx1,SOD, and Nrf2 were transcriptionally upregulated in
nHEKs following Iris-exosome treatment (Figure 3e). According to these results, it can be
said that Iris-exosomes appear to exert a protective effect against cellular oxidative stress
caused by H2O2.
Appl. Sci. 2023, 13, x FOR PEER REVIEW 9 of 19
Figure 3. Iris-exosome can reduce oxidative stress in nHEKs. (a) DPPH and (b) ABTS radical scav-
enging activity of Iris-exosome. Ascorbic acid (5 µg/mL) as a positive control. Cells were pretreated
with Iris-exosomes for 4 h, followed by treatment with 200 µM H2O2 for 30 min. After being incu-
bated at 37 °C in the dark for 30 min in culture medium containing 10 µM DCF-DA, intracellular
ROS generation was measured using (c) fluorescence microscopy (scale bar, 250 µm) and (d) a flu-
orescence microplate reader. (e) nHEKs were treated using Iris-exosomes for 4 h and examined to
assess the mRNA level of antioxidant-related genes (NQO-1, HO-1, CAT, GSS, GPx1, SOD, and Nrf2)
using RT-PCR, normalized with GAPDH and β-actin. Data are presented as mean ± SD of three in-
dependent experiments. *** p < 0.001 compared with untreated cells; ### p < 0.001 compared with
H2O2-treated cells.
3.4. Iris-Exosome Is Capable of Aenuating Cellular Senescence Induced by H2O2
In addition to assessing their radical scavenging activity in the intracellular environ-
ment, we investigated whether Iris-exosomes could protect nHEKs against oxidative-
stress-induced cellular senescence. nHEKs were treated with various concentrations of
Iris-exosomes and/or 200 µM H2O2, and senescence-associated β-galactosidase (SA-β-gal)
assay was conducted to examine the SA-β-gal-positive senescent cells. The results indicate
that H2O2 treatment only increased the number of senescent cells to 274.78% of the total
reached in the untreated control group (100.00%); however, Iris-exosome-pretreated
nHEKs reduced the number of senescent cells to 160.31%, 155.16%, and 138.37% (Figure
4a,b). It is well-known that p16 and p21 act as a cell cycle-dependent kinase inhibitor. RT-
PCR was used to confirm these results via the analysis of the expression levels of senescent
markers (p16 and p21). The results shown in Figure 4c,d indicate that the mRNA expres-
sion of p21 decreased concentration-dependently in the Iris-exosome-pretreated cells com-
pared to the H2O2-treated cells. These results suggest that Iris-exosomes protect nHEKs
against intracellular ROS production and cellular senescence induced by H2O2.
Figure 3.
Iris-exosome can reduce oxidative stress in nHEKs. (
a
) DPPH and (
b
) ABTS radical
scavenging activity of Iris-exosome. Ascorbic acid (5
µ
g/mL) as a positive control. Cells were
pretreated with Iris-exosomes for 4h, followed by treatment with 200
µ
M H
2
O
2
for 30 min. After
being incubated at 37
◦
C in the dark for 30 min in culture medium containing 10
µ
M DCF-DA,
intracellular ROS generation was measured using (
c
) fluorescence microscopy (scale bar, 250
µ
m)
and (
d
) a fluorescence microplate reader. (
e
) nHEKs were treated using Iris-exosomes for 4 h and
examined to assess the mRNA level of antioxidant-related genes (NQO-1,HO-1,CAT,GSS,GPx1,
SOD, and Nrf2) using RT-PCR, normalized with GAPDH and
β
-actin. Data are presented as mean
±
SD of three independent experiments. *** p< 0.001 compared with untreated cells; ### p< 0.001
compared with H2O2-treated cells.
Appl. Sci. 2023,13, 11681 9 of 18
3.4. Iris-Exosome Is Capable of Attenuating Cellular Senescence Induced by H2O2
In addition to assessing their radical scavenging activity in the intracellular envi-
ronment, we investigated whether Iris-exosomes could protect nHEKs against oxidative-
stress-induced cellular senescence. nHEKs were treated with various concentrations of
Iris-exosomes and/or 200
µ
M H
2
O
2
, and senescence-associated
β
-galactosidase (SA-
β
-gal)
assay was conducted to examine the SA-
β
-gal-positive senescent cells. The results indicate
that H
2
O
2
treatment only increased the number of senescent cells to 274.78% of the total
reached in the untreated control group (100.00%); however, Iris-exosome-pretreated nHEKs
reduced the number of senescent cells to 160.31%, 155.16%, and 138.37% (Figure 4a,b). It is
well-known that p16 and p21 act as a cell cycle-dependent kinase inhibitor. RT-PCR was
used to confirm these results via the analysis of the expression levels of senescent markers
(p16 and p21). The results shown in Figure 4c,d indicate that the mRNA expression of
p21 decreased concentration-dependently in the Iris-exosome-pretreated cells compared
to the H
2
O
2
-treated cells. These results suggest that Iris-exosomes protect nHEKs against
intracellular ROS production and cellular senescence induced by H2O2.
Appl. Sci. 2023, 13, x FOR PEER REVIEW 10 of 19
Figure 4. Anti-senescence effects of Iris-exosome against H2O2-induced cellular stress in nHEKs. (a)
Representative images of nHEKs assayed for senescence-associated β-galactosidase (SA-β-gal; blue)
(scale bar, 200 µm). nHEKs were treated with Iris-exosomes for 4 h and oxidative stress caused via
4 h treatment of 200 µM H2O2. The data were analyzed after 40 h incubation. (b) Senescent cells were
counted within the same cell number at least three times independently. The level of senescent cells
compared with total cells was visualized as a graph. (c) RT-PCR was performed as the previous
condition to analyze senescence-related genes (p16, p21, and p63). (d) The mRNA levels of the se-
nescence marker were analyzed via real-time qPCR. Each value was normalized to GAPDH and β-
actin as a loading control. The result is expressed as the average of the values quantified by GAPDH
and β-actin. *** p < 0.001 compared with untreated cells; ## p < 0.01 and ### p < 0.001 compared with
H2O2-treated cells.
3.5. Iris-Exosome Inhibits H2O2-Induced Cellular Oxidative Stress via p38 Mitogen-Activated
Protein Kinase (MAPK) Signaling Pathway
Regulation of p21, a senescence marker and cell cycle-dependent kinase inhibitor, is
mediated by p53 in keratinocytes. Therefore, considering the above results, we examined
the upstream regulators responsible for p21 regulation. Under the same conditions as the
previous experiments, immunobloing was used to evaluate the expression of p53 and p-
p53 (ser15). Similar to the previous RT-qPCR, the expression of p16 and Lamin B1, which
are known to be cellular senescence markers, did not change (Figure 5a). Similar to p21,
H2O2 treatment upregulated the expression of p53 and p-p53 (Figure 5a). Additionally, the
expression of p21, p53, and p-p53 was downregulated by H2O2 after pretreatment with
Iris-exosomes in a concentration-dependent manner (Figure 5a). According to these find-
ings, the changes in p21 caused by H2O2 and the Iris-exosome treatment resulted from p53
action. Thus, we determined that the MAPK signaling pathway was involved in the nHEK
treatment with H2O2 and Iris-exosomes. H2O2 significantly increased the phosphorylation
levels of p38, ERK, and JNK in the nHEKs (Figure 5b). However, the Iris-exosomes mark-
edly inhibited the H2O2-induced phosphorylation of Threonine180 and Tyrosine182 resi-
dues in p38 (Figure 5b). Similarly, the phosphorylation of JNK at threonine 183 and tyro-
sine 185 was also reduced by the Iris-exosomes (Figure 5b). However, the Iris-exosome
treatment did not inhibit the ERK activation induced by the H2O2 treatment (Figure 5b).
These results indicate the possibility that the Iris-exosomes regulated H2O2-induced cellu-
lar senescence responses by decreasing the p38- and JNK-mediated p53–p21 signaling
Figure 4.
Anti-senescence effects of Iris-exosome against H
2
O
2
-induced cellular stress in nHEKs.
(
a
) Representative images of nHEKs assayed for senescence-associated
β
-galactosidase (SA-
β
-gal;
blue) (scale bar, 200
µ
m). nHEKs were treated with Iris-exosomes for 4 h and oxidative stress caused
via 4 h treatment of 200
µ
M H
2
O
2
. The data were analyzed after 40 h incubation. (
b
) Senescent
cells were counted within the same cell number at least three times independently. The level of
senescent cells compared with total cells was visualized as a graph. (
c
) RT-PCR was performed as the
previous condition to analyze senescence-related genes (p16,p21, and p63). (
d
) The mRNA levels of
the senescence marker were analyzed via real-time qPCR. Each value was normalized to GAPDH and
β
-actin as a loading control. The result is expressed as the average of the values quantified by GAPDH
and
β
-actin. *** p< 0.001 compared with untreated cells; ## p< 0.01 and ### p< 0.001 compared with
H2O2-treated cells.
3.5. Iris-Exosome Inhibits H2O2-Induced Cellular Oxidative Stress via p38 Mitogen-Activated
Protein Kinase (MAPK) Signaling Pathway
Regulation of p21, a senescence marker and cell cycle-dependent kinase inhibitor, is
mediated by p53 in keratinocytes. Therefore, considering the above results, we examined the
upstream regulators responsible for p21 regulation. Under the same conditions as the previous
Appl. Sci. 2023,13, 11681 10 of 18
experiments, immunoblotting was used to evaluate the expression of p53 and p-p53 (ser15).
Similar to the previous RT-qPCR, the expression of p16 and Lamin B1, which are known to
be cellular senescence markers, did not change (Figure 5a). Similar to p21, H
2
O
2
treatment
upregulated the expression of p53 and p-p53 (Figure 5a). Additionally, the expression of
p21, p53, and p-p53 was downregulated by H
2
O
2
after pretreatment with Iris-exosomes in
a concentration-dependent manner (Figure 5a). According to these findings, the changes
in p21 caused by H
2
O
2
and the Iris-exosome treatment resulted from p53 action. Thus, we
determined that the MAPK signaling pathway was involved in the nHEK treatment with
H
2
O
2
and Iris-exosomes. H
2
O
2
significantly increased the phosphorylation levels of p38,
ERK, and JNK in the nHEKs (Figure 5b). However, the Iris-exosomes markedly inhibited the
H
2
O
2
-induced phosphorylation of Threonine180 and Tyrosine182 residues in p38 (Figure 5b).
Similarly, the phosphorylation of JNK at threonine 183 and tyrosine 185 was also reduced by
the Iris-exosomes (Figure 5b). However, the Iris-exosome treatment did not inhibit the ERK
activation induced by the H
2
O
2
treatment (Figure 5b). These results indicate the possibility
that the Iris-exosomes regulated H
2
O
2
-induced cellular senescence responses by decreasing
the p38- and JNK-mediated p53–p21 signaling pathways. Altogether, these results indicate
that Iris-exosomes may regulate H
2
O
2
-induced cellular senescence responses by decreasing
JNK- and p38-mediated p53–p21 signaling.
Appl. Sci. 2023, 13, x FOR PEER REVIEW 11 of 19
pathways. Altogether, these results indicate that Iris-exosomes may regulate H
2
O
2
-in-
duced cellular senescence responses by decreasing JNK- and p38-mediated p53–p21 sig-
naling.
Figure 5. Anti-senescence effect of Iris-exosome is associated with p38 and JNK phosphorylation.
(a) Protein levels of senescence markers (p16, p21, p53, p-p53(S15), and Lamin B1) analyzed using
Western blot. (b) The protein levels of MAPKinase (ERK, JNK, p38) were assessed using Western
blot. β-actin was used as a loading control. The results are presented as mean ± SD (n = 3). *** p <
0.001 compared with untreated cells; # p < 0.05, ## p < 0.01 and ### p < 0.001 compared with H
2
O
2
-
treated cells.
3.6. Iris-Exosome Improves Oxidative-Stress-Induced Impairment of Keratinocyte
Barrier Function
Keratinocytes play important roles in epidermal differentiation and skin regenera-
tion. Oxidative stress induced by hydrogen peroxide causes premature keratinocyte se-
nescence and abnormal terminal differentiation [25,27]. According to our findings, Iris-
exosomes protect nHEKs from oxidative stress and cellular damage. Therefore, we tested
whether Iris-exosome exerts protective effects against H
2
O
2
-mediated cell dysfunction
during differentiation and regeneration. First, to determine whether Iris-exosomes could
restore the abnormal differentiation caused by H
2
O
2
treatment in nHEKs, Western bloing
was performed to target differentiation markers in nHEKs treated with CaCl
2
(1.2 mM)
for 12 days. In the presence of CaCl
2
, a significant increase in the expression of the inter-
mediate differentiation markers K1, K10, and LOR was observed on days 8 and 12 after
treatment with CaCl
2
(Figure 6a). Interestingly, compared to the control cells (CaCl
2
-
treated only), we found that the H
2
O
2
-treated group showed early signs of upregulated
intermediate differentiation markers, K1, K10, and LOR proteins, on day 3 after treatment
with CaCl
2
(Figure 6a). Iris-exosomes, however, aenuated the early increase in the ex-
pression levels of the differentiation markers caused by H
2
O
2
to levels similar to those of
the control group (Figure 6a). Then, we tested the protective effects of exosomes against
H
2
O
2
-induced cellular damage using a wound-healing assay. We treated nHEKs with Iris-
exosomes for 4 h, treated them with 200 µM H
2
O
2
for 4 h, and then scratched them with a
sterile pipee tip. The change in the gap width after 48 h was used to determine wound-
Figure 5.
Anti-senescence effect of Iris-exosome is associated with p38 and JNK phosphorylation.
(
a
) Protein levels of senescence markers (p16, p21, p53, p-p53(S15), and Lamin B1) analyzed using Western
blot. (
b
) The protein levels of MAPKinase (ERK, JNK, p38) were assessed using Western blot.
β
-actin was
used as a loading control. The results are presented as mean
±
SD (n= 3). *** p< 0.001 compared with
untreated cells; # p< 0.05, ## p< 0.01 and ### p< 0.001 compared with H2O2-treated cells.
3.6. Iris-Exosome Improves Oxidative-Stress-Induced Impairment of Keratinocyte Barrier Function
Keratinocytes play important roles in epidermal differentiation and skin regeneration.
Oxidative stress induced by hydrogen peroxide causes premature keratinocyte senescence
and abnormal terminal differentiation [
25
,
27
]. According to our findings, Iris-exosomes
protect nHEKs from oxidative stress and cellular damage. Therefore, we tested whether
Iris-exosome exerts protective effects against H
2
O
2
-mediated cell dysfunction during dif-
ferentiation and regeneration. First, to determine whether Iris-exosomes could restore
Appl. Sci. 2023,13, 11681 11 of 18
the abnormal differentiation caused by H
2
O
2
treatment in nHEKs, Western blotting was
performed to target differentiation markers in nHEKs treated with CaCl
2
(1.2 mM) for
12 days. In the presence of CaCl
2
, a significant increase in the expression of the intermediate
differentiation markers K1, K10, and LOR was observed on days 8 and 12 after treatment
with CaCl
2
(Figure 6a). Interestingly, compared to the control cells (CaCl
2
-treated only),
we found that the H
2
O
2
-treated group showed early signs of upregulated intermediate
differentiation markers, K1, K10, and LOR proteins, on day 3 after treatment with CaCl
2
(Figure 6a). Iris-exosomes, however, attenuated the early increase in the expression levels
of the differentiation markers caused by H
2
O
2
to levels similar to those of the control
group (Figure 6a). Then, we tested the protective effects of exosomes against H
2
O
2
-induced
cellular damage using a wound-healing assay. We treated nHEKs with Iris-exosomes for
4 h, treated them with 200
µ
M H
2
O
2
for 4 h, and then scratched them with a sterile pipette
tip. The change in the gap width after 48 h was used to determine wound-healing abil-
ity. The results show that groups treated with Iris-exosomes had higher scratch closure
rates than those treated with H
2
O
2
alone (Figure 6b). Altogether, these results indicate
that Iris-exosome is a bioactive component found in Iris germanica L. rhizome, displays
limited cytotoxicity, and exhibits a protective effect on oxidative-stress-induced cellular
dysfunction in nHEKs.
Appl. Sci. 2023, 13, x FOR PEER REVIEW 12 of 19
healing ability. The results show that groups treated with Iris-exosomes had higher
scratch closure rates than those treated with H2O2 alone (Figure 6b). Altogether, these re-
sults indicate that Iris-exosome is a bioactive component found in Iris germanica L. rhi-
zome, displays limited cytotoxicity, and exhibits a protective effect on oxidative-stress-
induced cellular dysfunction in nHEKs.
Figure 6. Protective effects of Iris-exosome on H2O2-induced impairment of keratinocyte barrier
function in nHEKs. (a) Protein levels of differentiation markers (K1, K10, and LOR) were assessed
using Western blot. nHEKs were treated with Iris-exosomes for 4 h and with 200 µM H2O2 for 4 h.
They were then incubated for 0, 4, 8, 12 days in the medium including 1.2 mM CaCl2. (b) Evaluation
of scratch wound healing. Cells were treated using Iris-exosomes and/or 200 µM H2O2 for each 4 h
and then scratched with a sterile pipee tip after cell confluence. The results were analyzed using a
microscope and the ImageJ software. Data are expressed as the mean ± SD (n = 3). ** p < 0.01 and ***
p < 0.001 compared with untreated cells.
4. Discussion
This study shows that exosomes from the rhizomes of Iris germanica L. suppress oxi-
dative stress in keratinocytes. Interestingly, the exosomes were not very effective at scav-
enging radicals (Figure 3). Nevertheless, cell viability loss caused by H2O2 treatment was
suppressed via treatment with exosomes, suggesting that they are responsible for remov-
ing ROS from keratinocytes. The results of the WST-1 and DCF-DA assays show that ex-
osome treatment significantly reduced intracellular ROS generation caused by H2O2 (Fig-
ures 2 and 3). Thus, Iris-exosomes are considered to indirectly improve intracellular anti-
oxidant function rather than provide benefits directly.
It is possible to analyze the purity of human-derived exosomes by comparing mem-
brane protein content before and after separation using membrane proteins such as CD81,
Figure 6.
Protective effects of Iris-exosome on H
2
O
2
-induced impairment of keratinocyte barrier function
in nHEKs. (
a
) Protein levels of differentiation markers (K1, K10, and LOR) were assessed using Western
blot. nHEKs were treated with Iris-exosomes for 4 h and with 200
µ
M H
2
O
2
for 4 h. They were then
incubated for 0, 4, 8, 12 days in the medium including 1.2 mM CaCl
2
. (
b
) Evaluation of scratch wound
healing. Cells were treated using Iris-exosomes and/or 200
µ
M H
2
O
2
for each 4 h and then scratched
with a sterile pipette tip after cell confluence. The results were analyzed using a microscope and the
ImageJ software. Data are expressed as the mean
±
SD (n= 3). ** p< 0.01 and *** p< 0.001 compared
with untreated cells.
Appl. Sci. 2023,13, 11681 12 of 18
4. Discussion
This study shows that exosomes from the rhizomes of Iris germanica L. suppress
oxidative stress in keratinocytes. Interestingly, the exosomes were not very effective at
scavenging radicals (Figure 3). Nevertheless, cell viability loss caused by H
2
O
2
treatment
was suppressed via treatment with exosomes, suggesting that they are responsible for
removing ROS from keratinocytes. The results of the WST-1 and DCF-DA assays show
that exosome treatment significantly reduced intracellular ROS generation caused by H
2
O
2
(Figures 2and 3). Thus, Iris-exosomes are considered to indirectly improve intracellular
antioxidant function rather than provide benefits directly.
It is possible to analyze the purity of human-derived exosomes by comparing mem-
brane protein content before and after separation using membrane proteins such as CD81,
CD63, and CD9. However, in the case of plant exosomes, no clearly identifiable membrane
proteins have yet been identified; even in the case of some membrane proteins that have
been identified, significant differences are expected among species. Our group, therefore,
conducted additional follow-up studies to overcome this limitation (phospholipid material
analysis, unique marker membrane protein analysis, etc.). As a quantitative method, NTA
was used to verify plant exosomes, while oil red O and cryo-TEM were used as qualitative
methods. In the future, more sophisticated analytical methods should be developed to
identify the characteristics of plant-based exosomes in detail.
The results of our study indicate that p21–p53 molecules play a more important role in
protecting nHEK cells from H
2
O
2
-induced cellular senescence than p16 molecules. ROS pro-
duction is closely associated with cellular senescence. Antioxidant enzymes, such as CAT
and SOD, can alleviate cellular senescence by regulating intracellular ROS levels [
67
]. Be-
cause Iris-exosomes upregulate antioxidant enzymes, we examined whether Iris-exosomes
can counteract cellular senescence in H
2
O
2
-induced nHEKs. Treatment with Iris-exosomes
reduced the activity of SA-
β
-gal, a marker of senescence (Figure 4). Furthermore, the
expression of p21 mRNA decreased in nHEKs treated with Iris-exosomes rather than just
H
2
O
2
(Figure 4). Moreover, H
2
O
2
treatment increased the protein levels of p21, p53, and
p-p53, whereas Iris-exosome treatment decreased their levels in a concentration-dependent
manner (Figure 5). These results indicate that Iris-exosome inhibits cellular senescence
caused by H2O2via the activation of the p53–p21 pathway. Another interesting finding is
that the expression of p16 and Lamin B1, other known senescence markers, did not change.
The relationship between p16 expression and cellular senescence has been previously re-
ported in many research papers. With age, the epidermis and dermis accumulate p16-
and SA-
β
-gal-positive cells [
68
–
74
]. Sasaki et al. showed that ROS-induced senescence
may be triggered by the demethylation of the promoter region of p16 in normal epidermal
keratinocytes [
75
]. These findings indicate that p16 is a marker of cellular senescence in
keratinocytes. However, other studies have suggested that p16 is not a reliable senescence
marker in keratinocytes. From the
in vitro
study of skin equivalents, different investigators
have reported different results for p16 expression. Some have observed an inverse corre-
lation with the expression of Ki-67, a proliferation marker in skin biopsy, whereas others
have reported no change or an increase [
76
,
77
]. In addition, nHEKs treated with H
2
O
2
and/or Iris-exosomes did not exhibit any changes in p16 mRNA or protein levels.
A further investigation of the putative mechanism underlying Iris-exosome’s pro-
tective effect against oxidative stress revealed that the exosome inhibits p53 activation
by inhibiting the expression of p-p38 and p-JNK proteins, which are increased by H
2
O
2
,
thus reducing the expression of p21. MAPKs (ERK1/2, JNK, and p38) are activated by a
variety of stimuli, such as heat shock and hydrogen peroxide [
78
–
81
]. While these MAPK
proteins are activated by ROS produced via UV irradiation or H
2
O
2
addition, JNK and p38
appear to be more sensitive to ROS than ERK1/2. It has been reported that excessive ROS
production stimulates JNK activity, resulting in pathological conditions such as arthritis
and cancer [
79
]. Additionally, various human cell lines have shown that ROS activates
c-Jun N-terminal kinase (JNK) and/or p38 [
82
–
85
]. In human keratinocytes, the same cell
lines act independently in response to ROS production. These findings indicate that ROS-
Appl. Sci. 2023,13, 11681 13 of 18
mediated MAPKs are important mediators of ROS in the regulation of cellular function
and the fate of keratinocytes. Additionally, MAPKs have been reported to regulate p53–p21
pathways upstream. According to one study, oxidative stress induced by H
2
O
2
activates
both the p53/p21/Rb and p38MAPK/MAPKAPK-2 pathways involved in the premature
senescence of human endometrium-derived mesenchymal stem cells (hMESC) [
86
]. In
other studies, p53 was shown to interact with MAPKs (ERK1/2, JNK, and p38) and phos-
phorylate and activate p53 in response to stress [
87
–
90
]. According to these results, the
MAPK–p53 signaling pathway contributes to the oxidative stress response of keratinocytes.
Therefore, we examined how exosomes downregulate these senescence marker proteins.
H
2
O
2
treatment increased the phosphorylation of p38 and JNK, whereas Iris-exosomes
decreased this phosphorylation in a dose-dependent manner. In line with previous studies,
oxidative stress was found to be associated with p38 and JNK mitogen-activated protein
kinases (MAPKs). Consequently, it can be stated that Iris-exosome inhibits p38 and JNK
protein expression, thus preventing p53 activation.
As keratinocytes differentiate, the following four layers of structure form in the epi-
dermis: the basal layer, spinous layer, granular layer, and stratum corneum. They play
crucial roles in maintaining the skin barrier [
24
,
25
]. Therefore, it is important to main-
tain a normal level of keratinocyte differentiation if one is to sustain a healthy epidermal
layer. Studies have shown that ROS abnormally promotes keratinocyte differentiation,
resulting in an incomplete epidermal layer [
25
]. Additionally, UV irradiation accumulates
ROS levels in nHEKs and improves SA-
β
-gal activity, and 3D-reconstructed epidermis
from UV-irradiated nHEKs shows thinner cross-sectional structures and more extensively
differentiated cells [
76
]. These results indicate that oxidative stress disrupts the balance
between the proliferation and differentiation of keratinocytes, resulting in the degener-
ation of most cells into aged and differentiated cells, as well as the breakdown of the
skin barrier and a thinner epidermis. In this study, we found that the Iris-exosome offers
recovery from intracellular ROS and investigated whether it protects keratinocytes from
oxidative-stress-induced degeneration (differentiation). In our study, early-stage H
2
O
2
treatment resulted in significantly increased levels of intermediate differentiation markers,
K1, K10, and LOR (3 days after CaCl
2
treatment). However, the changes in the levels of the
differentiation markers were close to the control levels in the nHEKs that had undergone
pretreatment with Iris-exosomes. These findings suggest that Iris-exosome may exhibit a
potential protective effect against H
2
O
2
-induced degeneration of nHEKs. In the future, it is
expected that a 3D-reconstructed epidermis model can be used to observe the abnormal
differentiation caused by H2O2in the epidermis.
During wound healing, normal keratinocyte function is important for keratinocyte
migration and re-epithelialization [
91
]. Low concentrations of H
2
O
2
can facilitate wound
healing, but excessive ROS levels are detrimental [
92
–
94
]. We tested whether Iris-exosome
could protect keratinocytes from wound-healing damage caused by H
2
O
2
using wound-
healing assays. Therefore, the scratch on the nHEKs treated with Iris-exosomes closed more
than that on the group treated with H
2
O
2
only. These results suggest that Iris-exosome
treatment may eventually restore wound-healing function, which is impaired by oxidative
damage. Overall, it is concluded that Iris-exosomes are bioactive components found in the
rhizome of Iris germanica L., have relatively low cytotoxicity, and protect nHEKs against
oxidative-stress-induced cellular dysfunction.
5. Conclusions
Our study shows that Iris-exosomes have antioxidant and anti-senescence properties.
Iris-exosomes also possess the potential to reduce oxidative stress and cellular dysfunction
caused by H
2
O
2
. As shown in Figure 7,Iris-exosomes upregulate the levels of antioxidant
enzymes, such as HO-1,CAT,GSS,GPx1, and SOD, which, in turn, regulate intracellular
ROS levels. Moreover, Iris-exosomes mitigate oxidative stress induced by H
2
O
2
, leading to
enhanced wound-healing activity and normal differentiation in skin cells. Iris-exosomes
also regulate the p38/JNK MAPK pathway to reduce the levels of p53 and p21, thereby
Appl. Sci. 2023,13, 11681 14 of 18
effectively inhibiting cellular senescence. These results may lead to improved skin health
and antiaging effects. Iris-exosomes also regulate differentiation markers and accelerate the
re-epithelialization of wounds. Therefore, further investigation via human clinical trials is
required in order to verify the efficacy of these methods in facilitating skin regeneration.
Appl. Sci. 2023, 13, x FOR PEER REVIEW 15 of 19
Figure 7. In nHEKs, ROS produced by H2O2 activate the intrinsic pathway of oxidative stress. The
Iris-exosomes increase the levels of antioxidant enzymes, such as HO-1, CAT, GSS, GPx1, and SOD,
which regulate the levels of intracellular ROS. Furthermore, increased wound-healing activity and
normal differentiation of skin cells are associated with Iris-exosome treatment, which mitigates ox-
idative stress induced by H2O2. Additionally, Iris-exosomes inhibit cellular senescence via modulat-
ing the p38/JNK MAPK pathway, resulting in reducing the levels of p53 and p21.
Supplementary Materials: The following supporting information can be downloaded at:
www.mdpi.com/xxx/s1, Figure S1: Protective effect of Iris-exosome on cell viability against H2O2-
induced cytotoxicity in human dermal fibroblast (nHDFs). (a) Cell viabilities pretreated with Iris-
exosomes for 24 h and examined by WST-1 assay. (b) Cell viability of nHDFs with H2O2 for 24 h. (c)
nHDFs were treated with 300 µM H2O2 for 24 h after Iris-exosomes for 24 h and cell viability was
measured by MTT assay. Values are expressed as mean±SD of three independent experiments. ** p
< 0.01 and *** p < 0.001 compared with untreated cells; ### p < 0.001 compared with H2O2-treated
cells.
Author Contributions: Conceptualization, J.-S.K., H.-J.L., H.L., Y.J., Y.K., S.-J.P., J.K. and S.B.; meth-
odology, J.-S.K., H.-J.L. and S.B.; software, J.-S.K. and H.-J.L.; validation, J.-S.K., H.-J.L. and S.B.;
formal analysis, J.-S.K., H.-J.L., E.-J.Y., H.L., Y.J. and Y.K.; investigation, J.-S.K., H.-J.L. and E.-J.Y.;
resources, J.-S.K., H.L., S.-J.P. and S.B.; data curation, J.-S.K., H.-J.L. and S.B.; writing—original draft
preparation, J.-S.K. and S.B.; writing—review and editing, J.-S.K., Y.J., Y.K., J.K. and S.B.; visualiza-
tion, J.-S.K. and S.B.; supervision, S.B.; project administration, S.B.; funding acquisition, S.B. All au-
thors have read and agreed to the published version of the manuscript.
Funding: This work was supported by the development of new products subject to purchase con-
ditions (S3303942) funded by the Ministry of SMEs and Startups (MSS, Korea).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: The authors are grateful to the Department of Cosmetics Engineering, Konkuk
University, for supporting the use of their research facilities.
Conflicts of Interest: The authors declare no conflict of interest.
Figure 7.
In nHEKs, ROS produced by H
2
O
2
activate the intrinsic pathway of oxidative stress. The
Iris-exosomes increase the levels of antioxidant enzymes, such as HO-1,CAT,GSS,GPx1, and SOD,
which regulate the levels of intracellular ROS. Furthermore, increased wound-healing activity and
normal differentiation of skin cells are associated with Iris-exosome treatment, which mitigates oxidative
stress induced by H
2
O
2
. Additionally, Iris-exosomes inhibit cellular senescence via modulating the
p38/JNK MAPK pathway, resulting in reducing the levels of p53 and p21.
Supplementary Materials:
The following supporting information can be downloaded at:
https://www.mdpi.com/article/10.3390/app132111681/s1, Figure S1: Protective effect of Iris-
exosome on cell viability against H
2
O
2
-induced cytotoxicity in human dermal fibroblast (nHDFs). (a)
Cell viabilities pretreated with Iris-exosomes for 24 h and examined by WST-1 assay. (b) Cell viability
of nHDFs with H
2
O
2
for 24 h. (c) nHDFs were treated with 300
µ
M H
2
O
2
for 24 h after Iris-exosomes
for 24 h and cell viability was measured by MTT assay. Values are expressed as mean
±
SD of three
independent experiments. ** p< 0.01 and *** p< 0.001 compared with untreated cells; ### p< 0.001
compared with H2O2-treated cells.
Author Contributions:
Conceptualization, J.-S.K., H.-J.L., H.L., Y.J., Y.K., S.-J.P., J.K. and S.B.; method-
ology, J.-S.K., H.-J.L. and S.B.; software, J.-S.K. and H.-J.L.; validation, J.-S.K., H.-J.L. and S.B.; formal
analysis, J.-S.K., H.-J.L., E.-J.Y., H.L., Y.J. and Y.K.; investigation, J.-S.K., H.-J.L. and E.-J.Y.; resources,
J.-S.K., H.L., S.-J.P. and S.B.; data curation, J.-S.K., H.-J.L. and S.B.; writing—original draft preparation,
J.-S.K. and S.B.; writing—review and editing, J.-S.K., Y.J., Y.K., J.K. and S.B.; visualization, J.-S.K. and
S.B.; supervision, S.B.; project administration, S.B.; funding acquisition, S.B. All authors have read
and agreed to the published version of the manuscript.
Funding:
This work was supported by the development of new products subject to purchase condi-
tions (S3303942) funded by the Ministry of SMEs and Startups (MSS, Korea).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments:
The authors are grateful to the Department of Cosmetics Engineering, Konkuk
University, for supporting the use of their research facilities.
Conflicts of Interest: The authors declare no conflict of interest.
Appl. Sci. 2023,13, 11681 15 of 18
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