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FULL COMMUNICATION
Combined Mulberry Leaf and Fruit Extract Improved Early Stage
of Cutaneous Wound Healing in High-Fat Diet-Induced Obese Mice
Hyeyoon Eo and Yunsook Lim
Department of Food and Nutrition, Kyung Hee University, Seoul, Korea.
ABSTRACT Obesity is a pandemic that causes many health challenges, including difficulties in achieving proper wound
healing without complications. The current study investigated the role of NLRP3 inflammasome in the early stages of
cutaneous wound healing and the effect of combined mulberry leaf and fruit extract ( MLFE) on cutaneous NLRP in-
flammasome involvement in delayed wound healing mice with high-fat diet (HFD)-induced obesity. After obesity was
induced by HFD for 10 weeks, the mice were supplemented with MLFE (at a dose of 500 mg/kg containing 333.3 mg/kg of
mulberry leaf extract and 166.7 mg/kg of mulberry fruit extract) by gavage, 5 days/week for 12 weeks. MLFE supplementation
ameliorated delayed wound closure in obese mice. While wound size was positively correlated with fasting blood glucose
level during the early stage of wound healing, it was strongly correlated with body weight gain and body fat mass during the
later stage of wound healing. Under obese conditions, the levels of NLRP3 inflammasome and its related markers (pro-
caspase-1 and precursor/mature interleukin 1 beta) were increased at a basal level, but the NLRP3 inflammasome was
suppressed during the inflammatory stage of cutaneous wound healing. However, MLFE supplementation stimulated cuta-
neous NLRP3 inflammasome in HFD-induced obese mice (day 3). Taken together, stimulating the NLRP3 inflammasome
might be beneficial in the early inflammatory stage of cutaneous wound healing and MLFE could be a potential therapeutic
intervention in delayed wound healing through activation of the NLRP3 inflammasome in obesity.
KEY WORDS: delayed wound healing inflammasome inflammation mulberry obesity NLRP3
INTRODUCTION
In2009–2010, 35% of US adults were identified as obese,
and this trend has continued for the last decade.
1
How-
ever, it does not seem to be a phenomenon limited to the
United States, and this well-known epidemic of obesity af-
fects the world population.
1,2
Hence, this pandemic is now
addressed as ‘‘Globesity.’’
2
When obese people suffer
wounds due to surgery or trauma, they often experience
delayed wound healing.
3
For these reasons, achieving proper
wound healing without any complications is difficult for
many obese patients because these individuals undergo
physiological alterations in their body systems.
3
Wound healing procedure can be divided into three phases:
inflammation, proliferation, and remodeling.
4
In particular,
the first phase, ‘‘Inflammation’’ occurs within 72 h after
wounding, and this phase plays a pivotal role in wound heal-
ing.
5
However, the inflammatory phase can be dysregulated in
some metabolic diseases, such as obesity, with impaired im-
munity. Obesity is regarded as a ‘‘metaflammatory disease,’’
which is characterized by metabolic alterations and a low-
grade chronic inflammatory state.
6
Hence, delayed wound
healing under obese conditions is strongly associated with
altered inflammatory response.
Inflammasomes are multiprotein complexes and cytosolic
sensors that respond to inflammatory stress by initiating an
intracellular inflammatory cascade.
7,8
The inflammasomes
stimulate the secretion and maturation of interleukin 1 beta
(IL-1b), which is a proinflammatory cytokine. Among the
several different inflammasomes, the nucleotide-binding
domain–leucine-rich repeat containing family and pyrin
domain containing 3 (NLRP3, also called cryopyrin, CIAS1,
and NALP3) inflammasome have been well characterized.
9
The NLRP3 inflammasome consists of three different pro-
teins: (1) NLRP3, (2) adapter protein apoptosis-associated
speck-like protein (ASC), and (3) pro-caspase-1.
7
Previous
studies have focused on the role of NLRP3 inflammasomes
extensively in macrophages.
10,11
However, NLRP3 in-
flammasomes are also expressed in epithelial tissues, such as
skin, as a first line of defense against outside threats
10–12
and
consequently can be involved in the innate immunity of the
skin.
10,11
Interestingly, NLRP3 is highly expressed in the
skin of C57BL/6 mice.
13
Indeed, there have been a few
studies that elucidated the role of the NLRP3 inflammasome
during both the proliferation and remodeling stage of
Manuscript received 6 May 2015. Revision accepted 17 September 2015.
Address correspondence to: Yunsook Lim, PhD, Department of Food and Nutrition,
College of Human Ecology,Kyung Hee University, 26 Kyunghee-daero,Dongdaemun-
gu,Seoul 02447, Republic of Korea, E-mail: ylim@khu.ac.kr
JOURNAL OF MEDICINAL FOOD
J Med Food 00 (0) 2015, 1–9
#Mary Ann Liebert, Inc., and Korean Society of Food Science and Nutrition
DOI: 10.1089/jmf.2015.3510
1
cutaneous wound healing under diabetic conditions.
8,14
Previous studies have demonstrated that increased NLRP3
expression triggered sustained inflammatory cascades and
decreased angiogenesis during the proliferation stage of
wound healing in genetically diabetic mice.
8,14
Never-
theless, little is known about the role of the NLRP3 in-
flammasome as a target marker of inflammatory response in
the early stages of cutaneous wound healing under obese
conditions. Thus, it will be novel but crucial to investigate
the role of the NLRP3 inflammasome in regulating the early
stages of cutaneous wound healing to facilitate the discovery
of new approaches to impaired wound healing in obesity.
Mulberry (Morus alba L.) is widely cultivated in Korea,
China, and Japan and its leaves, fruit, bark, and roots have
been traditionally regarded as functional foods and medi-
cines.
15,16
It is reported that the mulberry leaf is rich in
functional components, such as rutin, quercetin, and
1-deoxynojirimycin.
17
Both the mulberry leaf and fruit have
been well documented for their bioactive properties, such
as antidiabetic,
18,19
antioxidative,
16,19–22
and antidyslipi-
demic
20,21
effects. Our group previously reported an antiobe-
sity effect in addition to antioxidative and anti-inflammatory
effects.
23,24
In particular, our group found that supplementa-
tion with a mixture of mulberry leaf and fruit extract (MLFE)
had greater antiobesity and anti-inflammatory effects than
single supplementation of mulberry leaf extract.
24
Therefore, we hypothesized that the NLRP3 inflamma-
some plays an important role in the early stage of cutaneous
wound healing and that supplementation with a combination
of MLFE can regulate the NLRP3 inflammasome for proper
wound healing in obesity-induced delayed wound healing.
MATERIALS AND METHODS
Animals and diets
Male 4-week-old C57BL/6 mice were obtained from
Orient Bio, Inc. (Gyeonggi, Korea), were housed at a con-
stant temperature (22 –1C) with a 12-h dark/12-h light
cycle, and given access to tap water and food ad libitum.
After a 1-week acclimation period, the animals were ran-
domly divided into two groups, a control diet group (CON)
and a high-fat diet group (HF). Each group was treated with
either the control diet (D12450B, 10% kcal fat; Research
Diets, New Brunswick, NJ, USA) or the high-fat diet
(D12451, 45% kcal fat; Research Diets) for 9 weeks, re-
spectively. All procedures used were in accordance with
animal protocols approved by the Kyung Hee University
Institutional Laboratory Animal Care and Use Committee
[KHUASP(SE)-10-022].
Experimental design
MLFE were performed as previously described.
23,24
As
mentioned above, mixed administration of mulberry leaf and
fruit [MLFE: 2:1 ratio of mulberry leaf ethanol extract
(MLE) and mulberry fruit extract (MFE) at a dose of 500 mg/
kg/day (333.3, 166.7 mg/kg/day, respectively) has been
shown to have strong antioxidant and antiobesity activities in
our previous studies.
23,24
As a follow-up study to our previ-
ous research, the current study utilized MLFE at a dose of
500 mg/kg/day (333.3 mg/kg/day of MLE and 166.7 mg/kg/
day of MFE).
After being fed with a HFD for 10 weeks to induce
obesity, the HFD mice were subdivided into two groups
(with or without supplementation). The supplemented group
was administrated with MLFE at a dose of 500 mg/kg/day
(333.3, 166.7 mg/kg/day, respectively) by oral administra-
tion 5 times a week for 12 weeks. MLF was freshly sus-
pended in distilled water for oral gavage. At the same time,
the HF and CON groups were treated similarly, but identical
volumes of distilled water were used during the same
treatment period. Body weight and food intake were mea-
sured twice a week.
Sample collection and preparation
Blood and wounds at the designated time point after
wounding [0 h (basal level), 24, 72 h, and endpoint (END)]
were collected. Mice were fasted for 12 h after the last
treatment and then euthanized by inhalation with isoflurane
(Baxter, Deerfield, IL, USA). The blood was collected by
cardiac puncture using a heparin-coated syringe (Sigma-
Aldrich, St. Louis, MO, USA). Plasma was obtained by
centrifugation at 500 ·gfor 15 min. The wounds were re-
moved after cardiac puncture; blood or foreign materials
were washed using saline. The wounds were weighed and
immediately frozen in liquid nitrogen. All the samples were
stored at -80C until they were used for experiments.
Plasma biochemical analysis
Levels of fasting blood glucose (FBG) were measured
with a glucometer (NOCODING1, Seoul, Korea).
Levels of plasma triglyceride (TG), total cholesterol (TC),
and high-density lipoprotein cholesterol were determined
using commercial kits (Bio-Clinical System, Gyeonggi,
Korea).
Both plasma aspartate aminotransferase (GOT) and ala-
nine aminotransferase (GPT) were determined using com-
mercial kits (Bio-Clinical System).
Measurement of hepatic lipid peroxidation
To investigate lipid peroxidation in the liver, levels of
malondialdehyde (MDA) were measured by the thiobarbi-
turic acid (TBA) method. Hepatic tissue of each mouse was
homogenized with 0.15 M KCl solution. Two hundred mi-
croliters of tissue homogenate was taken in each tube, and
200 lL of 8.1% sodium dodecyl sulfate (SDS), 3 mL of 20%
acetic acid-0.8% TBA solution, and 600 lL of distilled
water were added. The mixed solution was heated at 95C
for 1 h. After cooling in tap water, 1 mL distilled water and
5 mL mixture of n-butanol and pyridine (15:1, v/v) were
added and centrifuged at 4000 rpm for 10 min. The absor-
bance of the supernatant at 532 nm was determined using an
ELISA reader (BIO-TEK instruments, Winooski, VT, USA)
with 1,1,3,3-tetramethoxypropane as the standard.
2EO and LIM
Wound biopsy and measurement of wound closure rate
In our previous study, a wound biopsy model used in this
study was described.
25
In short, full-thickness excisional
wounds were made on the back of the mouse. The mice were,
briefly, anesthetized with isoflurane (Baxter), and the backs
were shaved and sterilized with 70% (v/v) ethanol. The full-
thickness excisional wounds were made on the folded skin by
a sterile biopsy punch (4 mm diameter; Kai medical, Gifu,
Japan). To avoid self-licking, wounds on the dorsum were
made below the shoulder blades of each mouse.
Wounds in an individual mouse were digitally photo-
graphed every day, beginning on the day of wounding (D0)
with a standard equivalent to the initial wound area placed
beside the wound site. Wound closure was quantified by
Canvas 11 software (Deneba, Miami, FL, USA). The rate of
wound closure was defined as the ratio of the wound size
to the initial wound size. A smaller wound ratio indicated
faster wound closure.
Histological analysis
Wounded tissues were fixed in 10% formaldehyde,
buffered with phosphate-buffered saline (PBS, pH7.2), and
then embedded in paraffin wax. Histological sections
(10 lm thickness) of tissues were sliced and deparaffinized
in xylene. The sections were rehydrated in alcohol gradients
and then stained with hematoxylin and eosin (H&E) for
10 min and 60 s, respectively. The stained sections were
mounted with the histological mounting medium (Histo-
Mount, Atlanta, GA, USA) after staining. All images were
acquired using an optical microscope (ECLIPSE Ci-S; Ni-
kon, Tokyo, Japan).
Western blot analysis
Skin samples were at 4Cinthelysisbuffer(20mM
Tri-HCl, 150 mM NaCl
2
, pH 7.5, 1% NP40, 0.5% Na-
deoxycholate stock, 1 mM EDTA, 0.1% sodium dodecyl
sulfate) and then centrifuged at 5000 rpm at 4Cfor10min.
Supernatants were used as cytosol extraction for western blot
analysis. Pelleted nuclei remnants were transferred to other
tubes, and the pellets were resuspended in a hypertonic buffer
(glycerol, 1M HEPES, 4 M NaCl, 1 M MgCl
2
, 500 mM EDTA,
1 M DTT, PMSF, 1 M benzamidine, pepstatin, leupeptin,
aprotinin, and distilled water). The lysed nuclei were stored in
small aliquots at -80C until being used for skin nuclear
fractions. The total protein concentrations of the fractions were
measured using a BioRad Protein Assay Kit (Bio-Rad, Her-
cules, CA, USA). A total of 40 g of protein samples were
separated with SDS-PAGE and then transferred onto poly-
vinylidene fluoride membranes (Millipore, Marlborough,
MA, USA). The membranes were blocked with 5% bovine
serum albumin in phosphate-buffered saline–0.1% Tween 20
(PBS-T) and incubated overnight with primary antibodies at
4C. The primary antibodies were directed against ASC and
nuclear factor kappa B (NFjB) (Cell Signaling Technology,
Inc., Danvers, MA, USA); Lamin B1 (Abcam, Cambridge,
MA, USA); caspase-1 (precursor/mature), cryopyrin, IL-1b
(precursor/mature), and b-actin (Santa Cruz Biotechnology,
Inc., SantaCruz, CA,USA). Then, the blots were washed four
times for 10 min with PBS-T. Thereafter, the membranes were
incubated with respective secondary antibodies for 1h and
washed with PBS-T again. Signals were then developed using
the ECL luminol reagent (Santa Cruz Biotechnology, Inc.) and
then recorded and quantified with the Syngene G box (Syn-
gene, Cambridge, UK, USA).
Statistical analyses
Data are expressed as the mean –standard error of the
mean. Results were analyzed by Student’s t-test for com-
parisons between the two groups. The significance of dif-
ferences among the four groups was determined by one-way
analysis of variance followed by Duncan’s test for multiple
comparisons. In particular, to examine the relationship be-
tween wound size and FBG levels, body weight gain, or
body fat mass, Pearson’s correlation coefficients were cal-
culated for each mouse. For all tests, statistical analyses
were performed using SPSS (21.0K for Windows; SPSS,
Inc., Chicago, IL, USA) and a probability level of P<.05
was considered to be statistically significant.
RESULTS
Effect of MLFE on body weight and food intake
After obesity was induced for 10 weeks, the HF group
showed a significantly higher body weight, body weight
gain, and body fat mass compared to the CON group. Body
weight changes in the MLFE group were not significantly
different from those of the HF group after 12 weeks of
supplementation. However, the MLFE group showed sig-
nificantly lower body fat mass compared to the HF group.
Food efficiency ratio was increased in both the HF group
and the MLFE group compared to the CON group (Table 1).
Effect of MLFE on plasma and hepatic biomarkers
Levels of FBG, plasma lipid profiles, hepatic function,
and lipid peroxidation are examined, as shown in Table 1.
FBG levels in the HF group were significantly higher than
those in the CON group. The MLFE group showed signifi-
cantly decreased FBG concentrations compared to the HF
group, but still higher than the CON group.
The HF group had significantly higher levels of plasma
TG and TC compared to the CON group. However, con-
centrations of plasma TG and TC in the MLFE group were
significantly reduced compared to those in HF.
To investigate the effects of MLFE supplementation on
hepatic function as well as the potential toxicity of admin-
istration, plasma GOT and GPT activities were examined in
the plasma. Plasma levels of both GOT and GPT in the HF
group were significantly higher compared to those in the
CON group. At the same time, GPT levels in MLFE were
significantly reduced compared to those in the HF group.
In the current study, the hepatic level of MDA was mea-
sured as a marker of lipid peroxidation. MDA levels in the HF
group were significantly increased compared to those in the
ROLE OF MULBERRY IN DELAYED WOUND HEALING 3
CON group, but there was no significant difference in hepatic
MDA levels between the HF group and the MLFE group.
Effect of MLFE on wound closure rate during cutaneous
wound healing in HFD-induced obese mice
Wound closure rate is presented in Figure 1A, and repre-
sentative wound images at every time point after wounding
are shown for each group in Figure 1B. Wound closure rates
at 24 h (D1) and 72 h (D3) in the HF group were significantly
faster than those in the CON group. The MLFE group showed
significantly smaller wound size at 72 h compared to the HF
group. Wound closure rates in the HF group were signifi-
cantly faster than those in the CON group from day 5 to the
endpoint (D14: END) after wounding. From day 12 (D12),
the MLFE group exhibited significantly accelerated wound
closure rate compared to HF until the endpoint.
In addition, we also presented histological sections in skin
tissues during wound healing (Fig. 2C) and inflammatory cell
recruitment into skin wounds of mice (Fig. 2D) by H&E staining.
Relationship between wound size and FBG level, body
weight, or body fat mass in HFD-induced obese mice
Relationship between wound size (individual wound size
adjusted by initial wound size) and FBG level was evaluated
by Pearson’s correlation coefficients (Table 2). The relative
wound size from day 2 to 11 postwounding was positively
correlated to the FBG levels. Interestingly, the wound size
was positively correlated to the body weight gain and body
fat mass during the later stage of wound healing. The more
wound healing progressed, the more correlation was ob-
served between wound size and body fat mass.
Protein levels of cutaneous NLRP3 inflammasome
and its related markers during the early stage of wound
healing in HFD-induced obese mice
To investigate the roles of cutaneous NLRP3 inflamma-
some and its related markers during the cutaneous wound
healing process, we measured the protein levels of NLRP3,
ASC, pro-caspase-1, and caspase-1 (Fig. 2A–C). The pro-
tein levels of NLRP3 and pro-caspase-1 in the HF group at
basal level were significantly higher than those in the CON
group. However, there was no significant difference in the
protein level of ASC between the CON group and the HF
group. At 24 and 72 h after wounding, the protein level of
NLRP3 was significantly decreased in the HF group,
whereas it was increased in the CON group. Protein levels of
ASC and caspase-1 in the HF group were significantly lower
than those in the CON group at 72 h. However, the MLFE
group showed significantly lower protein levels of NLRP3,
ASC, and pro-casepase-1 compared to the HF group at the
basal level (Fig. 2A–D). The protein levels of NLRP3 at 24
and 72 h in the MLFE group were higher than those in the
HF group. At 24 h, the protein level of caspase-1 (mature) in
the MLFE group was significantly higher than those in the
HF group. There was no significant difference in the protein
level of ASC between the HF group and the MLFE group at
24 h after wounding (Fig. 2B, E). However, the protein
levels of pro-caspase-1, caspase-1, and ASC in MLFE were
significantly higher than those in HF at 72 h.
Effect of MLFE on the protein levels of cutaneous
IL-1band nuclear NFkB during early stage
of wound healing in HFD-induced obese mice
We examined protein levels of inflammatory markers, in-
cluding precursor and mature forms of IL-1b(as shown in
Fig. 2D) and nuclear NFjB (as shown in Fig. 2E), which are
stimulated by the NLRP3 inflammasome. The HF group was
characterized by increased protein levels of precursor IL-1b,
mature IL-1b, and nuclear NFjBcomparedtotheCONgroup
at 0 h. However, those levels in the HF group were all sig-
nificantly reduced at 24 h contrary to those in the CON group.
There were no significant differences at 72h in the protein
levels of precursor IL-1band mature IL-1bbetween the CON
group and the HF group (Fig. 2D). The protein level of nu-
clear NFjB in the HF group was still lower compared to that
in the CON group at 72 h (Fig. 2E). Concurrently, the protein
levels of both precursor and mature IL-1bin the MLFE group
were significantly lower than those in the HF group at the
basal level (Fig. 2D). However, the protein level of mature
IL-1bin the MLFE group was significantly increased con-
trary to the HF group, which showed a decreased protein level
of IL-1bcompared to the CON group at 24 h after wounding.
There were no significant differences in the protein levels of
both precursor and mature forms of IL-1bbetween the HF
group and the MLFE group at 72 h. There was no difference
in the protein level of nuclear NFjB between the HF group
and the MLFE group at the basal level, but the protein levels
of NFjB at 24 and 72h in the MLFE group were higher than
those in the HF group (Fig. 2E).
Table 1. Effects of MLFE on Body Weight, Food Intake,
and Plasma Biochemical Indicators
Group
CON HF MLFE
Body weight
before supplementation
(g)
28.79 –0.44
a
38.88 –0.30
b
39.05 –0.28
b
after supplementation
(g)
32.97 –0.43
a
47.33 –0.27
b
47.30 –0.33
b
weight gain (g) 4.19 –0.24
a
8.45 –0.22
b
8.25 –0.24
b
(%) 14.73 –0.90
a
21.81 –0.67
b
21.18 –0.69
b
Food intake (g/day) 2.59 –0.02
a
2.44 –0.02
b
2.55 –0.05
a
Food efficiency ratio 1.61 –0.09
a
3.46 –0.08
b
3.27 –0.11
b
Body fat mass (g) 2.59 –0.14
a
7.62 –0.09
c
6.66 –0.13
b
(%) 7.72 –0.35
a
16.10 –0.13
c
14.10 –0.28
b
FBG (mg/dL) 154.50 –6.16
a
228.20 –10.82
c
199.26 –13.45
b
Lipid profiles (mg/dL)
Triglyceride 89.71 –5.12
a
186.54 –17.52
b
123.10 –17.16
a
Total cholesterol 139.06 –7.37
a
201.17 –8.62
c
173.70 –8.46
b
Hepatic Function
GOT (Karmen/mL) 51.80 –5.98
a
84.59 –12.90
b
64.63 –5.13
ab
GPT (Karmen/mL) 8.21 –1.57
a
17.39 –3.40
b
6.92 –1.71
a
Hepatic MDA (nM) 13.81 –1.61
a
22.23 –1.84
b
17.87 –1.43
ab
Values are mean –S.E.M. and n=6 for each group. Different letters within a
variable are significantly different at P<0.05. CON, mice fed the control diet;
FBG, fasting blood glucose; HF, mice fed high-fat diet; MDA, malondialde-
hyde; MLFE, mice supplemented with 500 mg/kg/day of MLFE.
4EO and LIM
DISCUSSION
To the best of our knowledge, this is the first study to
investigate (1) the role of NLRP3 inflammasome during the
early stages of cutaneous wound healing in obese mice and
(2) the effect of supplementation with MLFE on delayed
wound healing in obesity. Our results demonstrated that
MLFE supplementation reduced body fat mass, FBG levels,
plasma lipid profiles, and hepatic toxicity. In addition, the
present study demonstrated that there was an alteration in the
development of cutaneous NLRP3 inflammasome during the
early stages of wound healing in obese mice. Furthermore,
MLFE supplementation normalized the protein levels of the
NLRP3 inflammasome during the early stages of cutaneous
wound healing and consequently MLFE showed accelerated
wound closure in HFD-induced obese mice.
It is well known that excessive amounts of adipose tissue
result in increased circulating fatty acids and fat storage in
nonadipose tissues, such as the liver.
26
Excessive fat storage in
nonadipose tissues induces insulin resistance and dyslipidemia.
For these reasons, patients having excessive fat storage in
nonadipose tissues are more likely to have hyperglycemia
and cardiovascular diseases.
26
Thus, controlling excessive
body fat mass is the first target for controlling obesity-
related metabolic disease. In the current study, obese mice
showed the typical characteristics of obesity, including in-
creases in body fat mass, FBG level, and dyslipidemia
compared to lean control mice. However, the MLFE group
showed decreases in total body fat mass, and FBG, plasma
TG and TC levels compared to obese mice.
In our previous study, we demonstrated that regulation of
blood glucose levels makes it possible to accelerate delayed
wound healing in type 1 diabetic mice.
25
The present study
investigated the correlation between relative wound size and
body fat mass, as well as FBG level. According to our
results, wound closure in the early stages of cutaneous
FIG. 1. Effect of mulberry leaf and fruit extract (MLFE) supplementation on wound closure rate during cutaneous wound healing in HFD-induced
obese mice. (A) Wound closure rate. (B) Representative wounds on 0h (D0), 24 h (D1), 72 h (D3), and end (D11) after injuries were shown for each
group. (C) Histological sections of wounds, scale bar =0.5 mm, magnification: ·40. (D) Inflammatory cell recruitment into wound sites, scale
bar =0.01 mm, magnification: ·1000. Arrows in (D) indicate neutrophils infiltrated in wounded tissues. Values are mean –standard error of the mean
(SEM) (n=6). Mean values with unlike letters were significantly different (P<.05). CON, mice fed the control diet (negative control); HF, mice fed
high-fat diet ( positive control); MLFE, mice supplemented with 500 mg/kg/day of MLFE. Color images available online at www.liebertpub.com/jmf
ROLE OF MULBERRY IN DELAYED WOUND HEALING 5
wound healing was more correlated with FBG than body
weight gain or body fat mass. However, wound size and
body fat mass reached were highly correlated at 6 days after
wounding by the endpoints. The results suggest that an in-
crease in body fat mass is another factor inducing delayed
wound healing in obesity. These results are matched with
the data of wound closure rates as shown in Figure 1A.
Particularly, the major differences between the HF group
and the MLFE group were body fat mass as an anthro-
pometrical marker and FBG level as a biochemical marker.
Therefore, body fat mass and FBG could be potential indi-
cators of delayed wound healing in obesity.
As mentioned above, inflammation is the most important
stage in wound healing procedures. However, obese indi-
viduals often have some trouble in wound healing without
any complications because of an altered inflammatory re-
sponse in their whole body system.
3,6
In addition, the NLRP3
inflammasome is regarded as a key player in induction and
progression of inflammatory diseases such as obesity.
27
Recently, Weinheimer-Haus et al. suggested that the NLRP3
FIG. 2. Effect of MLFE supplemen-
tation on protein levels of NLRP3 in-
flammasome and its related markers
during cutaneous wound healing in
HFD-induced obese mice. (A) NLRP3,
(B) associated speck-like protein (ASC),
(C) pro-caspase-1 (upper panel)and
caspase-1 (lower panel), (D) precursor
interleukin 1 beta (IL-1b)(upper panel)
and mature IL-1b(lower panel), and (E)
nuclear factor kappa B (NFjB). The
protein samples measured by western
blot were pooled from six z per group. A
representative image of repeated exper-
iments is shown in the lower panel.The
bands show the intensity of the bands
that were densitometrically measured
and normalized to the band levels of a-
tubulin (cytosol) or Lamin B1 (nucleus).
Values are mean –SEM. Mean values
with unlike letters were significantly
different (P<.05). CON, mice fed the
control diet (negative control); HF,
mice fed high-fat diet ( positive control);
MLFE, mice supplemented with
500 mg/kg/day of MLFE.
6EO and LIM
pathway is a novel mechanism during the early stage of
wound healing.
28
Based on this previous evidence, we
hypothesized that controlling NLRP3 expression would lead
to proper immune response during the early stages of wound
healing and, consequently, would accelerate delayed wound
closure in obese mice.
In the current study, the HF group showed delayed wound
healing compared to the CON group. The HF group also
showed a different pattern by increasing the protein level of
NLRP3 in skin tissue compared to the CON group during
cutaneous wound healing. At the basal level, the protein
levels of NLRP3 inflammasome, including NLRP3 and
pro-caspase-1, were increased under obese conditions. The
protein levels of the NLRP3 inflammasome-related markers,
such as IL-1band NFjB in obese mice, were increased as
well. Several studies have well documented the inflammation
associated with NLRP3 inflammasome-caspase-1-IL-1baxis
at the molecular level.
17–19,29–32
IL-1bas a proinflammatory
marker increases NFjB activation by stimulating NFjB
translocation.
31
Conversely, the NFjB activation results in
the increase of pro-IL-1bmRNA expression. In addition, an
increase in NFjB translocation promotes mRNA expression
of NLPR3 and other inflammatory markers, including TNFa,
iNOS, and IL-6.
30
When NLRP3 is activated, NLRP3 inter-
acts with ASC and then recruits pro-caspase-1.
32
After
oligomerization of the NLRP3 inflammasome, self-cleavages
of pro-caspase-1 occur and then activated caspase-1 is gen-
erated. Consequently, active caspase-1 activates processing
and secretion of IL-1b.
7,32
As the HF group showed increased
protein levels of NLRP3 inflammasome and its related
markers, it could be inferred that obese mice had inflamma-
tion in the skin tissue.
However, HFD-induced obese mice also showed immu-
nosuppressive characteristics during cutaneous wound
healing in the current study. Previous studies have reported
obesity-related immunosuppression in animals and hu-
mans.
33–35
In the previous studies, obese individuals were
shown to have a diminished immune response characterized
by a decreased natural killer cell activity, a lower lympho-
cyte response to mitogens related to TNFa, and lower pro-
liferative responses to mitogens. In contrast, other studies
investigated the role of the NLRP3 inflammasome inten-
sively in innate immune responses.
36,37
It is demonstrated
that NLRP3 is an innate responder to pathogenic and danger
signals such as microbial pathogens.
38,39
Taken together, it
is possible that NLRP3 inflammasome can be dysregulated
due to diminished immune responses in obesity, especially
in the early stages of wound healing.
Under normal conditions, NLRP3 was increased after
wounding and maturation of IL-1bwas increased in the
earlier inflammatory phase (24 h after wounding) during
wound healing. In addition, the nuclear level of NFjB in the
CON group was higher than that in the HF group, both in the
early and late inflammatory phases. The pattern of the NFjB
level in the CON group was analogous to patterns of NLRP3
inflammasome. In other words, wound healing was pro-
ceeded by stimulating NLRP3 inflammasome and its related
inflammatory cytokines under normal conditions. It is in-
dicated that the skin responded appropriately to the early
stages of wound healing by regulation of NLRP3 in-
flammasome under healthy conditions. However, the HF
group showed suppression or no change in the protein levels
of NLRP3 inflammasome and its related markers in the
inflammatory phase of wound healing (24 and 72 h after
wounding), as shown in Figure 2. These results of the cur-
rent study are in line with Weinheimer-Haus et al. study,
which demonstrated the role of NLRP3 inflammasome
during early stage of wound healing.
28
Weinheimer-Haus et
al. elucidated that wounds from NLRP3 null mice showed
lower levels of the proinflammatory cytokines, including
IL-1band TNFacompared to wild-type mice, and NLRP3
null mice showed delayed angiogenesis because of NLRP3-
dependently reduced production in IL-1b.
28
Consequently,
NLRP3 null mice showed delayed wound healing.
28
Therefore, dysregulated NLRP3 inflammasome from
obesity-related immunosuppression caused an improper in-
flammatory response during the early stages of cutaneous
wound healing.
Table 2. Pearson’s Correlation Coefficients Between Biochemical and Anthropometrical Markers and Wound Size
Wound Size
D1
1
D2 D3 D4 D5 D6 D7 D8 D9 D10 D11 D12 D13 D14
FBG
2
Pearson’s correlation .387** .345** .354** .177 .244* .322** .304** .174 .156 .122 .101 .155 .214 .313**
Significance (two-tailed) .001 .002 .002 .126 .034 .004 .008 .133 .177 .296 .400 .337 .071 .007
BW gain (g)
Pearson’s correlation .263* .157 .247* .195 .325** .404** .378** .328** .419** .422** .367** .345** .384** .419**
Significance (two-tailed) .022 .176 .031 .091 .004 .000 .001 .004 .000 .000 .002 .003 .001 .000
Body fat mass (g)
Pearson’s correlation .285* .143 .321** .187 .302** .437** .422** .376** .420** .457** .494** .466** .519** .555**
Significance (two-tailed) .013 .217 .005 .106 .008 .000 .000 .001 .000 .000 .000 .000 .000 .000
Wound ratio represents the individual wound size adjusted by the initial wound size.
1
Days after wounding (e.g.,D5=5 days after wounding).
2
FBG level is measured after genistein supplementation for 2 weeks (the same as D0).
*Correlation is significant at the .05 level (two-tailed).
**Correlation is significant at the .01 level (two-tailed).
ROLE OF MULBERRY IN DELAYED WOUND HEALING 7
Furthermore, we investigated the effect of MLFE on al-
tered regulation of cutaneous NLRP3 inflammasome and its
related markers during delayed wound healing in HFD-
induced obese mice as well. Our group recently demon-
strated that resveratrol, which is highly concentrated in
mulberry, improved hepatic inflammation by regulating
NLRP3 inflammasome.
40
In our current study, the MLFE
group showed similar patterns of protein levels of NLRP3,
ASC, and caspase-1, as well as accelerated wound closure,
during the early phases of wound healing. The protein levels
of NLRP3 and nuclear NFjB in the MLFE group, after
wounding, were not significantly different from those in the
HF group. Nonetheless, NLRP3 and nuclear NFjB levels in
the MLFE group were higher than those in the HF group
after 24 and 72 h of wounding. The results suggest that when
MLFE supplementation stimulates protein levels of NLRP3
inflammasome and inflammatory markers, the levels might
have been sufficient to reach a certain level for onset of proper
inflammatory response. At the same time, patterns of cuta-
neous NLRP3 inflammasome in the MLFE group, after
wounding, were similar to those in the CON group, not to those
in the HF group. Although the exact activators or mechanisms
of NLRP3 inflammasome are still unclear, our data suggested
that MLFE normalized the level of NLRP3 inflammasome and
improved the improper cutaneous inflammatory response
during the early phases of wound healing in obesity.
Collectively, we have characterized altered activation of
the NLRP3 inflammasome during the inflammatory phase of
cutaneous wound healing in HFD-induced obese mice.
Furthermore, the current study demonstrated that MLFE
accelerated delayed wound healing by regulation of
NLRP3 inflammasome-related inflammatory response in
HFD-induced obese mice. These results suggest that NLRP3
inflammasome might be a novel target for facilitating the
early stages of wound healing in obesity, and MLFE might
have the potential as a nutraceutical to treat not only obesity
but also obesity-related complications.
ACKNOWLEDGMENT
This work was supported by the National Research
Foundation of Korea (NRF-2010-0006624 and NRF-
2012R1A1A2040217).
AUTHOR DISCLOSURE STATEMENT
No competing financial interests exist.
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ROLE OF MULBERRY IN DELAYED WOUND HEALING 9
