life

Article
Topical Application of Antrodia cinnamomea Ointment in
Diabetic Wound Healing
Ruey-Chih Su 1,2 , Jyh-Gang Leu 3,4 , Yuan-Hsin Chen 1 , Chao-Yi Chen 2 , Yi-Feng Yang 2 , Chih-Cheng Yen 2 ,
Shiu-Huey Chou 1,2 and Yao-Jen Liang 1,2, *

1 Department of Life Science, Fu-Jen Catholic University, New Taipei City 242062, Taiwan;
[email protected] (R.-C.S.); [email protected] (Y.-H.C.); [email protected] (S.-H.C.)
2 Graduate Institute of Applied Science and Engineering, Fu-Jen Catholic University,
New Taipei City 242062, Taiwan; [email protected] (C.-Y.C.); [email protected] (Y.-F.Y.);
[email protected] (C.-C.Y.)
3 School of Medicine, Fu-Jen Catholic University, New Taipei City 242062, Taiwan; [email protected]
4 Division of Nephrology, Department of Internal Medicine, Shin Kong Wu Ho-Su Memorial Hospital,
Taipei 111, Taiwan
* Correspondence: [email protected]; Tel.: +886-2-2905-3593

Abstract: The number of diagnosed diabetic patients is increasing worldwide. Many people with
diabetes develop wounds that are slow to, or never, heal, which can lead to serious health issues.
Diabetes causes long-term excessive blood glucose buildup in human body, which leads to an over-
reactive inflammatory response and excessive oxidative stress. As a result, varied wound healing
effects were observed according to different circumstances and stage of healing. We used two diabetic
wound animal models to analyze the wound healing effect of Antrodia cinnamomea ointment in either
topical application and/or oral administration, and explored its mechanism by Western blot analysis.
The results showed that topical Antrodia cinnamomea treatment can significantly promote wound



healing. The increased expressions of angiopoietin 1 and angiopoietin 2 protein and reduction of


CD68 expression were found around wound area. Simultaneous treatment of oral and topical Antrodia
Citation: Su, R.-C.; Leu, J.-G.; Chen,
Y.-H.; Chen, C.-Y.; Yang, Y.-F.; Yen,
cinnamomea ointment did not show an accelerated healing effect in our animal model. This study is
C.-C.; Chou, S.-H.; Liang, Y.-J. Topical the first report to demonstrate the effect of topical application of Antrodia cinnamomea ointment on
Application of Antrodia cinnamomea diabetic wounds healing, and its relationship with angiogenesis. This may also open a new field for
Ointment in Diabetic Wound Healing. future development and application of Taiwan Antrodia cinnamomea.
Life 2022, 12, 507. https://doi.org/
10.3390/life12040507 Keywords: Antrodia cinnamomea; diabetes; ointment; wound healing; angiogenesis
Academic Editor: R. Paul Robertson

Received: 29 January 2022

Accepted: 28 March 2022 1. Introduction
Published: 30 March 2022
If diabetes is not properly controlled, it may lead to serious complications. These
Publisher’s Note: MDPI stays neutral complications may also cause foot wounds that are unable to heal and develop into necrosis.
with regard to jurisdictional claims in In developed nations, diabetes is the leading cause of age-unrelated blindness, non-trauma-
published maps and institutional affil- related amputations, and dialysis. Studies indicate that even with careful management of
iations. diabetes, 15% patients still suffer from wounds that are unable to heal at normal rates [1].
In diabetic patients, T-cell overreaction can result in apoptosis and slow healing.
Diabetes can also reduce both macrophage activity and lymphatic vessels near wounds,
which slow healing process. Due to the slow wound-healing, necrosis may occur as
Copyright: © 2022 by the authors.
the worst outcome, along with oxidative stress to reduce tissue cell differentiation and
Licensee MDPI, Basel, Switzerland.
activity [2]. As a result, it may be difficult for adjacent cells to enter the wound area and
This article is an open access article
participate in repair. Diabetes is also found associated with narrowing and constriction
distributed under the terms and
conditions of the Creative Commons
of vasculature, which resulted in reduced blood oxygen. As a consequence, nerve ends
Attribution (CC BY) license (https://
lose their perceptual sensitivity, and reduced immune responses [3]. Oxidative stress
creativecommons.org/licenses/by/
during fibroplasias also causes fibroblasts to lose their functionality, resulting in diminished
4.0/).

Life 2022, 12, 507. https://doi.org/10.3390/life12040507 https://www.mdpi.com/journal/life

Life 2022, 12, 507 2 of 12

differentiation and migration capabilities, which compromises the capacity of these cells to
enter wound sites and create appropriate collagen matrices [4].
The high glucose conditions worsen inflammation and oxidative stress in wound
area. All of these factors combined make diabetic patients’ wounds encounter rotating
inflammation and fibroplasia, which lead to ulcers or even amputations. Furthermore,
diabetes can also affect DNA and many proteins, such as angiopoietin, which influences
angiogenesis [5]. Impeded angiogenesis during wound-healing processes, due to impaired
cellular signal transduction, results in disruption of stable new vasculature formation [6].
Finally, the diabetic patients’ wounds experience abnormal collagen homeostasis during
tissue remodeling, causing fibroblast dysfunction with concomitant reduction of myofi-
broblasts differentiation, which then lead to diminished wound contraction and decreased
formation of surface tissue [7].
Antrodia cinnamomea is a native Taiwanese basidiomycete with porous hymenium [8]
that grows only in the inner cavity of a decayed tree trunk of Cinnamomum kanehirai Hay
(Lauraceae). Empirical studies demonstrate that Antrodia cinnamomea exhibits several useful
properties, including anti-cancer effects that involve inhibition of cancer cell metastasis and
proliferation, protection of internal organs [9], anti-oxidation [10], and anti-inflammatory
effects [11]. With regard to diabetes efficacy, Antrodia cinnamomea reduces blood glucose,
including aberrantly high blood glucose levels [12], and it is currently available on the
Taiwanese market in Antrodia cinnamomea beverages, which are sold as a health supple-
ment product.
Erythropoietin (EPO) is a haemopoietic factor that is used to adjust production and
differentiation of red blood cell (RBC) precursor cells [13]. EPO relies on its specific
erythropoietin receptor (EPOR) to modulate blood production processes [14]. Once EPO
and EPOR combine, they activate different target genes, which results in an inhibition of
apoptosis [15]. EPO has been shown to stimulate vascular growth and is also effective in
repairing epithelial tissue [16]. Results from diabetic mouse models indicate that EPO at
the small wound site can accelerate mature capillary formation and reduce inflammation
through increased epithelialization [17]. Similar results have also been obtained in rat
models of diabetes for large wounds repairing [18]. The peroxisome proliferator-activated
receptor superfamily (PPARs) comprises a class of nuclear receptors that help regulate
multiple cellular pathways [19]. The anti-inflammatory effect of PPARδ had been shown in
several previous studies [20,21]. Another study, PPARδ agonists also significantly inhibited
high glucose (25 mM)-induced IL-6 and TNF-α production, NF-κB translocation, and
apoptosis [20].
Prior studies reported that Antrodia cinnamomea is efficacious for healing tumors and
aids in new vascular growth [22]. To evaluate the effects of Antrodia cinnamomea ointment
treatments in diabetic wound healing, we investigate the efficacy of ointment and orally
administered Antrodia cinnamomea to treat linear and large square wounds in diabetes
animal models.

2. Materials and Methods

2.1. Culture of Antrodia cinnamomea Mycelia
The purchased wild Antrodia cinnamomea fruiting bodies were identified before exper-
iment based on morphological features compared with a standard Antrodia cinnamomea
strain from the Food Industry Research and Development Institute, Hsinchu, Taiwan. They
were initially sterilized using 70% ethanol and antibiotics broth. After several times of
washing with sterilized distilled water, small pieces of fruiting bodies were put into Ep-
pendorf tubes with antibiotics broth and vortex. The supernatant was then transferred
to the solidified Malt extract-based culture medium (MEA). Mycelia colony germinated
from single spore was selected for subculture (Figure 1). The malt extract broth (MEB)
was prepared for liquid suspension culture. The MEA plates or MEB suspension culture
inoculated with Antrodia cinnamomea mycelia were kept in an incubator at 28 ◦ C in the dark.
In addition, the taxonomic identity of cultured mycelia was confirmed by isolation of ge-
 Life 2022, 12, x FOR PEER REVIEW 3 of 13

Life 2022, 12, 507 broth (MEB) was prepared for liquid suspension culture. The MEA plates or MEB suspen- 3 of 12
sion culture inoculated with Antrodia cinnamomea mycelia were kept in an incubator at 28
°C in the dark. In addition, the taxonomic identity of cultured mycelia was confirmed by
isolation
nomic DNAof and
genomic DNA
subjected to and subjected
sequencing to sequencing
using using
ITS as marker ITS asHigh-performance
sequence. marker sequence.
High-performance
liquid liquid
chromatography chromatography
(HPLC) method [23](HPLC)
was alsomethod
applied[23] was alsothe
to measure applied to meas-
triterpenoids
ure the triterpenoids
contents contents ofmycelia.
of Antrodia cinnamomea Antrodia cinnamomea mycelia.

Figure1.1.Mycelia
Figure Myceliacolony
colonygerminated
germinatedfrom
fromsingle
singlespore
sporewas
wasselected
selectedand
andsubcultured.
subcultured.

2.2.
2.2.Animals
Animals
The
TheBALB/c mice and
BALB/c mice and Wistar
Wistar rats
rats (4
(4 weeks
weeks old)
old) were
were obtained
obtained from
from National
National Labor-
Labo-
ratory Animal Center, Taipei, Taiwan and the BioLASCO Taiwan Co.,
atory Animal Center, Taipei, Taiwan and the BioLASCO Taiwan Co., Ltd., Yilan, Taiwan, Ltd., Yilan, Taiwan,
respectively.
respectively. TheThe BALB/c mice and
BALB/c mice and Wistar
Wistar rats
rats were
were kept
kept inin polycarbonate
polycarbonate cagescages and
and
◦
housed
housedin inwell
wellaerated
aeratedrooms
roomswith
withaa12-h
12-hlight/12-h
light/12-h dark
dark cycle
cycle at 25 ±±22°C,
at 25 C,fed
fedwith
withstand-
stan-
dard rodent diet
ard rodent diet and
and water
water adad libitum
libitum in
in Fu
Fu Jen
Jen Laboratory
Laboratory Animal
Animal Center.
Center. All
Allprocedures
procedures
involving animals care in this study were carried out in accordance
involving animals care in this study were carried out in accordance with the with the recommenda-
recommen-
tions set set
dations forth by by
forth thethe
University
University Committee
Committee IACUC
IACUC ininFuFuJen
JenLaboratory
LaboratoryAnimal
AnimalCenter
Center
(FJU A10526). Since this study also tested whether the effect of
(FJU A10526). Since this study also tested whether the effect of simultaneoussimultaneous oral
oraladminis-
admin-
tration
istration Antrodia
of of cinnamomea
Antrodia cinnamomea would
wouldbe better thanthan
be better the wound
the wound healing effecteffect
healing of ointment
of oint-
alone, the rat cutaneous wound model was selected because the volume
ment alone, the rat cutaneous wound model was selected because the volume of the solu- of the solution
for tube feeding Antrodia cinnamomea was required to be larger. It was also observed that
tion for tube feeding Antrodia cinnamomea was required to be larger. It was also observed
the wound healing effect of Antrodia cinnamomea in different species of animals would
that the wound healing effect of Antrodia cinnamomea in different species of animals would
be different. Diabetic Mice or rats only treated vehicle ointment topically as Vehicle DM
be different. Diabetic Mice or rats only treated vehicle ointment topically as Vehicle DM
group. Rats in DMC group did not receive either oral or topical treatment application.
group. Rats in DMC group did not receive either oral or topical treatment application.
Rats treated with both topical and oral Antrodia cinnamomea treatment as ACYY group,
Rats treated with both topical and oral Antrodia cinnamomea treatment as ACYY group,
and those accepted topical Antrodia cinnamomea treatment alone as ACYN group. Vehicle
and those accepted topical Antrodia cinnamomea treatment alone as ACYN group. Vehicle
group was normal rats that fed with water and topical vehicle treatment.
group was normal rats that fed with water and topical vehicle treatment.
2.3. Induction of Diabetic Animal
2.3. Induction of Diabetic Animal
Diabetes was induced by giving intraperitoneal injections of Streptozotocin (STZ,
Diabetes
200 mg/kg body was induced
weight by giving
in mice; intraperitoneal
75 mg/kg body weightinjections
in rats)of[24]
Streptozotocin
in BALB/c mice (STZ,and200
mg/kg body weight in mice; 75 mg/kg body weight in rats) [24] in BALB/c
Wistar rats. After one week, those with blood glucose levels higher than 200 mg/dl were mice and Wistar
rats. After
judged one
to be week, those
diabetic. The with
bloodblood
sampleglucose levels higher
was obtained than 200
by lancet mg/dl
(facial were
vein judged
sampling
tomice
in be diabetic.
and tailThe
vein blood sample
sampling inwas
rats)obtained by lancet
and checked (facial vein
by glucose sampling
oxidase methodin mice
using and
a
tail vein sampling in rats) and checked by glucose oxidase method
pre-calibrated Glucometer (TD-4227 Glucometer, Fora Care Taiwan, Taipei, Taiwan). The using a pre-calibrated
Glucometer (TD-4227
STZ-induced Glucometer,
diabetic mouse and ratFora
wereCare
thenTaiwan,
surgically Taipei, Taiwan).
wounded in theThebackSTZ-induced
area (1 cm
diabetic mouse and rat were2
linear wound in mice; 1 cm square wound in rats), which penetrate the epidermiswound
then surgically wounded in the back area (1 cm linear to the
in mice;
fascia. 1 cm
They 2 square wound in rats), which penetrate the epidermis to the fascia. They
were treated by daily application of various ointments in different groups
were treatedthe
throughout bystudy,
daily application
including theof various
vehicle ointments
group andinAntrodia
different groups throughout
cinnamomea group. The the
study,ofincluding
group the vehicle of
oral administration group and cinnamomea
Antrodia Antrodia cinnamomea
filtrate wasgroup. Thein
applied group of oral
2 mg/kg ad-
(total
ministration
0.5 mL in PBS)of perAntrodia
day by cinnamomea filtrateWhen
oral tube feeding. was applied in 2 mg/kg
the experimental time (total 0.5was
point mLreached,
in PBS)
the mice or rats were euthanasia sacrificed to obtain skin tissue samples.
Life 2022, 12, 507 4 of 12

2.4. Ointment Preparation

The Antrodia cinnamomea ointment was formulated by vendors to facilitate wound
smear, and completely cover the wound. Firstly, the Antrodia cinnamomea medicament pow-
der was dissolved with 70% alcohol and added ddH2O up to 45 mL. Followed by adding of
glycerin (Glycerin, First Chemical, Taipei, Taiwan) and emulsifier (Creagel, First Chemical,
Taipei, Taiwan) in series. Rapidly stir the mix solution until solidification. The final concen-
tration of drug paste is 1 mg/g. After applying the ointment, we attached a transparent
bandage to the outside of the wound to allow the ointment to be completely absorbed.

2.5. Measurement of Wound Area

We use the Dino Capture photomicrography lenses (Dino-Lite; Hsinchu, Taiwan) with
fixed magnification observation to record, and subject to measurement and calculation
of wound size using manufacturer’s software (details on https://www.dinolite.us/en/
dinocapture/ (accessed on 5 May 2021)). To quantify the healing effect of various treatment
groups, the measured wound area of each animal on different days after treatment were
divided by that of the first day, and the results were compared between them.
Histological procedure and Semi-Quantitative evaluation of histopathological obser-
vation are described as before and according to the published reference on Veterinarni
Medicina [25] with some modification, a Five-phase scoring system was used in this study
(Table 1):

Table 1. Five-phase scoring system.

Grading Epithelization PMN Fibroblast Collagen

0 Thickness of cut edges Absent Absent Absent
Migration of cells
1 Mild ST Mild-ST Minimal-GT
(<50%)
Migration of cells
2 Mild DL/GT Mild-GT Mild-GT
(>50%)
3 Bridging the excision Moderate DL/GT Moderate-GT Moderate-GT
4 Keratinization Marked DL/GT Marked-GT Marked-GT
PMN: polymorphonuclear leucocyte, ST: surrounding tissue, DL: demarcation line. GT: granulation tissue.

2.6. Immunocytochemical Stain

The paraffin-embedded blocks were cut into 5-mm-thick sections to perform im-
munohistochemical staining using the Ventana BenchMark XT automated stainer (Ventana,
Tucson, AZ, USA). Staining for the primary antibodies of Cytokeratin (Ready to use, Ven-
tana, Tucson, AZ, USA) and Myeloperoxidase (MPO) (Ready to use, Ventana, Tucson,
AZ, USA) were carried out. In each run of staining, the phosphate buffered solution was
included as the negative control, and those samples that strongly express markers served
as the positive controls.

2.7. Western Blot Analysis

A small piece of tissue was put into an Eppendorf tube with a spoonful of zirconia
beads (1 mm diameter), and homogenized in cell lysis buffer (PRO-PREP). Protein concen-
tration was determined by Bio-Rad Protein Assay Kit with Bovine Serum Albumin (BSA) as
a standard. Equal amount of protein from each sample was separated by SDS-PAGE using
10% polyacrylamide gel. The separated proteins were then transferred to polyvinylidene
difluoride membranes (PVDF) (Immobilon-P transfer membrane, Millipore; Burlington,
MA, USA). Membranes were blocked with 5% non-fat milk for total protein in 0.1% PBST
(PBS, 0.01% Tween-20) for 1 h. The membranes were then incubated with primary anti-
bodies, CD68 (H-255), Ang-1 (C-19), Ang-2 (F-1), VEGF (VG-1), RAGE(D-5), EPOR (H-194)
(1:10,000 dilution, Santa Cruz Biotechnology; Santa Cruz, CA, USA), PPAR (PP-K9436-10)
(1:10,000 dilution, R&D Systems; Minneapolis, MN, USA), or actin (C-4). One hour after
incubation, the membrane was washed with 1X PBST. The membranes were then incubated
Life 2022, 12, 507 5 of 12

with a secondary antibody, i.e., rabbit anti-mouse, or goat anti-rabbit, or donkey anti-goat
(1:10,000 dilution, Santa Cruz Biotechnology; Santa Cruz, CA, USA) for 1 h. After incuba-
tion, specific protein bands were detected using Immobilon Western Chemiluminescent
HRP Substrate (Millipore; Burlington, MA, USA) and recorded by Kodak XAR-5 films. The
signal intensity was determined by densitometry using the TotalLab TL100 v2006 software,
and results are shown relative to the control value which was set to be 1 [26].

2.8. Statistical Analysis

All data were presented as the mean ± standard error of mean (SEM). To test statistical
significance, data were subjected to unpaired one-way ANOVA by the Sigma Stat 3.5
software statistical package (Systat SigmaStat V3.5.0.54 Software; San Jose, CA, USA).
Differences between groups were assessed with Fisher Least Significant Difference (LSD
test) as indicated. The significance level was set at p-value < 0.05; * p ≤ 0.05; ** p ≤ 0.01;
# p ≤ 0.05; ## p ≤ 0.01.

3. Results
3.1. Effects of Antrodia cinnamomea Broth Filtrate on Linear Diabetic Wound Closure
Before ointment preparation, AC extract was tested for the potential endotoxin con-
tamination using the RAW264.7 cell culture, i.e., a mouse macrophage cell line. There was
no nitrate production observed when applying AC extract compared to the RAW264.7
cells stimulated with LPS or LPS plus interferon-γ, which resulted in various amount of
activation (Supplement Figure S1). Simultaneously, the RAW264.7 cell proliferation did not
significantly alter after co-treatment with various doses of AC extracts, which also suggests
no cytotoxic effect of AC extracts on RAW264.7 cells (Supplement Figure S2). We have
excluded the possible endotoxin contamination in our AC extract preparation.
After application of Antrodia cinnamomea (AC) ointment, the wounds in the treatment
groups clearly contracted more than that of the vehicle group from day one through day
seven (71.5 ± 12.6 vs. 45.3 ± 10.8, p < 0.05) (Figure 2A). Compared with the vehicle
group on Day 7, the linear wounds in the Antrodia cinnamomea filtrate group significantly
Life 2022, 12, x FOR PEER REVIEW
contract more (Figure 2B). These results suggested that Antrodia cinnamomea 6filtrate
of 13
ointment
accelerate diabetic wound healing in linear wound.

Figure Effects
Figure 2.2.Effects of Antrodia
of Antrodia cinnamomea
cinnamomea (AC) ointment
(AC) ointment on lineardiabetic
on linear cutaneous cutaneous
wound. diabetic
(A) wound.
(A) Full-thickness
Full-thickness woundswounds were induced
were induced for vehiclefor or Antrodiatreatment
vehicle cinnamomea
or Antrodia cinnamomea treatment
in diabetic mice. in diabetic
Images of representative mice from each group taken on post-surgery days are shown. (B) Mean
wound area at indicated time points in each group show that healing rate in linear wound healing
in diabetic mice. Each value represents the mean ± SEM; n = 6 for each group. * p < 0.05 when AC
group compared to vehicle group.

The effect of AC on wound inflammation was presented by the physical appearance

 Life 2022, 12, 507 6 of 12

mice. Images of representative mice from each group taken on post-surgery days are shown. (B) Mean
wound area at indicated time points in each group show that healing rate in linear wound healing
in diabetic mice. Each value represents the mean ± SEM; n = 6 for each group. * p < 0.05 when AC
group compared to vehicle group.

The effect of AC on wound inflammation was presented by the physical appearance

of the healing wounds at day 7. By use of hematoxylin-eosin staining and observed un-
der 400× magnification, the wound site appears as an open wound with mild epithelial
hyperplasia. There were also some infiltrating cells surrounding the peripheral tissues of
the wound (Figure 3). The result of Masson Trichrome stain showed that collagen fibers of
control group stained blue and were abundantly found in the dermis of the dorsal skin.
However, the reduction of collagen fibers was prominently ameliorated in the dermis of the
AC group. When stained with cytokeratin, the wound site of control group showed epithe-
lial hyperplasia, subsided outer edges portions of keratinized epithelium, and aggregated
infiltrating cells distributed between epithelial hyperplasia. Results from myeloperoxidase
Life 2022, 12, x FOR PEER REVIEW stain of control group indicated that there is little aggregated distribution of infiltrating
7 of 13
leukocytes. The Semi-Quantitative evaluation of histopathological observations is summa-
rized in Table 2.

Figure 3. Post-wound skin tissue with specific stained in day 7. The specific stained skin sections from
Figure 3. Post-wound skin tissue with specific stained in day 7. The specific stained skin sections
AC-treated
from diabetic
AC-treated mice
diabetic were
mice examined
were microscopically.
examined Observation
microscopically. under
Observation 400×
under magnification.
400× magnifica-
tion.
Table 2. The semi-quantitative scoring of the king in control and AC treatment.
Table 2. The semi-quantitative scoring of the king in control and
Control ACAC treatment. p Value
Epithelization Control
2.8 ± 0.13 AC
3.7 ± 0.15 p Value
<0.001
PMN infiltration
Epithelization 2.0 ± 0.21
2.8 ± 0.13 3.7 ± ±
0.4 0.16
0.15 <0.001
<0.001
Fibroblast 2.0 ± 0.15 0.5 ± 0.17 <0.001
PMN infiltration 2.0 ± 0.21 0.4 ± 0.16 <0.001
Collagen 1.9 ± 0.10 0.6 ± 0.16 <0.001
Fibroblast 2.0 ± 0.15 0.5 ± 0.17
PMN = Polymorphonuclear leukocytes. Data are shown as mean ± SE.
<0.001
Collagen 1.9 ± 0.10 0.6 ± 0.16 <0.001
PMN
3.2. =The
Polymorphonuclear leukocytes.
Relationship between AntrodiaData are shown Broth
cinnamomea as mean ± SE. and Angiogenesis
Filtrate
The vascular growth factors of diabetic patients are dysfunction, therefore, we ex-
3.2. The Relationship between Antrodia Cinnamomea Broth Filtrate and Angiogenesis
amined the effects of treatments on various vascular growth factors. Compared with the
The vascular
diabetes growththose
vehicle group, factors of diabetic
treated patientscinnamomea
with Antrodia are dysfunction,
filtratetherefore,
gave risewe
to aexam-
signifi-
ined the effects of treatments on various vascular growth factors. Compared with
cant increase in Ang-1 (Figure 4A) and Ang-2 (Figure 4B) in day 7 (Figure S3). In Figure the di-4C,
abetes
VEGFvehicle group,
protein those treated
expression did notwith
reachAntrodia cinnamomea
the significant filtrate(pgave
difference rise toThese
= 0.053). a signif-
data
icant increase in Ang-1 (Figure 4A) and Ang-2 (Figure 4B) in day 7 (Figure S3). In Figure
4C, VEGF protein expression did not reach the significant difference (p = 0.053). These data
indicated that Antrodia cinnamomea filtrate treatment could balance the angiogenesis pro-
cess in diabetic wound healing.
Life 2022, 12, 507 7 of 12

Life 2022, 12, x FOR PEER REVIEW 8 of 13

indicated that Antrodia cinnamomea filtrate treatment could balance the angiogenesis process
in diabetic wound healing.

Figure 4. Angiogenesis and anti-inflammatory effects in diabetic wound area after topical Antro-
Figure 4. Angiogenesis
dia cinnamomea andon
treatment anti-inflammatory effectsrelated
day 7. Angiogenesis in diabetic wound
protein, (A)area after topical Antrodia
Angiopoietin-1 (Ang-1),
cinnamomea treatment
(B) Angiopoietin-2 on day
(Ang-2) 7. Angiogenesis
expressions related on
in skin tissues protein,
Day 7 (A)
postAngiopoietin-1
injury. (C) VEGF(Ang-1),
protein(B) An-
expres-
giopoietin-2 (Ang-2) expressions in skin tissues on Day 7 post injury. (C) VEGF protein expressions
sions did not reach statistically significant. (D) CD68 protein expressions after the cutaneous wound
did not reach statistically significant. (D) CD68 protein expressions after the cutaneous wound in
in diabetic mice. Each value represents the mean ± SEM; n = 6 for each group. * p < 0.05 when AC
diabetic mice. Each value represents the mean ± SEM; n = 6 for each group. * p < 0.05 when AC group
group compared to vehicle group. (n = 6).
compared to vehicle group. (n = 6).
3.3. Anti-Inflammatory Effect of Antrodia cinnamomea Broth Filtrate
3.3. Anti-Inflammatory Effect of Antrodia Cinnamomea Broth Filtrate
CD68 is a macrophage receptor. When present at wound sites, macrophages are the
CD68 is cells
first immune a macrophage
to respondreceptor. When present
to inflammation. at wound
However, sites, macrophages
if the inflammation aredoes
response the
first immune
not cease, cells to respond
macrophages to inflammation.
will continue to accumulateHowever, if the site.
at the wound inflammation response
Thus, macrophages
does
servenotas acease,
meanmacrophages
of monitoringwill continue toresponses
inflammatory accumulate at the
[27]. wound
In Figure site.
4D, theThus, macro-
Antrodia cin-
phages serve as a mean of monitoring inflammatory responses [27]. In Figure
namomea filtrate application group showed a significant decline in the CD68 inflammation 4D, the An-
trodia
factor,cinnamomea
compared with filtrate
theapplication groupgroup.
diabetes vehicle showed a significant decline in the CD68 in-
flammation factor, compared with the diabetes vehicle group.
3.4. Combined Oral and Topical Antrodia cinnamomea Broth Filtrate Treatment
3.4. Combined
This studyOral
alsoand Topical
aims Antrodia
to clarify Cinnamomea
whether changesBroth Filtrate
in wound Treatment
healing and growth factors
resulted in the beneficial effect on blood glucose reduction.
This study also aims to clarify whether changes in wound healing and After oral administration
growth factorsof
the Antrodia cinnamomea filtrate, no decrease in blood glucose levels was
resulted in the beneficial effect on blood glucose reduction. After oral administration of observed in the
diabetic
the micecinnamomea
Antrodia (data not shown).
filtrate,We
no analyzed
decrease inwhether combined
blood glucose oral was
levels topical Antrodia
and observed in the
cinnamomea
diabetic micefiltrate treatments
(data not shown).influence the diabetic
We analyzed whetherwound
combined healing, n = topical
oral and 6 in each group.
Antrodia
cinnamomea filtrate treatments influence the diabetic wound healing, n = 6 in each group.
The diabetic rats treated with topical Antrodia cinnamomea ointment only as Vehicle DM
group. ACYN represents the rat group that was treated with topical Antrodia cinnamomea
Life 2022, 12, 507 8 of 12
Life 2022, 12, x FOR PEER REVIEW 9 of 13

The diabetic rats treated with topical Antrodia cinnamomea ointment only as Vehicle DM
group. ACYN represents
filtrate ointment the rat
only without group Antrodia
feeding that wascinnamomea
treated withfiltrate. Antrodia
topicalACYY cinnamomea
represents the
filtrate ointment only without feeding Antrodia cinnamomea filtrate. ACYY represents
group that was treated with both oral and topical Antrodia cinnamomea filtrate. The vehicle the
group
group that
was was treated
diabetic ratswith bothwith
treated oral vehicle
and topical Antrodia
ointment. cinnamomea
After one-wayfiltrate.
ANOVA Theanalysis,
vehicle
group was diabetic rats treated with vehicle ointment. After one-way
the wound areas of ACYN and ACYY groups significantly decreased from day 7 to day ANOVA analysis,
the
11 inwound
Figureareas
5A. Inofaddition,
ACYN and oralACYY groups
Antrodia significantly
cinnamomea filtratedecreased
treatments from
did day 7 to day
not result in
11 in Figure 5A. In addition, oral Antrodia cinnamomea filtrate treatments
any observable enhancement of wound healing Figure 5B. These results demonstrated did not result in
any
that observable enhancement
Antrodia cinnamomea of wound
filtrate healing
accelerated theFigure
diabetic5B.wound
These healing
results demonstrated that
without lowering
Antrodia cinnamomea filtrate accelerated the diabetic wound healing without
blood glucose; and combined oral and topical use of Antrodia cinnamomea filtrate are notlowering blood
glucose;
potent inand combined
wound healingoral and topical
model use of
of diabetic Antrodia cinnamomea filtrate are not potent in
rats.
wound healing model of diabetic rats.

Figure 5.5.Wound
Woundhealing after
healing topical
after and/or
topical oral Antrodia
and/or cinnamomea
oral Antrodia treatment
cinnamomea on diabetic
treatment on wounds.
diabetic
(A) Images
wounds. of Images
(A) diabeticof
mice from mice
diabetic diabetic
fromvehicle control
diabetic (Vehicle),
vehicle combined
control topical
(Vehicle), and oral
combined Antrodia
topical and
oral Antrodia
cinnamomea cinnamomea
treatment treatment
(ACYY) (ACYY)
and topical and topical
Antrodia Antrodia
cinnamomea cinnamomea
treatment treatment
alone (ACYN) on alone
post-
(ACYN)
injury on are
days post-injury
shown (B) days arepictures
Gross shown from
(B) Gross pictures from
both combined both
topical andcombined topical
oral Antrodia and oral
cinnamomea
Antrodia cinnamomea treatment (ACYY) group and topical Antrodia cinnamomea treatment
treatment (ACYY) group and topical Antrodia cinnamomea treatment alone (ACYN) group of diabetic alone
(ACYN) group of diabetic cutaneous wound from day 9 to day 11 in diabetic rats. Each value rep-
cutaneous wound from day 9 to day 11 in diabetic rats. Each value represents the mean ± SEM; n = 6
resents the mean ± SEM; n = 6 for each group. * p < 0.05 when treatment group compared to control
for each group. * p < 0.05 when treatment group compared to control group.
group.
3.5. The Effects of Antrodia cinnamomea Broth Filtrate on the EPOR and PPARδ Expressions in
3.5. TheArea
Wound Effects of Antrodia Cinnamomea Broth Filtrate on the EPOR and PPARδ Expressions
in Wound
EPO Area
is a haemopoietic factor which can influence production and differentiation
of progenitor red blood cell factor
EPO is a haemopoietic and stimulate
which can the growth production
influence of new vasculature, while it also
and differentiation of
displays
progenitorpositive effects
red blood forand
cell epithelial
stimulatetissue
the repair.
growthInofthenewwound healing rat
vasculature, model,
while EPOR
it also dis-
(EPO
plays receptor) at thefor
positive effects wound site will
epithelial tissue not only In
repair. increase
the woundthe formation
healing ratofmodel,
matureEPORnew
capillaries, but also
(EPO receptor) at theincrease
woundepithelial growth
site will not only and shorten
increase the inflammation. Therefore,
formation of mature new we ca-
examined thealso
pillaries, but EPOR levelsepithelial
increase of woundgrowth
site after
andtopical
shorten treatment alone (ACYN
inflammation. group)
Therefore, we ex-or
combined
amined thewithEPOR orallevels
treatment
of wound(ACYY sitegroup), and the
after topical results showed
treatment a trendgroup)
alone (ACYN of EPOR or
increments
combined with but no
oralsignificant
treatmentdifference amongand
(ACYY group), various groupsshowed
the results (Figurea6A) (Figure
trend of EPORS3).
These resultsbut
increments also
noindicated
significant that EPOR signaling
difference may notgroups
among various be the main factor
(Figure 6A)contributing
(Figure S3).
to the beneficial effect of Antrodia cinnamomea filtrate. On the other hand,
These results also indicated that EPOR signaling may not be the main factor contributing PPARδ has been
reported that participates in embryonic development and bone homeostasis
to the beneficial effect of Antrodia cinnamomea filtrate. On the other hand, PPARδ has been [28,29], and
is correlated
reported thatwith skin wound
participates healing, cell
in embryonic growth [30],and
development andbone
inflammation
homeostasisresponses
[28,29], [31].
and
Our results demonstrate
is correlated with skin wound that topical Antrodia
healing, cinnamomea
cell growth filtrate
[30], and treatment responses
inflammation (ACYN group)[31].
Our results demonstrate that topical Antrodia cinnamomea filtrate treatment (ACYN group)
Life 2022, 12, x FOR PEER REVIEW 10 of 13
Life 2022, 12, 507 9 of 12

significantly increased the PPARδ expressions. When the oral and topical treatments were
significantly increased the PPARδ expressions. When the oral and topical treatments were
combined (ACYY group), PPARδ displayed more significant changes than the other
combined (ACYY group), PPARδ displayed more significant changes than the other groups
groups (Figure 6B). The vehicle group was fed with water and topical vehicle treatment.
(Figure 6B). The vehicle group was fed with water and topical vehicle treatment. Rats in
Rats in DMC group did not receive oral or topical treatment application. In sum, these
DMC group did not receive oral or topical treatment application. In sum, these results
results
suggestsuggest
that thethat the beneficial
beneficial effects ofeffects ofcinnamomea
Antrodia Antrodia cinnamomea filtratemay
filtrate treatment treatment may
relate to the
relate to the signaling of
signaling of PPARδ pathway.PPARδ pathway.

Figure 6. Expression of EPOR and PPARδ proteins in diabetic wound areas after Antrodia cinnamomea
Figure 6. Expression of EPOR and PPARδ proteins in diabetic wound areas after Antrodia cin-
treatment.
namomea The diabetic
treatment. rats without
The diabetic any treatment
rats without were DMC
any treatment group.group.
were DMC Vehicle group
Vehicle was was
group only
topically
only treated
topically with vehicle
treated ointment.
with vehicle (A) EPOR
ointment. proteinprotein
(A) EPOR expressions after Antrodia
expressions cinnamomea
after Antrodia cin-
treatment.
namomea (B) PPARδ
treatment. (B) protein
PPARδ expressions after combined
protein expressions topical and
after combined Antrodia
oraland
topical cinnamomea
oral Antrodia cin-
namomea
treatmenttreatment
(ACYY) and(ACYY) and
topical topical cinnamomea
Antrodia Antrodia cinnamomea
treatmenttreatment alone Each
alone (ACYN). (ACYN).
valueEach value
represents
represents
the mean ±the mean
SEM; n =±6SEM; n =group.
for each 6 for each
* p < group. * p <treatment
0.05 when 0.05 when treatment
group comparedgroup compared
to control to
group.
control group.
4. Discussion
4. Discussion
Wound healing is a very complex process. For diabetic patients, exposure to long-term
elevated
Woundblood glucose,
healing is a exacerbated
very complex inflammatory
process. For responses, and high
diabetic patients, oxidative
exposure stress
to long-
are factors
term thatblood
elevated causeglucose,
difficulty for woundinflammatory
exacerbated healing [32]. responses,
Our study andfound thatoxidative
high external
application
stress of the
are factors Antrodia
that cinnamomea
cause difficulty forfiltrate
woundointment resulted
healing [32]. in smaller
Our study found wound sites
that exter-
compared to the control group. The Antrodia cinnamomea filtrate did not
nal application of the Antrodia cinnamomea filtrate ointment resulted in smaller woundexhibit potential
for reducing
sites compared blood
to theglucose;
controlhowever, it may
group. The affect cinnamomea
Antrodia angiogenesisfiltrate
and inflammation.
did not exhibit po-
With regard to growth factors, we found that Antrodia cinnamomea
tential for reducing blood glucose; however, it may affect angiogenesis and filtrate treatment
inflammation.
resulted in enhanced Ang-1 and Ang-2 levels. The Ang-1 can protect against VEGF-
Life 2022, 12, 507 10 of 12

induced vascular permeability and maintains vascular integrity [33]. The Ang-2 can
stimulate endothelial cell matrix turnover, which allows the endothelial cells to migrate and
differentiate to promote angiogenesis [34]. Ang-2 substantially increases when tissues are
hypoxic (while Ang-1 does not vary in this way), and excessive levels of Ang-2 can cause
apoptosis, resulting in unstable angiogenesis [35]. We postulate that the control group,
which received only oral administration of water, had unstable angiogenesis compared to
their counterparts; when the treatment groups had already passed the onset of angiogenesis,
the control group may still have remained at the initial phase of wound healing. These may
explain why Ang-2 but not VEGF protein significantly increased in the wound area tissue
on the day 7, and how Antrodia cinnamomea filtrate ointment can promote wound healing.
EPO can stimulate angiogenesis and exhibits positive effects on epithelial tissue re-
pair [16], therefore, we also investigated if EPOR is involved. However, Antrodia cinnamomea
filtrate did not appear to be efficacious for stimulating EPOR activity. Therefore, it indi-
cates that Antrodia cinnamomea filtrate does not act on EPOR pathways in stimulating
wound healing. On the other hand, PPARδ was found to be related to glucose hemosta-
sis, inflammation, cell survival, and proliferation [36,37]. Recent study has showed that,
PPARδ agonists suppressed inflammation and promoted neovascularization in the corneal
wound [37]. These effects were key mechanism of skin regeneration. In the present study,
we found that Antrodia cinnamomea filtrate could activate PPARδ expressions, inferring that
may contribute to wound healing. However, more studies targeting pathway downstream
of PPARδ were needed.
Combined treatment with the ointment and oral administration was tested with the
rationale of stimulating diabetic wound healing both internally and externally. Moreover,
this dual approach has been previously examined in studies of combined administration
of oral and topical ointments to treat fungal infections of feet [38]. For each combined
treatment group, the wound sites tended to become smaller in size compared to the diabetes
control group and those received oral administration of Antrodia cinnamomea filtrate without
applying topical ointment.
Based on CD68 measurements, the group that received oral administration of water
without concomitant application of topical ointment had significantly increased inflam-
mation compared to their counterparts, which imply that Antrodia cinnamomea filtrate
has anti-inflammatory efficacy. However, there was little appreciable variation in EPOR,
indicating that the effects of Antrodia cinnamomea filtrate may not occur via EPOR path-
ways. Additional study of the involved mechanisms is, thus, warranted. However, in
this study, we are the first to show that oral Antrodia cinnamomea filtrate can activate the
PPARδ expressions. In conclusion, our study provides evidence that Antrodia cinnamomea
filtrate treatment enhances wound healing. Antrodia cinnamomea filtrate is primarily ac-
tive in vasculature formation and anti-inflammatory efficacy. In combined administration
through oral and topical pathways, the efficacy was not apparently better than through
either route individually. However, overall, Antrodia cinnamomea filtrate showed a tendency
for anti-inflammatory and angiogenesis capabilities, which suggests that it may emerge as
a new pharmaceutical for diabetic wound healing.

Supplementary Materials: The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/life12040507/s1, Figure S1: Addition of the AC extract alone did
not activate macrophages, Figure S2: The effect of AC extracts on the proliferation of RAW 264.7 cell,
Figure S3: Original western blot figures angiogenesis, CD68, EPOR, and PPAR.
Author Contributions: Conceptualization, R.-C.S. and J.-G.L.; methodology, Y.-J.L. and Y.-H.C.;
software, C.-Y.C.; validation, C.-Y.C., Y.-F.Y. and C.-C.Y.; formal analysis, Y.-F.Y.; investigation, C.-C.Y.;
resources, C.-Y.C.; data curation, C.-Y.C.; writing—original draft preparation, Y.-H.C.; writing—
review and editing, J.-G.L.; visualization, Y.-J.L.; supervision, R.-C.S.; project administration, Y.-J.L.;
validation and investigation, S.-H.C.; funding acquisition, J.-G.L. All authors have read and agreed to
the published version of the manuscript.
Life 2022, 12, 507 11 of 12

Funding: This study was partially supported by Shin Kong Wu Ho-Su Memorial Hospital (J.-G.L.,
SKH-8302-104-DR-07).
Institutional Review Board Statement: All procedures involving animals care in this study were
carried out in accordance with the recommendations set forth by the University Committee IACUC
in Fu Jen Laboratory Animal Center (FJU A10526).
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available on request from the
corresponding author.
Conflicts of Interest: The authors declare no conflict of interest.

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