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Spatholobus suberectus Dunn Water Extract Ameliorates Atopic Dermatitis–Like Symptoms by Suppressing Proinflammatory Chemokine Production In Vivo and In Vitro

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S. patholobus suberectus Dunn, a traditional Chinese herbal medicine, has various pharmacological activities, such as anti-inflammatory properties. However, to the best of our knowledge, its therapeutic effect on atopic dermatitis (AD) has not been investigated. In this study, we explored the effect of S. suberectus Dunn water extract (SSWex) on AD in vivo and in vitro. In Dermatophagoides farina extract (DfE)–treated NC/Nga mice, the oral administration of SSWex alleviated AD-like symptoms, such as ear thickness, dermatitis score, epidermal thickness, immune cell infiltration, and levels of AD-related serum parameters (immunoglobulin E, histamine, and proinflammatory chemokines). In HaCaT cells, the production of proinflammatory chemokines induced by interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) was inhibited by SSWex pretreatment. SSWex treatment inhibited the phosphorylation of mitogen-activated protein kinase and activation and translocation of transcriptional factors, such as signal transducer and activator of transcription 1 and nuclear factor kappa B in IFN-γ/TNF-α–stimulated HaCaT cells. These results indicate that SSWex may be developed as an efficient therapeutic agent for AD.
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Spatholobus suberectus Dunn Water
Extract Ameliorates Atopic
DermatitisLike Symptoms by
Suppressing Proinammatory
Chemokine Production In Vivo and In
Vitro
Hyun-Kyung Song
1
, Sun Haeng Park
1
, Hye Jin Kim
1
,
2
, Seol Jang
1
and Taesoo Kim
1
*
1
Herbal Medicine Research Division, Korea Institute of Oriental Medicine, Daejeon, South Korea,
2
College of Pharmacy, Seoul
National University, Seoul, South Korea
S. patholobus suberectus Dunn, a traditional Chinese herbal medicine, has various
pharmacological activities, such as anti-inammatory properties. However, to the best
of our knowledge, its therapeutic effect on atopic dermatitis (AD) has not been
investigated. In this study, we explored the effect of S. suberectus Dunn water extract
(SSWex) on AD in vivo and in vitro.InDermatophagoides farina extract (DfE)treated NC/
Nga mice, the oral administration of SSWex alleviated AD-like symptoms, such as ear
thickness, dermatitis score, epidermal thickness, immune cell inltration, and levels of AD-
related serum parameters (immunoglobulin E, histamine, and proinammatory
chemokines). In HaCaT cells, the production of proinammatory chemokines induced
by interferon-γ(IFN-γ) and tumor necrosis factor-α(TNF-α) was inhibited by SSWex
pretreatment. SSWex treatment inhibited the phosphorylation of mitogen-activated protein
kinase and activation and translocation of transcriptional factors, such as signal transducer
and activator of transcription 1 and nuclear factor kappa B in IFN-γ/TNF-αstimulated
HaCaT cells. These results indicate that SSWex may be developed as an efcient
therapeutic agent for AD.
Keywords: Spatholobus suberectus DUNN, atopic dermatitis, NC/Nga mice, HaCaT cells, proinammatory
chemokines
INTRODUCTION
Atopic dermatitis (AD) is a chronic and allergic inammatory skin disease caused by various
environmental factors, including mite dust, smoking, and allergens. It is characterized by skin
hypersensitivity, itching, eczema, erythema, and relapsed skin lesions (Langan et al., 2020). The
prevalence of AD in adults globally, in 2018, ranged from 2.1% to 4.9% and is still increasing
(Barbarot et al., 2018). Moreover, AD is the initial stage of the atopic march,leading to food allergy,
asthma, and allergic rhinitis, which negatively affect the quality of life of patients (Spergel and Paller,
2003). In AD, levels of immunoglobulin (Ig) E, histamine, and several proinammatory mediators,
such as cytokines and chemokines, and inltration of inammatory cells, including mast cells and
T cells, are elevated in the serum (Mansouri and Guttman-Yassky, 2015).
Edited by:
Jian Gao,
Shanghai Childrens Medical Center,
China
Reviewed by:
Guanghai Yan,
Yanbian University Medical College,
China
Shamshad Alam,
University at Buffalo, United States
*Correspondence:
Taesoo Kim
xotn91@kiom.re.kr
Specialty section:
This article was submitted to
Inammation Pharmacology,
a section of the journal
Frontiers in Pharmacology
Received: 13 April 2022
Accepted: 16 May 2022
Published: 20 June 2022
Citation:
Song H-K, Park SH, Kim HJ, Jang S
and Kim T (2022) Spatholobus
suberectus Dunn Water Extract
Ameliorates Atopic DermatitisLike
Symptoms by Suppressing
Proinammatory Chemokine
Production In Vivo and In Vitro.
Front. Pharmacol. 13:919230.
doi: 10.3389/fphar.2022.919230
Frontiers in Pharmacology | www.frontiersin.org June 2022 | Volume 13 | Article 9192301
ORIGINAL RESEARCH
published: 20 June 2022
doi: 10.3389/fphar.2022.919230
NC/Nga mice are the most extensively studied animal models
for AD, and they exhibit symptoms similar to those of human
patients with AD, for example, elevated serum levels of
inammatory factors and the inltration of immune cells into
AD-like skin lesions (Matsuda et al., 1997). These symptoms are
induced by several environmental allergens. Among them, D.
farina mites in house dust are the most common allergens that
cause asthma, allergic rhinitis, and AD (Platts-Mills and
Chapman, 1987;Holm et al., 1999). Repeated exposure of NC/
Nga mice to the D. farina extract (DfE) effectively induces AD-
like skin lesions in the skin when the mice are maintained under
specic pathogen-free conditions (Oshio et al., 2009).
Keratinocytes are the major form of epidermal cells and
maintain skin homeostasis through regulation of immune cell
recruitment via proinammatory chemokine production
(Albanesi et al., 2005). Several studies have reported that
interferon-γ(IFN-γ) and tumor necrosis factor-α(TNF-α)
activate inammatory reactions in keratinocytes (Ju et al.,
2009;Yang et al., 2018;Song et al., 2021). These keratinocytes
produce proinammatory chemokines, such as interleukin (IL)-
8, monocyte chemoattractant protein-1 (MCP-1), thymus and
activation-regulated chemokines (TARC), macrophage-derived
chemokines (MDCs), and regulated on activated normal T-cell
expressed and secreted (RANTES) (Werfel, 2009;Yang et al.,
2013;Cha et al., 2019). Thus, controlling the production of these
proinammatory chemokines in keratinocytes can contribute to
the treatment of inammatory skin in AD.
Corticosteroids, emollients, and antihistamines are commonly
used in AD. However, the long-term use of these drugs can have
serious side effects (Waljee et al., 2017). Therefore, alternative
medicines, including herbal extracts, have been considered in the
development of novel treatment agents for AD. S. suberectus Dunn is
a traditional Chinese herbal medicine and is used to treat anemia,
rheumatism, and menoxenia (Qin et al., 2019). It has various
pharmacological activities, including antitumor (Wang et al.,
2011), antifungal, and antibacterial effects (Zhang et al., 2013). In
addition, it has been reported to have anti-inammatory, antioxidant,
and antirheumatic effects (Li et al., 2003;Ravipati et al., 2012;Ha et al.,
2013). However, to the best of our knowledge, the effects of S.
suberectus Dunn water extract (SSWex) on AD skin lesions have not
been studied. Therefore, in this study, we hypothesized that SSWex
could have anti-atopic and anti-inammatory effects on AD-like
symptoms in vivo and in vitro. We investigated whether SSWex could
alleviate AD-related symptoms on the skin and inammatory
reactions in the DfE-treated NC/Nga mouse model and IFN-γ/
TNF-αstimulated HaCaT cells.
MATERIALS AND METHODS
Preparation of S. suberectus Dunn Water
Extract
SSWex was purchased from KOC Biotech (Daejeon, South
Korea). Dried S. suberectus Dunn (1 kg) was mixed with
distilled water in a 1:10 ratio (v/w) and reuxed at 100 ± 2°C
for 3 h. The extract was ltrated through a 53-μm mesh lter and
nally dried in a lyophilizer to obtain a freeze-dried powder. The
SSWex preparation procedure was performed as recommended
by KOC Biotech. SSWex powder was stored in the herbarium of
the Herbal Medicine Research Division at 20°C until further use.
For in vivo and in vitro experiments, the SSWex powder was
dissolved in water at a concentration of 100 mg/ml and then
diluted according to each experimental condition.
Mice and Induction of Atopic Dermatitis
Eight-week-old male NC/Nga mice were purchased from Central
Lab Animal Inc. (Seoul, South Korea). All mice were maintained
in a specic pathogen-free environment at the Experimental
Animal Center of Korea Institute of Oriental Medicine
(KIOM). All animal experiments were performed in
accordance with the standard of the Institutional Animal Care
and Use Committee at the KIOM (approval No. 20-069). The
mice were provided with ad libitum tap water and a standard
laboratory diet (Purina 38057, Cargill Agri Purina Inc., Sungnam,
South Korea). The mice were allowed to acclimatize for 1 week
and further divided randomly into experimental groups (six
mice/cage). The day before the start of AD induction, the fur
on the back of all the mice was shaved. Thereafter, 150 μlof4%
sodium dodecyl sulfate (SDS; Sigma-Aldrich, St. Louis, MO,
United States) was sprayed onto an area of over 4 cm
2
(2 cm
2
×2cm
2
) of the dorsal skin and ear surface for disruption of skin
barrier. After 1 h, 100 mg of DfE (Biostir Inc., Kobe, Japan)
ointment was applied onto the same areas (Pal et al., 2016;
Park et al., 2020). The mice were treated with DfE twice a
week for 3 weeks (Figure 1). Furthermore, 7 days after the rst
DfE application, SSWex (30, 100, and 300 mg/kg) or
dexamethasone (Dexa; 3 mg/kg; positive control) dissolved in
water was orally administered daily for 2 weeks. The NC/Nga
mice were randomly divided into four groups: normal
(untreated), DfE (DfE only), SSWex (DfE +3 0, 100, or
300 mg/kg SSWex), and Dexa (DfE + 3 mg/kg Dexa).
Measurement of Ear Thickness, Dermatitis
Score, and Body Weight
Ear thickness, dermatitis score, and body weight of NC/Nga mice
were recorded twice a week. A digital caliper (CAS
©
, Seoul, South
FIGURE 1 | Experimental scheme for the Dermatophagoides farinae
extract (DfE)treated NC/Nga mouse model. The mice were sensitized with
150 μl 4% sodium dodecyl sulfate (SDS) and 100 mg DfE ointment on their
shaved backs twice a week for 3 weeks. The S. suberectus Dunn water
extract (SSWex) or dexamethasone (Dexa) was administrated orally daily from
day 9 to day 22.
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Song et al. Spatholobus suberectus Dunn in AD
Korea) was used to evaluate the ear thickness. The individual
scores graded as 0 (no symptoms), 1 (mild), 2 (moderate), or 3
(severe) were calculated for each of the four symptoms (scarring/
dryness, erythema/hemorrhage, edema, and excoriation/erosion).
All dermatitis severity scores were quantied as the sum of all
individual scores. The body weights of the mice were measured
for monitoring.
Analysis of Serum Immunoglobulin E,
Histamine, and Proinammatory
Chemokines
The serum samples were collected from NC/Nga mice that were
killed on the last day of the experiment and then stored at 80°C
until analysis. The LBIS Mouse Immunoglobulin E (IgE) ELISA
Kit (Fujilm, Shibukawa, Japan) was used to determine the total
levels of IgE. Histamine levels were measured using Histamine
Research ELISA(LDN, Nordhorn, Germany). The serum levels
of total TARC, MDC, and RANTES were detected using mouse
ELISA kits from R&D Systems (Minneapolis, MN, United States).
These experiments were conducted according to the
manufacturers instructions.
Histopathological Analysis and
Immunohistochemistry
The ear and back skin tissues of the mice were removed, xed
with 10% formaldehyde, embedded in parafn, and serially
sectioned into 46-μm sections using a microtome (Leica
Biosystems, Newcastle, United Kingdom). The tissues were
mounted on slides and stained with hematoxylin/eosin
solution (Sigma-Aldrich) or toluidine blue (Toluidine Blue
Stain Kit, VitroVivo Biotech, Rockville, MA, United States).
For immunohistochemical staining, the slide-mounted tissues
were incubated overnight at 4°C with CD3 primary antibodies
(1:150 dilution; cat.no. ab16669, Abcam, Cambridge,
United Kingdom) and processed using a XT System
Benchmark autostainer (Ventana Medical System, Tucson, AZ,
United States) according to the manufacturers instruction. The
cells positive for CD3 and toluidine blue staining as measured
from the tissue sections were analyzed using Solution for
Automatic Bio-Image Analysis software (Ebiogen, Seoul, South
Korea).
Cells and Reagents
Human keratinocyte HaCaT cells were purchased from
Elabscience (Catalog No. EP-CL-0090, Houston, TX,
United States). The cells were cultured in a high-glucose-
containing Dulbeccos modied Eagles medium (Hyclone,
Logan, UT, United States) supplemented with 1% penicillin/
streptomycin (Gibco-BRL, Gaithersburg, MD, United States)
and heat-inactivated 10% fetal bovine serum (Gibco-BRL) in a
humidied incubator containing 5% CO
2
at 37°C. Recombinant
human IFN-γ, TNF-α, Alexa 594 goat anti-rabbit antibody
(cat.no. A-11037), Alexa 488 goat anti-rabbit antibody (cat.no.
A-11034), and Alexa 488 goat anti-mouse antibody (cat.no. A-
10680) were purchased from Thermo Fisher Scientic (Waltham,
MA, United States). DRAQ5, p-IκBα(cat.no. 2859), IκBα
(cat.no. 9242), p65 (cat.no. 8242), β-actin (cat.no. 3700),
p-JNK (cat.no. 9251), JNK (cat.no. 9252), p-ERK (cat.no.
9101), ERK (cat.no. 9102), p-p38 (cat.no. 9211), p38 (cat.no.
9212), p-STAT1 (Tyr) (cat.no. 9167), p-STAT1 (Ser) (cat.no.
9177), and STAT1 (cat.no. 9172) antibodies were purchased from
Cell Signaling Technology (Beverly, MA, United States). PCNA
(cat.no. sc-56), p50 (cat.no. sc-8414), secondary horseradish
peroxidase (HRP)conjugated anti-mouse (cat.no. sc-2357),
and anti-rabbit (cat.no. sc-516102) antibodies were purchased
from Santa Cruz Biotechnology (Santa Cruz, CA, United States).
Cell Cytotoxicity
Cell cytotoxicity was determined using the CellTiter 96
®
AQueous One Solution Cell Proliferation Assay kit (Promega,
Madison, WI, United States). HaCaT cells were allowed to attach
to 96-well plates overnight, treated with various concentrations of
SSWex (0500 μg/ml), and incubated for 24 h. Furthermore, a
prewarmed 20 μl assay reagent was added to each well, and the
cells were incubated at 37°C for 1 h. The absorbance was detected
at 490 nm using a Synergy HTX Multi-Mode Reader (BioTek,
Winooski, VT, United States).
Analysis of Secreted Proinammatory
Chemokines
The cells in the 96-well plate were preincubated with SSWex at
indicated concentrations (50, 100, and 300 μg/ml) for 1 h and
stimulated with 10 ng/ml of IFN-γ/TNF-αfor 24 h at 37°C. After
stimulation, the supernatant was collected and centrifuged at
4,000 rpm for 5 min to remove the particulate matter. The levels
of secreted proinammatory chemokines, such as MDC and
RANTES, in stimulated HaCaT cells were detected using human
ELISA kits (R&D Systems), in accordance with the manufacturers
instructions. The released TARC, MCP-1, and IL-8 were calculated
using the LEGENDplexHuman Proinammatory Chemokine
Panel (BioLegend, San Diego, CA, United States). The bead-based
immunoassay was performed according to the manufacturers
instructions. The stained-bead samples were detected using a BD
LSRFortessaFlow Cytometer (BD Biosciences, San Jose, CA,
United States) and analyzed using BD CellQuestsoftware. Data
were formalized using LEGENDplexSoftware v8.0 (VigeneTech
Inc., Carlisle, MA, United States).
Western Blot Analysis
HaCaT cells were pretreated with 300 μg/ml SSWex for 1 h and
incubated with 10 ng/ml IFN-γ/TNF-αat 37°C for the indicated
times (030 min). The cell lysates were prepared using ice-cold
radioimmunoprecipitation assay buffer (Biosesang, Seongnam,
South Korea) containing HaltProtease and Phosphatase
Inhibitor Cocktail (Thermo Fisher Scientic). The lysates were
centrifuged at 13,000 rpm for 10 min at 4°C. Quantication of the
collected proteins in supernatants was performed using the
PierceBCA assay kit (Thermo Fisher Scientic). Total
proteins (20 μg) were separated using 4%15% Mini-
PROTEAN TGX Precast Protein Gels (Bio-Rad, Hercules, CA,
United States) by electrophoresis and further transferred to
Frontiers in Pharmacology | www.frontiersin.org June 2022 | Volume 13 | Article 9192303
Song et al. Spatholobus suberectus Dunn in AD
Fluoro Trans
®
PVDF Membrane (Pall Corporation, Dreieich,
Germany). The membranes were blocked with 5% skim milk
(Sigma-Aldrich) or 3% bovine serum albumin (BSA; MP
Biomedicals, Irvine, CA, United States) in tris-buffered saline
with 1% tween-20 (TBST) for 2 h at 4°C, followed by overnight
incubation with primary antibodies (1:1,000 dilution) in blocking
solutions. After incubation with secondary antibodies
(horseradish peroxidaseconjugated anti-IgG) diluted in
blocking buffer at a ratio of 1:2500 for 1 h, the signals on the
membranes were developed using the Super Signal West Femto
Chemiluminescent Substrate (Thermo Fisher Scientic). Protein
detection was performed using the ChemiDoc Imaging System
(Bio-Rad).
Nuclear Fraction
HaCaT cells were pretreated with various concentrations of
SSWex for 1 h, followed by stimulation with 10 ng/ml IFN-γ/
TNF-αfor 1 h at 37°C. The stimulated cells were immediately
washed twice with ice-cold PBS and harvested. Nuclear proteins
were isolated using NE-PER
®
Nuclear and Cytoplasmic
Extraction Reagents (Pierce Biotechnology, Rockford, IL,
United States) according to the manufacturers protocol.
Immunouorescence Staining
HaCaT cells (6 × 10
5
/dish) were seeded in a 12-mm Nunc Glass Base
dish (Thermo Fisher Scientic). The cells were preincubated with
300 μg/ml of SSWex for 1 h, followed by stimulation with 10 ng/ml
IFN-γ/TNF-αfor 1 h at 37°C. The stimulated cells were washed twice
with PBS and xed in 3% paraformaldehyde (diluted with PBS) for
20 min at 4°C. The xed cells were washed four times with 0.1%
TritonX-100 buffer for 10 min, blocked with 3% BSA (diluted with
0.1% TritonX-100 buffer) for 1 h at room temperature, and
incubated with the primary antibodies (1:500) overnight at 4°C.
Theprimaryantibodiesusedherewerethesameasthoseusedin
Western blot analysis. Subsequently, the cells were incubated with
Alexa Fluor 594 or 488 anti-rabbit or mouse IgG secondary antibody
(1:500) for 2 h at 4°C. The nuclei were stained using DRAQ5in
blocking buffer. After incubation of 15 min, the cells were acquired
using an FV10i confocal microscope (Olympus, Tokyo, Japan).
High-Performance Liquid Chromatography
To perform simultaneous determination and quantitative analysis of
the four reference compounds [gallic acid, (+)-catechin, procyanidin
B2, and epicatechin] in SSWex, a Waters e2695 liquid
chromatography system (Waters Co., Milford, MA,
United States) equipped with a Waters 2998 photodiode array
detector was used. Empower software (version 3, Waters Co.)
was used for data acquisition and processing. The four
compounds were separated on a Phenomenex Luna C18 column
(250 mm × 4.6 mm; particle size 5 μm; Phenomenex, Torrance, CA,
United States) and detected at 280 nm. The mobile phase was 0.1%
aqueous acetic acid (A) and acetonitrile (B) in a gradient elution
mode. The gradient elution was set as follows: 010 min, 5%15% B;
1030 min, 15%20% B; and 3040 min, 20%30% B. After each
analysis, an 8-min wash with acetonitrile was performed, and a
further equilibration time of 8 min was observed to return to the
initial mobile phase composition. The ow rate of the mobile phase
was 1.0 ml/min, and the injection volume was 20 μl. SSWex was
dissolved in methanol (10 mg/ml) and ltered through a 0.2-μm
syringe lter to prepare the sample solutions for quantitative analysis
of the four compounds. The four reference compounds with purity >
98% were used for analysis. Gallic acid was purchased from
ChemFaces Biochemical (Wuhan, China), and all other
compounds [(+)-catechin, procyanidin B2, and epicatechin] were
obtained from Biopurify Phytochemicals (Chengdu, China).
Statistical Analyses
All results from more than three independent experiments are
indicated as the means ± standard error of mean (S.E.M.).
Statistical analyses were performed using GraphPad Prism
version 8.0 (GraphPad Software, San Diego, CA,
United States) by ordinary one-way analysis of variance.
Between-group comparisons were performed using Tukeys
post hoc test to calculate statistical signicance (p<0.05).
RESULTS AND DISCUSSION
S. suberectus Dunn Water Extract Inhibits
the Clinical Severity of Atopic
DermatitisLike Skin Symptoms and
Histological Features in D. farina
ExtractTreated NC/Nga Mice
To investigate the effects of SSWex on AD-like symptoms in NC/Nga
mice, DfE or Dexa was administrated twice a week for 3 weeks.
Pathological symptoms of AD, such as skin swelling, erythema,
cornication, exudation, dry skin, and increased ear thickness, were
observed in DfE-treated NC/Nga mice (Yang et al., 2013;Kang et al.,
2017). The application of SSWex signicantly relieved AD-like
symptoms, including ear thickness and dermatitis score, in DfE-
treated NC/Nga mice. No difference in body weight was observed
among the DfE-treated groups (Figures 2A,B). Dexa 3 mg/kg was used
as a positive control (Kim et al., 2009). Repeated application of DfE to
NC/Nga mice causes various symptoms of AD, such as epidermal
thickening of the skin (Kang et al., 2017).Theincreaseinepidermal
thickness was due to epidermal proliferation, which is pathologically
activated by the differentiation of keratinocytes in inammatory skin
lesions (Limandjaja et al., 2017). To histologically evaluate the effects of
SSWex on AD-like skin lesions, we performed skin histological analysis
using hematoxylin and eosin staining. Epidermis thickness increased in
the DfE group compared with that in the normal group; however,
SSWex treatment signicantly reduced the epidermis thickness in the
dorsal skin and ear tissue (Figures 2C,D). These results indicated that
SSWex may alleviate AD-like symptoms and histological features of
skin and ear lesions in DfE-treated NC/Nga mice.
S. suberectus Dunn Water Extract Reduces
the Inltration of Mast Cells and T Cells in
Skin Lesions in D. farina ExtractTreated
NC/Nga Mice
Patients with AD have increased inltration of immune cells,
such as mast and T cells, in AD skin lesions (Leung et al., 2004;
Frontiers in Pharmacology | www.frontiersin.org June 2022 | Volume 13 | Article 9192304
Song et al. Spatholobus suberectus Dunn in AD
Peng and Novak, 2015). Mast cells are innate immune cells that
are thought to be involved in allergic diseases, including AD; they
recognize specic antigens through high-afnity receptors for IgE
(FcεRI) (Hofmann and Abraham, 2009). Mast cells are believed to
be involved in the pathogenesis of AD through a wide range of
proinammatory mediators secreted from FcεRI-activated mast
cells, along with an elevation in IgE levels and the number of mast
cells (Kawakami et al., 2009). In addition, the sensory nerve
density of the epidermis and dermis increases in AD-like skin
lesions (Gupta and Harvima, 2018). Therefore, we examined the
number of mast cells in the skin and ear of DfE-treated NC/Nga
mice. Toluidine blue staining revealed that the number of mast
FIGURE 2 | Effect of SSWex on DfE-induced clinical and histopathological features of the AD-like skin lesions in NC/Nga mice. (A) Photographic images of the
backs of mice from each group on the last day of the experiment. (B) Body weight, ear thickness, and dermatitis scores were measured twice a week for 3 weeks. (C)
Skin and ear tissue sections were stained with hematoxylin and eosin (H&E). The stained sections were observed under a microscope at ×100 magnication. Scale bar =
100 μm. (D) Skin and ear epidermis thicknesses of the DfE-treated NC/Nga mice were analyzed and represented as a graph. Values are presented as the mean ±
S.E.M (n=6).
#
p<0.05,
##
p<0.005,
###
p<0.0005, and
####
p<0.0001 vs. the normal group; *p<0.05, **p<0.005, ***p<0.0005, and ****p<0.0001 vs. the DfE group.
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Song et al. Spatholobus suberectus Dunn in AD
cells increased in the DfE group compared with that in the normal
group, whereas SSWex administration reduced the number of
mast cells in AD-like lesions in the dorsal skin and ear (Figure 3).
In addition, to investigate the effect of SSWex on T-cell
inltration in AD-like skin lesions, we analyzed the number of
CD3
+
(T-cell marker) cells using immunohistochemistry. We
observed an increased number of CD3
+
cells in AD-like skin
lesions, consistent with previous studies (Park et al., 2012;Yang
et al., 2014a). SSWex treatment decreased T-cell inltration in
DfE-induced AD on the skin (Figure 3). AD is caused by an
imbalance between T-helper (Th) 1 and Th2 cells (Bieber, 2010).
Among the inltrating T cells, Th2 cells are one of the major cell
types involved in AD development (Purushothaman et al., 2018).
Although Th2-mediated responses are more prominent in the
acute AD phase, Th1-mediated responses are more prominent in
the chronic AD phase (Leung et al., 2004). Therefore, reducing
the inltration of mast and T cells in skin lesions in AD is
important. Our results demonstrated that SSWex treatment could
FIGURE 3 | Effect of SSWex on the inltration of mast and T cells in skin lesions of DfE-treated NC/Nga mice. (A,B) Sectioned dorsal skin and ears were stained with
toluidine blue to evaluate mast cell inltration. Dorsal skin tissue sections were immunostained with antiCD3 antibodies to conrm T-cell inltration. The stained sections
were observed under a microscope at ×400 magnication. Scale bar = 100 μm. Data are represented as the mean ± S.E.M. (n=6).
#
p<0.05,
##
p<0.005,
###
p<
0.0005, and
####
p<0.0001 vs. the normal group; *p<0.05, **p<0.005, ***p<0.0005, and ****p<0.0001 vs. the DfE group.
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Song et al. Spatholobus suberectus Dunn in AD
reduce this inltration and thus alleviate skin lesions in DfE-
treated NC/Nga mice.
S. suberectus Dunn Water Extract Inhibits
D. farina ExtractInduced Serum
Immunoglobulin E, Histamine, and
Proinammatory Chemokine Production in
NC/Nga Mice
IgE is an important component of allergic diseases and is closely
associated with Th2 immune response (Leung, 1993). It leads to
the secretion of various allergic mediators, including histamines
and cytokines, by binding to mast cells (OMahony et al., 2011).
Particularly, the IgEmast cellhistamine axis has been well-
understood for decades, and this pathway is known to cause
AD-associated itching disorders (Yang and Kim, 2019). The
inhibitory effects of SSWex on AD-induced serum IgE and
histamine release were measured using ELISA. DfE-induced
AD-like skin inammation was accompanied by an increase in
serum IgE and histamine levels; however, SSWex treatment
inhibited this increase in DfE-treated NC/Nga mice
(Figure 4A).
Proinammatory chemokines play a major role in various
processes of AD progression, such as immune cell activation,
differentiation, and inltration of inammatory sites (Sebastiani
et al., 2002). Proinammatory chemokines can be produced by
various immune cells, including mast cells, T cells, dendritic cells,
keratinocytes, and eosinophils, activated by various stimuli in AD
(Nedoszytko et al., 2014;Pucheu-Haston et al., 2015). Among
these chemokines, TARC and MDC are CC chemokine receptor
type (CCR) 4 ligands, which play important roles in the
inltration of Th2 cells into AD skin lesions (Oshio et al.,
2009). In addition, RANTES is one of the CCR ligands that
play an active role in regulating the inltration and activation of
immune cells, including Th2 and mast cells, in AD-related
inammatory tissues (Alam, 1997). TARC, MDC, and
RANTES are highly expressed in AD patients and mouse
models (Hashimoto et al., 2006;Oshio et al., 2009;Lim et al.,
FIGURE 4 | Effect of SSWex on serum immunoglobulin E (IgE), histamine, and proinammatory chemokine levels in DfE-induced atopic dermatitis (AD)like skin
lesions in NC/Nga mice. The serum samples were collected on the last day of the experiment. The serum samples were diluted ×100 times and analyzed. (A) Total IgE
and histamine levels in serum were detected using ELISA. (B) Levels of proinammatory chemokines (MDC, RANTES, and TARC) were determined using ELISA. Data
are expressed as the mean ± S.E.M. (n=6).
#
p<0.05,
##
p<0.005,
###
p<0.0005, and
####
p<0.0001 vs. the normal group; *p<0.05, **p<0.005, ***p<0.0005,
and ****p<0.0001 vs. the DfE group.
Frontiers in Pharmacology | www.frontiersin.org June 2022 | Volume 13 | Article 9192307
Song et al. Spatholobus suberectus Dunn in AD
2014;Ko et al., 2019). Thus, the role of SSWex in inhibiting the
production of these proinammatory chemokines in DfE-treated
NC/Nga mice was evaluated. SSWex treatment signicantly
reduced the serum levels of TARC, MDC, and RANTES
(Figure 4B). These results support the inhibitory action of
SSWex on the inltration of immune cells, including mast and
T cells. In this study, in vivo data demonstrated that SSWex
administration improved AD-like skin lesions in NC/Nga mice
via alleviating multiple DfE-induced events.
S. suberectus Dunn Water Extract
Suppresses the Production of
Proinammatory Chemokines in IFN-γ/
TNF-αStimulated HaCaT Cells
Epidermal keratinocytes in AD skin lesions play a major role in
the immune response through the secretion of inammatory
mediators, such as proinammatory chemokines. The
inammatory responses of synergistically activated
FIGURE 5 | Effects of SSWex on proinammatory cytokines and chemokines in IFN-γ-/TNF-αinduced HaCaT cells. (A) HaCaT cells treated with SSWex at
different concentrations (0500 μg/ml) for 24 h. Cell viability was determined using a cell proliferation assay kit. (B) HaCaT cells were pretreated with SSWex for 1 h and
then stimulated with 10 ng/ml of IFN-γ/TNF-αfor 24 h. Secreted MDC and RANTES were measured via ELISA. Secreted TARC, MCP-1, and IL-8 were detected using
the bead-based immunoassay. The data are shown as the mean ± S.E.M. of three independent experiments.
#
p<0.05,
##
p<0.005,
###
p<0.0005, and
####
p<
0.0001 vs. untreated; *p<0.05, **p<0.005, ***p<0.0005, and ****p<0.0001 vs. IFN-γ/TNF-α.
Frontiers in Pharmacology | www.frontiersin.org June 2022 | Volume 13 | Article 9192308
Song et al. Spatholobus suberectus Dunn in AD
keratinocytes by IFN-γ/TNF-αare mainly used in studies for
inammatory skin diseases, including AD (Yang et al., 2015;Lee
et al., 2018;Lee et al., 2020). The keratinocytes activated by IFN-
γ/TNF-αrelease proinammatory chemokines, such as TARC,
MDC, and RANTES, which play an important role in the
inltration of Th2 cells into AD skin lesions (Yang et al.,
2015). In addition, co-stimulation of IFN-γ/TNF-αis known
to release monocyte- and neutrophil-specic chemokines, such as
MCP-1 and IL-8, in inamed keratinocytes. The release of these
chemokines is considered to play a major role in the recruitment
and accumulation of inammatory cells in skin inammatory
diseases (Sebastiani et al., 2002;Cha et al., 2019). Therefore,
inhibition of the release of these proinammatory chemokines in
keratinocytes of AD skin lesions is considered important in AD
treatment.
To determine the cytotoxicity of SSWex, HaCaT cells were
incubated with SSWex in a dose-dependent manner for 24 h.
SSWex appeared to be non-toxic till a level of 500 μg/ml
(Figure 5A). To investigate the inhibitory effects of SSWex on
IFN-γ/TNF-αinduced proinammatory chemokine production,
HaCaT cells were pretreated with SSWex for 1 h and further
stimulated with IFN-γ/TNF-αfor 24 h. The proinammatory
chemokines released in the cell culture medium supernatant were
analyzed using ELISA or bead-based immunoassay. SSWex
(50300 μg/ml) signicantly inhibited the production of IFN-
FIGURE 6 | Effect of SSWex on MAPK/NF-κB/STAT1 activation by IFN-γ-/TNF-αin HaCaT cells. The cells were preincubated with SSWex for 1 h and further
stimulated with IFN-γ/TNF-αfor various time intervals (0, 5, 15, and 30 min). Using a Western blot analysis, (A) the phosphor ylatedand total MAPK (p38, ERK, and JNK)
protein levels were quantied and (B) phosphorylation or degradation of IκBαand STAT1 proteins was detected in the cells. Quantied Western blot data are shown as
the means ± S.E.M. of three separate experiments. *p<0.05, **p<0.005, ***p<0.0005, and ****p<0.0001 vs. IFN-γ/TNF-α.
Frontiers in Pharmacology | www.frontiersin.org June 2022 | Volume 13 | Article 9192309
Song et al. Spatholobus suberectus Dunn in AD
FIGURE 7 | Effect of SSWex on IFN-γ-/TNF-αinduced NF-κB and STAT1 translocation to the nucleus in HaCaT cells. The cells were pretreated with 50, 100, or
300 μg/ml SSWex for 1 h and further incubated with 10 ng/ml IFN-γ/TNF-αfor 1 h. (A) For Western blot analysis, nuclear fractionation was performed to conrm
p-STAT1, STAT1, and NF-κB (p65 and p50) subunits in the nucleus. β-Actin and PCNA were used as loading controls. (B,C) Quantied Western data are shown as the
mean ± S.E.M. of three independent experiments.
#
p<0.05,
##
p<0.005,
###
p<0.0005, and
####
p<0.0001 vs. untreated; *p<0.05, **p<0.005, ***p<0.0005,
and ****p<0.0001 vs. IFN-γ/TNF-α. The nuclear translocation of p-STAT1 (Tyr; green and Ser; red) and NF-κB (p65; red and p50; green) was detected by
immunouorescence combined with DRAQ5(blue) staining for the nucleus (scale bar = 20 μm).
Frontiers in Pharmacology | www.frontiersin.org June 2022 | Volume 13 | Article 91923010
Song et al. Spatholobus suberectus Dunn in AD
γ/TNF-αinduced TARC, MDC, RANTES, MCP-1, and IL-8 in
HaCaT cells (Figure 5B). Therefore, the in vitro anti-atopic
effects of SSWex were conrmed.
S. suberectus Dunn Water Extract Inhibits
IFN-γ/TNF-αInduced MAPK/STAT1/NF-κB
Activation in HaCaT Cells
IFN-γand TNF-αactivate the extracellular signalregulated
kinases (ERK), c-Jun N-terminal kinases (JNK), and p38
mitogenactivated protein kinase (MAPK) pathways in various
cell types, including keratinocytes (Sung et al., 2012;Jeong et al.,
2016). The MAPKs regulate the synthesis of proinammatory
molecules, such as chemokines, in AD through their intracellular
signaling pathways, including signal transducer and activator of
transcription 1 (STAT1) and nuclear factor kappa B (NF-κB)
(Kwon et al., 2012;Park et al., 2015a;An et al., 2017). STAT1 and
NF-κB are important transcription factors in the immune system
and are stimulated by IFN-γ/TNF-αto produce proinammatory
chemokines in HaCaT cells (Park et al., 2015b;Yang et al., 2018).
Upon stimulation with IFN-γ/TNF-α, STAT1 is activated,
phosphorylated, and further translocated to the nucleus where
it can activate the target genes by binding to promoters. Similarly,
activation of NF-κB by the phosphorylation and degradation of
FIGURE 8 | (A) HPLC chromatograms of the standard mixture (a) and SSWex (b) at 280 nm. (B) Chemical structures of the four compounds in SSWex: gallic acid
(1), (+)-catechin (2), procyanidin B2 (3), and epicatechin (4).
TABLE 1 | Calibration curves and contents of the four compounds in Spatholobus suberectus Dunn.
Compound Linear range (μg/ml) Regression equation r
2
Content (mg/g)
Mean SD
Gallic acid 2.50160.00 y= 40290x68434 0.9997 0.409 0.001
(+)-Catechin 3.44220.00 y= 14186x20060 0.9998 6.129 0.112
Procyanidin B2 3.44220.00 y= 14714x6332.8 0.9999 1.884 0.009
Epicatechin 3.91250.00 y= 29502x3311.9 0.9999 3.951 0.005
Frontiers in Pharmacology | www.frontiersin.org June 2022 | Volume 13 | Article 91923011
Song et al. Spatholobus suberectus Dunn in AD
inhibitor kappa B-alpha (IκB-α) causes translocation of NF-κB
(p65 and p50) to the nucleus where it can activate the target genes
by binding to promoters (Sung et al., 2012). Thus, inhibition of
the MAPK/STAT1/NF-κB signaling pathway can alleviate the
symptoms of AD and is considered an important strategy in the
development of novel therapeutics for AD.
To investigate the molecular mechanism of the inhibitory effect
of SSWex in IFN-γ-/TNF-αstimulated HaCaT cells, we rst
determined whether SSWex inhibits the activation of the MAPK
signaling pathway using Western blot analysis. SSWex 300 μg/ml
inhibited the IFN-γ-/TNF-αinduced phosphorylation of p38, ERK,
and JNK in HaCaT cells (Figure 6A). In addition, SSWex treatment
inhibited the IFN-γ-/TNF-αinduced phosphorylation of STAT1
and IκBαand degradation of IκBαin HaCaT cells (Figure 6B).
Furthermore, we studied the effect of SSWex on the nuclear
translocation of STAT1 and NF-κB using Western blot after
nuclear fractionation. SSWex treatment suppressed the IFN-γ-/
TNF-αinduced nuclear translocation of p-STAT1, p65, and p50
in a dose-dependent manner (Figure 7A). This inhibitory effect
of SSWex was also demonstrated by immunouorescence
staining (Figures 7B,C). These results demonstrated that
SSWex inhibited the activation of keratinocytes by inhibiting
proinammatory chemokine production via regulation of the
MAPK/STAT1/NF-κB signaling pathway.
High-Performance Liquid Chromatography
Analysis of Four Compounds in the S.
suberectus Dunn Water Extract
High-performance liquid chromatography (HPLC) analysis was
performed with an analytical method established to separate the
marker compounds in SSWex. The four compounds in SSWex
were detected simultaneously: gallic acid, (+)-catechin,
procyanidin B2, and epicatechin with the retention times of
7.11, 14.85, 16.15, and 17.99 min, respectively. The
representative HPLC chromatogram is shown in Figure 8A,
and the chemical structure of each compound is shown in
Figure 8B. The coefcients of determination (r
2
) calculated
from the calibration curves of the four compounds were all
0.9997, indicating good linearity. The quantitative analysis
revealed that the amounts of gallic acid, (+)-catechin,
procyanidin B2, and epicatechin in SSWex were 0.409, 6.129,
1.884, and 3.951 mg/g, respectively (Table 1).
Gallic acid, one of the most important polyphenols, has been
found in many plants and fruits, such as grapes, strawberries,
bananas, gallnuts, and green tea (Zuo et al., 2002;Yeganeh Ghotbi
and bin Hussein, 2010;Bhat et al., 2016). It possesses antitumorigenic
and anti-inammatory activities (Faried et al., 2007;Kaur et al., 2009).
Moreover, it can exhibit anti-allergic inammatory activity on crucial
effector cells in allergic inammation, such as basophils, eosinophils,
and dendritic cells (Liu et al., 2013;Chan et al., 2015;Tsang et al.,
2016). Catechins, including (+)-catechin and epicatechin, are widely
present in foods and herbs, such as berries, grapes, cacaos, and apples.
They are particularly present in high amounts in tea (Isemura, 2019).
Catechins have many benecial properties for human health, such as
antimicrobial, anticancer, ROS regulatory, anti-aging, antioxidant,
anticardiovascular disease, and anti-inammatory activities (Isemura,
2019;Nakano et al., 2019). Procyanidin B2 is a phenolic compound
and is mainly found in grapes, apples, blueberries, cocoa, and tea (Su
et al., 2018;Xiao et al., 2018). Procyanidin B2 is reported to exhibit
various pharmacological activities, including antioxidant, antitumor,
and anti-inammatory properties (Sakano et al., 2005;Suda et al.,
2013;Yang et al., 2014b). Among these activities of procyanidin B2,
anti-inammatory effects occur through regulation of various
inammatory mediators, including cytokines, chemokines, and
nitric oxide. Procyanidin B2 regulates these inammatory
mediators via the regulation of MAPK/NF-κB activity (Martinez-
Micaelo et al., 2012a;Martinez-Micaelo et al., 2012b). Therefore, the
anti-inammatory effects of these four compounds could be
responsible for the anti-inammatory action of SSWex. In
addition, the anti-atopic effects of gallic acid (Hu and Zhou, 2021)
and epicatechin (Song et al., 2021) have already been reported. Since
there are no reports of the anti-atopic effects of (+)-catechin or
procyanidin B2, we will evaluate these in subsequent studies.
CONCLUSION
The topical application of SSWex suppressed AD symptoms in
skin lesions in DfE-treated NC/Nga mice. SSWex inhibited
immune cell inltration in AD-like skin lesions and increase
in AD-related serum parameters, such as IgE, histamine, and
proinammatory chemokines. Moreover, we observed that
SSWex regulated the expression of proinammatory
chemokines via the MAPK/STAT1/NF-κB pathway in IFN-γ-/
TNF-αstimulated HaCaT cells. Our study provides evidence of
the potential of SSWex as a novel agent or food supplement for
the prevention and treatment of AD.
DATA AVAILABILITY STATEMENT
The raw data supporting the conclusion of this article will be
made available by the authors, without undue reservation.
ETHICS STATEMENT
All animal experiments were conducted under the approval (No.
20-069) of the Institutional Animal Care and Use Committee at
the Korea Institute of Oriental Medicine (KIOM).
AUTHOR CONTRIBUTIONS
TK: conceptualization and supervision. H-KS, SP, HK, and SJ:
experimentation. H-KS: data analysis and manuscript writing.
FUNDING
This work was supported by the Korea Institute of Oriental
Medicine, Ministry of Education, Science and Technology,
Republic of Korea (Grant No. KSN2021330).
Frontiers in Pharmacology | www.frontiersin.org June 2022 | Volume 13 | Article 91923012
Song et al. Spatholobus suberectus Dunn in AD
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Frontiers in Pharmacology | www.frontiersin.org June 2022 | Volume 13 | Article 91923014
Song et al. Spatholobus suberectus Dunn in AD
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