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Evaluation of in vitro
pharmacological activities of
medicinal mushrooms in the
context of dry eye disease
Alexander Areesanan
1
, Andreas Wasilewicz
2
, Sven Nicolay
1
,
Ulrike Grienke
2
, Amy M. Zimmermann-Klemd
1
,
Judith M. Rollinger
2
and Carsten Gründemann
1
*
1
Translational Complementary Medicine, Department of Pharmaceutical Sciences, University of Basel,
Basel, Switzerland,
2
Division of Pharmacognosy, Department of Pharmaceutical Sciences, Faculty of Life
Sciences, University of Vienna, Vienna, Austria
Introduction: Ethnic groups worldwide use mushrooms, particularly polypores (a
group of fungi with woody fruiting bodies), to manage inflammatory conditions.
In this study, the in vitro anti-inflammatory potential and mycochemical
composition of six polypore extracts derived from the fruit bodies of Fomes
fomentarius (L.) Fr. (FF), Ganoderma lucidum (Fr.) P. Karst. (GL), Ganoderma
tsugae Murrill (GT), Gloeophyllum odoratum (Wulfen) Imazeki (GO),
Laricifomes officinalis (Vill.) Kotl. and Pouzar (LO), and the sclerotium of
Inonotus obliquus (Fr.) Pilát (IO) were analyzed for their relevance to treat dry
eye disease (DED).
Methods: Ethanolic extracts of the fungal materials were prepared and
chemically characterized by UHPLC-ELSD/MS and TLC analyses before
investigating the extracts’cytotoxic, antioxidant, anti-inflammatory, and lipid-
stimulating properties. Radical scavenging and intracellular reactive oxygen
species (ROS) assays were carried out in UVB-exposed human corneal
epithelial (HCE-T) and immortalized human meibomian gland epithelial
(IHMGEC) cells to evaluate antioxidant capacities. To examine the influence of
the extracts of the inflammatory processes, associated with DED, a secretion
assay for pro-inflammatory cytokines was conducted in UVB-exposed HCE-T
and LPS-stimulated monocytic THP-1 cells. The lipid droplets secreted by
IHMGECs were analyzed to determine the extracts’lipid-stimulating properties.
Results: Extracts of GT, GL, GO, and IO found to have high radical scavenging
abilities. They significantly reduced intracellular ROS in UVB-exposed HCE-T and
iHMGEC cells. GO and GL extracts inhibited cytokine secretion in HCE-T cells
even at low concentrations. All tested extracts significantly inhibited the secretion
of pro-inflammatory cytokines (IP10, IL-6, IL-8, and α) in LPS-stimulated
monocytic THP-1 cells.
OPEN ACCESS
EDITED BY
Fabio Boylan,
Trinity College Dublin, Ireland
REVIEWED BY
Angela Bisio,
University of Genoa, Italy
Valdir Veiga,
Military Institute of Engineering, Brazil
*CORRESPONDENCE
Carsten Gründemann,
carsten.gruendemann@unibas.ch
RECEIVED 08 January 2025
ACCEPTED 05 February 2025
PUBLISHED 05 March 2025
CITATION
Areesanan A, Wasilewicz A, Nicolay S,
Grienke U, Zimmermann-Klemd AM,
Rollinger JM and Gründemann C (2025)
Evaluation of in vitro pharmacological activities
of medicinal mushrooms in the context of dry
eye disease.
Front. Pharmacol. 16:1557359.
doi: 10.3389/fphar.2025.1557359
COPYRIGHT
© 2025 Areesanan, Wasilewicz, Nicolay,
Grienke, Zimmermann-Klemd, Rollinger and
Gründemann. This is an open-access article
distributed under the terms of the Creative
Commons Attribution License (CC BY). The use,
distribution or reproduction in other forums is
permitted, provided the original author(s) and
the copyright owner(s) are credited and that the
original publication in this journal is cited, in
accordance with accepted academic practice.
No use, distribution or reproduction is
permitted which does not comply with these
terms.
Abbreviations: DED, dry eye disease; DPPH, 2,2-diphenyl-1-picrylhydrazyl; EGF, epidermal growth
factor; FF, F. fomentarius; GO, G. odoratum; GL, G. lucidum; GT, G. tsugae; HBSS, hank’s balanced Salt
solution; HCE-T, human corneal epithelial cell-transformed; H2DCFDA, 2′,7′-
dichlorodihydrofluorescein diacetate; IHMGECs, Immortalized human meibomian gland epithelial
cells; IL, interleukin; NF-κB, nuclear factor-kappa B; IO, I. Obliquus;LO,L. officinalis; PBS,
phosphate-buffered saline; THP-1, human leukemia monocytic cell line; UVB, ultraviolet B; WST-1,
water-soluble tetrazolium salt.
Frontiers in Pharmacology frontiersin.org01
TYPE Original Research
PUBLISHED 05 March 2025
DOI 10.3389/fphar.2025.1557359
Conclusion: Several extracts of the investigated fungal materials exhibit
multifaceted pharmacological in vitro activities. Due to low cytotoxic activity on
HCE-T, iHMGEC, and THP-1 cells, extracts from GL and GO are particularly
pertinent to the treatment of DED, even at low concentrations.
KEYWORDS
dry eye disease, antioxidant, inflammation, reactive oxygen species, proinflammatory
cytokines, polypore, medicinal mushroom
1 Introduction
Dry eye disease (DED) is a widespread multifactorial condition
that affects 5%–50% of the population worldwide, especially elder
women. Prevalence is steadily increasing, and the condition is rising,
becoming one of the top public health concerns (Papas, 2021). DED
is characterized by either hyper-evaporative tears (10% of cases), or a
lack of the quality of the tear film (80% of cases), or a combination of
both (10% of cases) (Rouen and White, 2018;Tsubota et al., 2017).
Symptoms of DED can occur individually or in combination and
include visual disturbance, grittiness, itchiness, and burning
sensation. DED symptoms make it difficult for the patients to
perform any tasks efficiently (Sakane et al., 2013) and thus
severely impair the quality of life (QOL). Without proper
treatment, symptoms tend to worsen (Baudouin et al., 2014). The
total economic burdens due to reduced productivity and medical
care as a result of DED has been estimated to be over $55 billion in
the United States (Yu et al., 2011) and over $100 billion in China
(Yang et al., 2021).
It is believed that inflammation, which is linked to
hyperosmolarity and tear film instability, is the key cause of DED
pathogenesis (Wei and Asbell, 2014). Unlike other mucosal surfaces
in the body, the microenvironment of the ocular surface is
constantly exposed to noxious insults such as pathogens,
pollution, and harmful substances that can disrupt the ocular
homeostasis. Such conditions severely stress the ocular surface by
increasing hyperosmolarity and tear film instability, and lead to
reactive oxygen species (ROS) formation, setting off a cascade of
several inflammatory cytokines followed by apoptosis (Bron et al.,
2017). Studies have demonstrated that DED is characterized by
inflammatory cytokines, specifically interleukin (IL)-1, 6 and 8, and
tumor necrosis factor (TNF)-α, on the ocular surface. These
cytokines can disrupt the corneal epithelial barrier, reduce its
physical protection and trigger further apoptosis (Massingale
et al., 2009;Wu et al., 2020). Thus, the removal of ROS and an
increase in antioxidant levels can be an approach for treating DED.
It has been found that therapeutic remedies, such as
corticosteroid, anti-inflammatory treatment, and artificial tears,
can alleviate DED pathologic symptoms (Barabino et al., 2012;
Lin and Yiu, 2014). However, side effects occurred in over 65%
of treated patients and relief was not long-lasting (Barabino et al.,
2012). Owing to the previously described reasons, a novel
therapeutic DED treatment is needed.
For centuries, mushrooms have been an important food source
for animals and microorganisms in the ecosystem. A vast number of
them also have important pharmacological effects due to their
unique constituents (Bhambri et al., 2022). The main constituents
of medicinal mushrooms are polysaccharides, phenols, and terpenes,
which have antibacterial, antidiabetic, antioxidant, anticancer, anti-
inflammatory, and immunomodulatory properties (Elkhateeb
et al., 2019).
In this study, six ethanolic extracts were prepared from the
medicinal mushrooms Fomes fomentarius, Ganoderma lucidum,
Ganoderma tsugae, Gloeophyllum odoratum, Inonotus oliquus,
Laricifomes officinalis (also known as Fomitopsis officinalis). Most
of them are widely known for their traditional use (Supplementary
Table ST1). The continuously growing body of pharmacological
studies highlights the biological effects of fungal extracts, thereby
substantiating the traditional medicinal uses of mushrooms: F.
fomentarius and its bioactive compounds have been reported in
the literature to have anti-bacterial (Dresch et al., 2015), anti-cancer
(Chen et al., 2008;Kim et al., 2015;Zang et al., 2013), anti-diabetic
(Lee, 2005), and anti-inflammatory properties (Park et al., 2004).
The use of G. lucidum is widespread in Asian countries and it has a
long medical history in China for the prevention and treatment of
various diseases (Wang et al., 2020). A large number of commercial
G. lucidum products in various forms, such as powders, dietary
supplements and teas made from different parts of the fungus
(mycelium, spores and fruiting bodies), are available on the
market (Wachtel-Galor et al., 2012). According to the
Pharmacopoeia Commission China, the fungus is said to provide
relief from nervousness, coughing, insomnia and anorexia
(Committee, 2000). It can also be used to regulate blood sugar
levels and the immune system, as well as to protect the liver
(Wachtel-Galor et al., 2012). G. tsugae is closely related to G.
lucidum. However, while G. lucidum grows wild only in Asia and
parts of Europe (on broad-leaved trees), G. tsugae occurs naturally
only in North America on conifers (Luangharn et al., 2021;Zhao
et al., 2019). It has been described to possess anti-cancer (Chien
et al., 2015;Wang et al., 1993), immunomodulating (Lai et al., 2001;
Lin et al., 2006;Won et al., 1992), and wound-healing effects (Su
et al., 1999;Yap et al., 2023). The fungus G. odoratum has not been
widely studied in terms of traditional use and biological-therapeutic
potential. However, it has been shown that its thrombin-inhibiting
effect is beneficial in preventing blood clotting (Cateni et al., 2015). It
has also been demonstrated to neutralise the H3N2 influenza virus
(Grienke et al., 2019), scavenge free radicals and suppress
phagocytosis (Doskocil et al., 2016). Moreover, G. odoratum is
rich in fumaric acid (Tel-Çayan, 2019), which is known for its
strong antioxidant and antimicrobial properties (Barros et al., 2013;
Ribeiro et al., 2008). The sclerotium of Inonotus obliquus contain a
high level of antioxidants (Lee et al., 2007). The therapeutic effects of
the fungus regarding anti-tumour (Mizuno et al., 1999;Taji et al.,
2008), immunomodulating (Chen et al., 2019;Kim, 2005), and anti-
inflammatory effects (Debnath et al., 2013;Hu et al., 2016;Javed
et al., 2019) have been demonstrated in vitro and in vivo.L. officinalis
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Areesanan et al. 10.3389/fphar.2025.1557359
is considered one of the most commonly used medicinal mushroom
in European folk medicine (Grienke et al., 2014). Since 2019, L.
officinalis has been classified as endangered fungal species by the
International Union for Conservation of Nature (IUCN) (Kałucka
and Svetasheva, 2019). Studies have found a wide range of
therapeutic benefits including antiviral (Stamets, 2005),
antioxidant (Sha, 2016;Wu et al., 2004), and antimicrobial
(Girometta, 2019;Grienke et al., 2014) effects.
2 Material and methods
2.1 Fungal material and extraction
The fungal materials were either collected and identified by U.
Peintner (University of Innsbruck, Austria) or purchased from
commercial providers (Supplementary Table ST2). Voucher
specimens are deposited at the Division of Pharmacognosy,
Department of Pharmaceutical Sciences, University of Vienna,
Austria. The taxon names were verified by Mycobank (https://
www.mycobank.org/, accessed on 02/04/2024). For each fungal
material, approximately 300 mg were extracted twice with 10 mL
of 96% (v/v) ethanol in an ultrasonic bath. The obtained extracts
were dried using a rotary evaporator at 40°C.
2.2 Mycochemcial analysis
UHPLC analysis was conducted on a Waters Acquity UPLC
H-Class system equipped with a sample manager, quaternary
solvent manager, column manager, PDA detector, ELSD, an
isocratic solvent manager, and a quadrupole MS detector (Waters
Acquity QDa) with electrospray ionization (ESI) source. For the
chromatographic runs, a BEH C
18
column (1.7 µm, 2.1 × 100 mm,
Waters) at 40°C was used. The flow rate was set to 0.3 mL/min with
water +0.1% formic acid (A) and acetonitrile + 0.1% formic acid (B)
as mobile phases. The following gradient was used: 5% B at 0.0 min,
5%–98% B in 12 min and 98% B for 4 min. Mass detection was
performed in positive (cone voltage: 15 V, capillary voltage: 0.8 kV)
and negative (cone voltage: 30 V, capillary voltage: 0.8 kV) modes
covering a mass range of 100–1,200 Da. A mixture of methanol:
water (9:1) + 10 mM ammonium formate was used as a make-up
solvent to increase the ionization of investigated metabolites. The
instrument was controlled by the software Empower 3.
TLC was performed on Merck silica gel 60 PF254 plates. The
mobile phase consisted of dichloromethane:methanol:water (10:1:
0.25, v/v/v) and was analyzed at visible light after derivatization with
vanillin (1% in methanol)/sulfuric acid (5% in methanol).
2.3 HCE-T cell culture
The human corneal epithelial cell-transformed (HCE-T) cell line
was obtained from the Riken Cell Bank (RCB2280). Following the
provider’s instruction as reported previously (Areesanan et al.,
2023). HCE-T cells were cultured in DMEM/F-12 medium
(Sigma-Aldrich) supplemented with 5% FCS (BioConcept), 5 μg/
mL insulin, 10 ng/mL human epidermal growth factor (EGF), 0.5%
DMSO, and 1% penicillin-streptomycin (all from Sigma-Aldrich) at
37°C with 5% CO
2
. The cells were trypsinised with 0.05% trypsin-
EDTA solution (Thermo Scientific) at ≥85% confluence. Unless
stated otherwise, cells were incubated with the culture medium in a
96-well plate at 2 × 10
4
cells/well for 24 h to reach over 90%
confluence before each experiment. After washing, the medium
was substituted with a phenol red-free, non-supplemented,
serum-free medium for 24 h to starve the cells before an
experiment was conducted.
2.4 Free-radical scavenging activity
The free-radical scavenging capacity of the mushroom extracts
was tested using a cell-free colorimetric assay based on 2,2 diphenyl-1-
picrylhydrazyl (DPPH; Sigma-Aldrich). DPPH changes the colour
from violet to pale yellow when the DPPH radical reacts with a
hydrogen atom source or electron donor. The study was conducted
according to Brand-Williams and colleagues’protocol with minor
modifications (Brand-Williams et al., 1995). To achieve a final
concentration of 100 nM of DPPH in a 96-well plate, ethanolic
DPPH was thoroughly mixed with a range of concentrations of
the mushroom extracts (final concentrations: 1–100 μg/mL). The
absorbance was measured spectrophotometrically at 517 nm using a
TECAN infinite M Plex after incubation for 30 min at RT in the dark,
and the scavenging efficiency was estimated as follows:
Free radical scavenging proficiency %
()
blank −sample
blank × 100%
where blank is the DPPH reagent absorbance without any test
sample. A rich water-soluble vitamin E derivative, Trolox
(100 μM, Tocris Bioscience), was used as a positive control.
2.5 Cell viability assay for HCE-T cells
The viability of HCE-T cells treated with the mushroom extracts
was determined based on the relative mitochondrial function, as
previously reported (Areesanan et al., 2023). The assay is based on
the conversion of a tetrazolium salt (WST-1; Sigma-Aldrich) to
formazan by cellular mitochondrial dehydrogenases, which is
measured by a colour change. Once the HCE-T cells were
seeded, grown, and starved in a 96-well plate as mentioned
above, the cells were incubated with various concentrations of
samples (0.3–100 μg/mL) for 24 h at 37°C with 5% CO
2
.
Afterwards, the cells were washed with phosphate-buffered saline
(PBS; Sigma-Aldrich), incubated with WST-1 (1:10) for 2 h at 37°C
and then analyzed spectrophotometrically at 450 nm, with 650 nm
as reference using a TECAN infinite M Plex. Triton X-100 (5% v/v,
Sigma-Aldrich), a detergent used to lyse cells, was used as a
positive control.
2.6 HCE-T intracellular ROS measurement
The intracellular ROS levels of UVB-exposed HCE-T cells were
determined by the emitted signal of 2′,7′-dichlorodihydrofluorescein
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diacetate (H
2
DCFDA; Invitrogen) using the method, as previously
described (Areesanan et al., 2023). Briefly, HCE-T cells were
incubated with 25 μMH
2
DCFDA probe for 30 min at 37°C,
washed, and treated with the mushroom extracts (1–30 μg/mL) for
10 min at 37°C. Subsequently, the treated cells were exposed to UVB
radiation at 312 nm, an intensity of 5.45 mW/cm
2
,andadoseof
50 mJ/cm
2
(Vilber Lourmat) and incubated for a further 30 min at
37°C. The fluorescence intensity (488/530 nm) was measured
spectrophotometrically using a TECAN infinite M Plex. Trolox
(100 μM, Tocris), a potent antioxidant, was used as a positive control.
2.7 HCE-T wound healing assay
In this study, HCE-T cells were scratched and treated with the
fungal extracts to determine the efficacy of wound healing,
according to the reported method (Areesanan et al., 2023). HCE-
T cells (2 × 10
4
cells/well of 96-well plate) for 24 h in the culture
medium until confluence was reached. The medium was then
replaced for 24 h with a supplement-reduced medium (DMEM/
F-12 medium supplemented with 1% penicillin-streptomycin and
5% FCS) to delay the cell migration. Using AutoScratch (BioTek),
consistently cell-free (wound) gaps were made across each well. The
wells were washed twice to remove detached cells before treating the
cells overnight with the supplemented-reduced medium mixed with
different concentrations of the samples (3–30 μg/mL). The images of
the gap areas were taken at 17 h post-scratch using an inverted
microscope (Zeiss) and the gap closure was quantified using I Fiji
ver. 2.14.0/1.54f (Schindelin et al., 2012) with an open-source
Wound Healing Size Tool plugin (Suarez-Arnedo et al., 2020). An
alkyl hydroperoxide, tert-Butyl hydroperoxide (25 μM, Sigma-
Aldrich), was used as a control that impedes wound healing.
2.8 HCE-T TEER and fluorescein
permeability assay
HCE-T cells (3 × 10
4
cells) were inoculated with 400 μLof
culture medium onto a translucent polyester (PET) membrane
transwell insert (0.4 μm pore size; Greiner bio-one), and 1 mL of
culture medium was added into the basolateral chamber. The cells
were kept for 96 h at 37°C. Following washing twice, the cells were
treated with the samples cultured in a phenol red-free non-
supplemented serum-free medium. Over the course of 72 h,
transepithelial electrical resistance (TEER, CellZscope) was
measured every 24 h. Afterwards, the cells were washed twice
with Hank’s Balanced Salt solution (HBSS; Gibco), before 50 μM
of sodium fluorescein (Sigma-Aldrich) was added into the inserts
and 1 mL of HBSS was added into the basolateral chamber. After
incubating the cells for 2 h at 37°C, 200 μL of the basolateral side’s
media was taken and spectrometrically quantified at 490 nm
(TECAN unlimited M Plex).
2.9 HCE-T secreted cytokine quantification
After a 24-h fasting period in a 96-well plate containing 2 × 10
4
cells/well, the HCE-T cells were treated with samples (3–30 μg/mL)
for 10 min at 37°C. The cells were then subjected to a UVB lamp
(312 nm, 5.45 mW/cm2, 50 mJ/cm2, Vilber Lourmat) and were
incubated for an additional 24 h at 37°C. The supernatants were
collected and stored at −80°C. To quantify the secreted mediators
(IL-6, TNF-α, and IL-8), the experiment was conducted following
the manufacturer’s instructions for the multiplex bead-based assay
kit (LEGENDplex™, BioLegend), which based on the principle of
capture bead-analyte-detection antibody sandwiches bound with
streptavidin-phycoerythrin (SA-PE). The bead captures signified the
types of the mediators and the fluorescent intensity was measured
through SA-PE. The analysis was conducted using a flow cytometer
(Beckman Coulter) and FlowJo software. Dexamethasone (10 μM,
Sigma-Aldrich) was used as a positive control to inhibit
cytokine secretion.
2.10 THP-1 NFκB-eGFP cell culture
Human monocytic THP-1 NFκB-eGFP reporter cells (Merck)
were used to depict the mushroom extracts’effects on immune cells.
The cell line was cultured in RPMI-1640 medium supplemented
with 2 mM L-glutamine (Sigma-Aldrich), 10% heat-inactivated FCS
(BioConcept), and 1% penicillin-streptomycin (Sigma-Aldrich). The
cells were maintained at 37°C with 5% CO
2
atmosphere.
2.11 Cell viability assay for THP-1 cells
WST-1 assay was used to determine the impact of the
mushroom extracts on the viability of the THP-1 cells. In a 96-
well plate, THP-1 (5 × 10
4
cells/well) were seeded together with the
extracts (0.3–100 μg/mL) in a serum-free media for 24 h at 37°C. The
treated cells were centrifuged at 300 g for 5 min to remove the
supernatants, washed twice, and the cells were further incubated
with 1:10 WST-1 for 2 h at 37°C. The absorbance was then measured
(450 nm/650 nm as a reference; TECAN infinite M Plex). Triton-X-
100 (1% v/v, Sigma-Aldrich) was used as a positive control.
2.12 THP-1 secreted cytokine quantification
In serum-free media together with lipopolysaccharide (LPS,
1μg/mL), THP-1 cells (5 × 10
4
cells/well of 96-well plate) were
incubated with the samples (3–30 μg/mL) for 48 h at 37°C. The
supernatants were collected and stored at −80°C until further
analysis. Detection of supernatant’s mediators (IP10, IL-8, TNF-
α, and IL-6) was achieved through a multiplex bead-based flow
cytometry assay (LEGENDplex, BioLegend) similar to the analysis
of the HCE-T supernatants. Dexamethasone (DEX, 10 μM, Sigma-
Aldrich) was used as a positive control.
2.13 IHMGECs cell culture
Immortalized human meibomian gland epithelial cells were
incubated at 37°C with 5% CO
2
in culture medium (Keratinocyte
Serum Free Serum (FM) (1X), EGF Human Recombinant, Bovine
Pituitary Extract; all from Thermo Fisher Scientific). Before each
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experiment, the cells were induced to a mature stage. To induce
differentiation, cells were grown in a serum-containing medium
which consisted of DMEM/F-12 medium (Sigma- Aldrich),
supplemented with 4% FCS (BioConcept), and 10 ng/mL human
EGF (Sigma-Aldrich) for 24 h at 37°C with 5% CO
2
.
2.14 Cell viability assay for IHMGECs
The cytotoxicity of the samples was evaluated using the WST-1
colorimetric assay. IHMGECs (ATCC; 2 × 10
4
cells/well) were seeded
in a 96-well plate and incubated at 37°Cwith5%CO
2
for 72 h in the
culture media. After the cells were washed, the cells were incubated
with the serum-containing medium mixed with the samples
(0.3–100 μg/mL) for 24 h followed by 2 h with WST-1 (Sigma-
Aldrich) at 37°C. The metabolic activity was quantified via scanning at
450 nm with 650 nm as a reference for the spectrometric analysis.
Triton-X-100 (5% v/v, Sigma-Aldrich) was used as a positive control.
2.15 IHMGECs intracellular ROS
measurement
IHMGECs (2 × 10
4
cells/well) were cultured in a 96-well plate for
72 h in the culture medium. Afterwards, the cells were washed and
incubated in the 4% differentiation medium for 24 h at 37°C. The
cells were washed at incubated with 10 μMH
2
DCFDA (Invitrogen)
for 30 min at 37°C in the 0% FCS differentiation medium, before
treating with or without the fungal extracts for 10 min at 37°C. The
cells were then irradiated with the UVB (312 nm, 5.45 mW/cm
2
,
50 mJ/cm
2
; Vilber Lourmat). After further incubation for 30 min, the
emitted signal was spectrophotometrically measured (488/530 nm;
TECAN infinite M Plex). Trolox (100 μM, Tocris) was used as a
positive control.
2.16 Lipid staining of meibomian gland cells
With a few minor modifications, LipidTOX (Invitrogen) was
utilised to monitor the effects of the samples on the cells and lipid
synthesis, following the description of Liu and colleagues (Liu et al.,
2014) with modifications. IHMGECs (3.5 × 10
4
cells/well) were
incubated on a sterile glass coverslip in each well of a 24-well plate
for 72 h at 37°C with the growth medium. The cells were treated for
24 h (3 and 30 μg/mL or 1 and 10 μg/mL) in the 4% FCS
differentiation medium after being washed with PBS. The control
groups were incubated in a differentiation medium containing 10%
FCS to strongly stimulate lipid synthesis. After a 24 h period, the
cells were rinsed and fixed with cold 4% paraformaldehyde (PFA,
Electron Microscopy Sciences) for 15 min at room temperature, and
then counter-stained for 30 min at RT in the dark using a mixture of
LipidTox (1:500, Invitrogen) and DAPI (400 nM, Invitrogen). Once
the cells had been thoroughly rinsed, the coverslips were taken off
the wells and ProlongTM Diamond Antifade Mountant (Thermo
Fisher Scientific) was used to mount the cells on a glass slide. The
cells were then kept in storage at 4°C. Each sample was subjected to a
total of 5 random photographs using a widefield fluorescence
microscope (Nikon Ti2).
Using Fiji (ver. 2.14.0/1.54f), the lipid droplets:cells ratio and
their size were measured to evaluate the lipid synthesis of IHMGECs
(Schindelin et al., 2012). In summary, each sample’s Z-position
pictures were combined into a single image using the maximum
intensity pixels criterion and stored in the 16-bit.tif format at
5.45 DPI. Rolling ball background subtraction algorithms were
applied with a radius of 8 pixels for the droplets and a radius of
4 pixels for the background to create images of reduced background
noise and a blurred background, respectively, in order to adjust the
uneven background intensity and remove areas with spatial intensity
variations. To clearly distinguish between the two, the decreased
background noise image was subtracted from the blurred
background image. The reduced background noises image was
subtracted from the blurred background image to provide an
intensity value of 0 for the droplets, which allowed them to be
easily distinguished from the background. After that, the invert
function was used to give the droplets more attention, resulting in all
of the droplets having larger values than the others. This was
followed by the implementation of a second rolling ball function
with 4-pixel radius. Gaussian blur function then was used to remove
any dust-effect particles followed by another rolling ball function
with 8-pixel radius. To generate a mask, the auto-threshold function
default was used. Then, with three erosion cycles, the clumped lipid
droplets were separated using the Watershed Irregular Features
algorithm. Ultimately, the analyze Particles function was used to
count and analyze each droplet. Similarly, to count the number of
cells in each sample’s picture and to separate clumped DAPI-stained
cells, a mask was made using the Watershed function.
2.17 Statistical analysis
Statistical data analysis was performed using PRISM version
10.3.1 (GraphPad software). Every experiment was carried out
separately at least three times, and the values that are given are
the means ± standard deviation. One-way ANOVA was used to
assess statistical significance, along with Dunnett’s test and the
Brown-Forsythe test to ensure homogeneity of variance.
Statistical significance was assumed at *p <0.05, **p <0.01,
***p <0.001, ****p <0.0001.
2.18 Declaration of generative AI and AI-
assisted technologies in the writing process
During the preparation of this work the authors used DeepL
Translator in order to improve language. After using this tool/
service, the authors reviewed and edited the content as needed and
take full responsibility for the content of the publication.
3 Results
3.1 Mycochemical profiling of
fungal extracts
The six fungal extracts of the fruit bodies of F. fomentarius (FF),
G. lucidum (GL), G. tsugae (GT), G. odoratum (GO),L.officinalis
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Areesanan et al. 10.3389/fphar.2025.1557359
(LO), and the sclerotium of I. obliquus (IO) were analyzed by
UHPLC-ELSD/-MS and TLC to gain insights into their
mycochemical composition (Figure 1;Supplementary Figure
SF1). The main constituents found in the ELSD chromatograms
were characterized based on the recorded MS data and literature
(Table 1). In extract GL, highly oxygenated lanostane triterpenes
(1–6) were tentatively annotated as the main constituents which are
well-known secondary metabolites of G. lucidum fruit body in
literature (Wu et al., 2017). In contrast, extracts GT, GO, IO and
LO consist of more lipophilic constituents which is reflected by the
later RTs of these compounds. For extract GT, three compounds
were tentatively annotated as acetoxylated triterpenes (7–9) and
their potential molecular formulas were proposed. One triterpene
was found in the extracts GO and IO and was annotated as
trametenolic acid B (11)(Grienke et al., 2019). A second
compound co-eluting with the peak of trametenolic acid B was
dereplicated as inotodiol (12) in the extract IO. Both triterpenes are
known to be the major compounds in I. obliquus fruit body (Kim
et al., 2020). In addition, another triterpene (10) was tentatively
annotated in extract GO for which a molecular formula was
established. Considering its rather short retention on the column,
it might be a more hydrophilic triterpene but could not be
dereplicated by literature. In extract LO, malonylated triterpenes
(13–15) were annotated based on MS data and literature (Han et al.,
2016). No triterpenes were detected in extract FF which only showed
a large peak in the beginning of the ELSD chromatogram as it is the
case for GL and LO. Within that peak, m/z values of 365.1 [M + Na]
+
in positive mode and 341.1 [M-H]
-
in negative mode were detected
and assigned to a disaccharide which might be derived from
polysaccharides upon MS ionisation. Besides low molecular
weight secondary metabolites such as triterpenes, polysaccharides
are among the most investigated chemical compounds of polypores
with various reported biological properties (Grienke et al., 2014). In
addition, the TLC fingerprint analysis revealed purple spots after
spraying with vanillin/sulfuric acid in each extract, suggesting the
presence of terpenoids at least as minor constituents in all extracts.
3.2 Toxicity, antioxidant properties and
wound healing capacity of the
mushroom extracts
In the first step, cytotoxic concentrations of the fungal extracts
were determined. For this purpose, a viability test was performed
FIGURE 1
UHPLC-ELSD chromatograms of fungal extracts containing annotated constituents.
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after 24 h of incubation of HCE-T cells with the fungal extracts.
Triton-X 100 (1% v/v) was used as a positive control that
significantly reduced the viability of the HCE-T cells. FF, GL,
and GT significantly reduced the viability of HCE-T cells at a
concentration of 100 μg/mL (Figure 2A). Among them, GT
showed the highest toxicity with cell viability comparable to
Triton-X 100 (Figure 2A). LO is highly toxic to HCE-T cells as
at concentrations of 30–100 μg/mL, the viability of HCE-T cells was
significantly below 50% (Figure 2A). Therefore, the concentration
range of LO was adjusted hereinafter and lower concentrations were
used for the functional tests. GT and IO showed no toxicity over the
entire concentration range (0.3–100 μg/mL) (Figure 2A).
Exposure to UVB can lead to an accumulation of ROS and
subsequently to corneal damage and inflammation. Therefore, the
ability of the mushroom extracts to reduce ROS as well as their
radical scavenging capacity was investigated. All mushroom extracts
exhibited free radical scavenging capacity in a concentration-
dependent manner (Figure 2B). However, for the extracts FF, GL
and LL, this increase was minor and without significant effects. For
GT, a significant free radical scavenging capacity was observed at
concentrations of 30 and 100 μg/mL while for GO and IO, the free
radical scavenging capacity was significant in the concentration
range of 3–100 μg/mL.
All mushroom extracts decreased the generation of ROS in
HCE-T cells concentration-dependently compared to the
untreated control (Figure 2C). In a dose-dependent manner, the
strongest ROS reduction was observed in IO followed by GT, GO,
and GL. Significantly lower ROS levels were obtained for 30 μg/mL
of GT and 30 and 10 μg/mL of IO (Figure 2C).
Since corneal damage is one of the symptoms of DED, the
influence of the extracts on the wound healing capacity of HCE-T
cells was also investigated below. None of the extracts significantly
promoted wound healing compared to the untreated control (NC)
(Figure 2D). However, slight concentration-dependent positive
effects on wound healing were observed for GL and GT
(Figure 2D). Nevertheless, these effects did not become
significant.
3.3 Effects of the fungal extracts on the TEER
of HCE-T cells
One of the symptoms of DED is the appearance of corneal
membrane damage. To evaluate the use of mushroom extracts in
DED, their effect on the tight junctional connection of HCE-T cells
was measured by TEER. Therefore, cells were incubated with the
extracts for 72 h and the TEER was measured every 24 h.
Benzalkonium chloride (BAC) disrupts tight junctions and was
therefore used as a control. The influence of the fungal extracts
on the TEER of HCE-T cells is somewhat divergent. While GL
(Figure 3A) and GO (Figure 3B) slightly decreased the TEER
compared to the untreated control (NC), treatment with LO
(Figure 3C) led to a significantly increased TEER. The TEER
after treatment with FF (Figure 3A), GT (Figure 3B) and IO
(Figure 3C) was comparable to the untreated control (NC). The
fluorescein permeability of HCE-T cells (Figure 3D) was largely
comparable to that of the untreated control (NC). Only GL and GO
showed slightly increased values.
TABLE 1 Tentative MS-based annotation of constituents detected in the investigated fungal extracts (as labelled in Figure 1).
No.
RT
[min]
Positive mode [m/z] Negative mode [m/z] Molecular
formula
Putative
annotation
17.04 497.3 [M + H-2x H
2
O]
+
, 515.3 [M + H-H
2
O]
+
, 555.3 [M
+Na]
+
495.4 [M-H-2x H
2
O]
-
, 513.4 [M-
H-H
2
O]
-
C
30
H
44
O
8
ganoderic acid G
27.14 499.3 [M + H-H
2
O]
+
, 517.2 [M + H]
+
, 539.3 [M + Na]
+
497.4 [M-H-H
2
O]
-
, 515.3 [M-
H]
-
C
30
H
44
O
7
ganoderic acid A or B
37.38 573.3 [M + H]
+
, 595.3 [M + Na]
+
553.4 [M-H-H
2
O]
-
C
30
H
44
O
9
ganoderic acid H
47.44 499.3 [M + H-H
2
O]
+
, 539.3 [M + Na]
+
515.4 [M-H]
-
C
30
H
44
O
7
ganoderic acid A or B
57.95 497.3 [M + H-H
2
O]
+
, 515.3 [M + H]
+
, 537.3 [M + Na]
+
495.3 [M-H-H
2
O]
-
C
30
H
42
O
7
ganoderic acid C or D
68.14 571.3 [M + H]
+
, 593.3 [M + Na]
+
551.38 [M-H-H
2
O]
-
C
32
H
42
O
9
ganoderic acid F
712.42 493.4, 535.3 [M + H- CH
3
COOH-H
2
O]
+
, 630.5 [M +
NH
4
]
+
635.4 [M + Na]
+
-C
36
H
52
O
8
threefold acetoxylated
triterpene
813.12 435.4 [M + H-2x CH
3
COOH]
+
, 495.5 - C
34
H
50
O
6
twofold acetoxylated
triterpenes
913.43 [M + H-CH
3
COOH]
+
, 577.4 [M + Na]
+
10 7.54 467.4, 485.3, 525.3 [M + Na]
+
501.4 [M-H]
-
C
30
H
46
O
6
highly oxygenated
triterpene
11 13.65 439.4 [M + H-H
2
O]
+
, 457.3 [M + H]
+
-C
30
H
48
O
3
trametenolic acid B
12 13.65 425.4 [M + H-H
2
O]
+
-C
30
H
50
O
2
inotodiol
13 10.60 571.4 [M + H-H
2
O]
+
, 611.4 [M + Na]
+
543.5, 587.5 [M-H]
-
, 609.5 C
35
H
56
O
7
malonylated triterpene
14 11.75 585.4 [M + H-H
2
O]
+
, 625.4 [M + Na]
+
601.5 [M-H]
-
C
35
H
53
O
8
officimalonic acid E
15 12.73 567.4 [M + H-H
2
O]
+
, 607.4 [M + Na]
+
-C
34
H
48
O
8
officimalonic acid B
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3.4 Effects of mushroom extracts on the
cytokine secretion of HCE-T cells
In the following, the influence of the mushroom extracts on
the secretion of the pro-inflammatory cytokines IL-6, TNF-αand
IL-8 by HCE-T cells was investigated. In addition to its
inflammatory effect, IL-6 accelerates wound healing of HCE-T
cells and TNF-αalso promotes re-epithelialisation and cell
proliferation depending on IL-6 and IL-8. The HCE-T cells
were exposed to UVB and treated with the fungal extracts for
24 h. Subsequently, IL-6, TNF-αand IL-8 levels were determined
by Legendplex™assay.
In contrast to other extracts, FF-treated HCE-T increased the
pro-inflammatory cytokines levels in a concentration-dependent but
not significant manner (Figure 4A). IO increased IL-6 secretion only
at the highest concentration (30 μg/mL) tested, while GL and GO
inhibited the IL-6 secretion of HCE-T cells (Figure 4A). For GO, this
inhibition was significant at concentrations of 3 and 30 μg/mL
(Figure 4A). Moreover, inhibition of IL-8 secretion was observed
after treatment with GL and GO (Figure 4B). For GO, this inhibition
was concentration-dependent with significances at concentrations
of 3 and 30 μg/mL (Figure 4B). In addition, FF increased TNF-α
secretion in a concentration-dependent manner, but not
significantly (Figure 4C), whereas GL, GO, and IO inhibited the
secretion of TNF-α(Figure 4C). Significant effects were observed for
10 μg/mL of GL, 30 μg/mL of GO, and 3 and 10 μg/mL of
IO (Figure 4C).
3.5 Effects of mushroom extracts on the
cytokine secretion of THP-1 cells
Since the clinical picture of DED is characterized by
inflammation of the cornea, the influence of the fungal extracts
on the THP-1 immune cell line was investigated below. First,
cytotoxic concentrations were determined analogously to the
HCE-T cells. For GL and GT, the concentrations 30 μg/mL and
100 μg/mL proved to be significantly cytotoxic for the THP-1 cells
(Figure 5A). Significant cytotoxic effects also occurred for FF and
GO at a concentration of 100 μg/mL (Figure 5A). In THP-1 cells, LO
showed a concentration-dependent reduction of cell viability
between 10 and 100 μg/mL (Figure 5A). However, none of the
tested concentrations was significantly cytotoxic (Figure 5A). IO was
well-tolerated in the entire concentration range (Figure 5A).
FIGURE 2
Effects of fungal extracts on the viability (A), radical scavenging capacity (B), intracellular ROS level (C), and wound healing (D) of HCE-T cells. (A)
HCE-T cells were incubated for 24 h with medium (NC), Triton X-100 (Triton-X; 1% v/v), DMSO (vehicle) or the fungal extracts. The number of viable cells
was determined and normalised to the untreated control and expressed as mean ± standard deviation. (B) In a cell-free assay, DMSO (vehicle) and the
mushroom extracts were incubated with DPPH (100 µM) for 30 min and the free radical scavenging capability was quantified. Trolox was used as
positive control. The mean fluorescence intensity was determined and plotted ±standard deviation. (C) HCE-T cells were treated with H
2
DCFDA (25 µM)
for 30 min and incubated with the fungal extracts for 10 min before exposure to a UV lamp. The fluorescence intensity was measured and the values were
normalised to the control (UVB) and presented as mean ± standard deviation. (D) HCE-T cells were scratched and incubated overnight with different
concentrations of mushroom extracts. Images were taken with a digital camera and analysed. The wound size was normalised to the control (untreated)
and expressed as mean ± standard deviation. All experiments were independently repeated (n = 3) and *p <0.05, **p <0.01, ***p <0.001, ****p <0.0001.
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NF-κB is important for the signal transduction required for the
inflammatory function of THP-1 cells. For this reason, the influence
of the fungal extracts on the expression of NF- κB in THP-1 cells was
evaluated. Except for IO, all extracts inhibited NF-κB expression in a
concentration-dependent manner (Figure 5B). For FF, GO and LO,
this inhibition was significant at a concentration of 30 μg/mL
(Figure 5B). A significant inhibition of NF-κB expression was
observed for GT at concentrations of 10 and 30 μg/mL
(Figure 5B). However, at a concentration of 30 μg/mL, this
extract also had significant cytotoxic effects on THP-1
cells (Figure 5B).
In order to analyze a possible effect of the mushroom extracts
in inflammation of the cornea associated with DED, the cytokine
secretion of THP-1 cells was also investigated in analogy to the
HCE-T cells. For this purpose, the cells were treated with the
extracts for 24 h, the supernatants were collected and the IP-10,
IL-6, TNF-α, and IL-8 levels were determined. All mushroom
extracts strongly and significantly inhibited the secretion of all
cytokines in a concentration-dependent manner with
significantly reduced levels for the entire concentration range
(3–30 μg/mL) (Figures 5C–F). However, treatment of the cells
with 30 μg/mL IO extract increased TNF-α(Figure 5D)andIL-8
(Figure 5F)levels,while3and10μg/mL of IO extract lowered
TNF-α(Figure 5D)andIL-8(Figure 5F) secretions. FF showed
the weakest inhibitory effects at the lowest concentration tested
(3 μg/mL). This was most noticeable in the results of the IL-6
measurement.
3.6 Effects of mushroom extracts on the
viability and lipid droplets of immortalized
human meibomian gland epithelial
cells (IHMGECs)
DED is characterized, in part, by a deficient quality of the tear
film produced by the meibomian gland cells. Therefore, the effects of
mushroom extracts on IHMGECs viability, their intracellular ROS
level, lipid-to-cell ratio, lipid size, cell number and cell size were
investigated in the following. To differentiate the IHMGECs into a
highly lipid-producing form, the cells were incubated with a
differentiation medium containing 10% inactivated fetal calf
serum (iFCS). To stimulate lipid production to a lesser extent or
not at all, a differentiation medium with 4% iFCS or no iFCS was
used. Cells incubated in the culture medium (CM) were used to
represent the undifferentiated form.
GT and LO significantly reduced cell viability at a concentration
of 100 μg/mL (Figure 6A). The lower concentrations tested had no
effect on cell viability (Figure 6A). 100 μg/mL of GL also reduced the
viability of iHMGECs; however, this inhibition was not significant
due to a very high standard deviation (Figure 6A). All other extracts
had no effect on the viability of IHMGECs in the entire
concentration range (0.3–100 μg/mL) (Figure 6A). The ROS level
was reduced in a concentration-dependent manner by all fungal
extracts (Figure 6B), particularly for FF, GT, GO, and GL. This
inhibition was significant for GT at concentrations of 10 and 30 μg/
mL, for IO in the entire concentration range (1–30 μg/mL) and for
FIGURE 3
Effects of fungal extracts on the TEER (A, B, C) and fluorescein permeability (D) of HCE-T cells. (A, B, C) HCE-T cells were cultured on a translucent
membrane of a transwell insert for 72 h and treated with mushroom extracts. The TEER was measured every 24 h. (D) After TEER measurement, cells were
washed and exposed to fluorescein for 2 h to determine permeability. BAC was used as a positive control. Data were normalised to 4% FCS and presented
as mean ± standard deviation. n = 3; *p <0.05, **p <0.01, ***p <0.001.
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LO in the concentration range of 0.three to three μg/mL (Figure 6B).
Nevertheless, the ROS effect of LO was observed in the toxic
concentrations. The lipid-to-cell ratio, cell number and cell size
of IHMGECs were not affected by any of the extracts in the tested
concentrations (Figures 6C–F).
4 Discussion
DED is a complex, prevalent ocular surface disease, and its
aetiology is still poorly understood. However, significant oxidative
stress and inflammatory processes appear to play an important role
(Wei and Asbell, 2014). Medicinal mushrooms embody a valuable
ethnopharmacological source and therefore the pharmacological
effects of six polypore extracts were investigated in inflammatory
DED-relevant in vitro assays.
Overall, the extracts proved to be safe in HCE-T cells and
IHMGECs at concentrations up to 30 μg/mL. Only FF showed
slight cytotoxic effects across the whole concentration range, and LO
was considerably cytotoxic to HCE-T cells at a concentration of
30 μg/mL. Moreover, cytotoxicity was detected in THP-1 cells at
lower concentrations (≥30 μg/mL) than in HCE-T cells and
IHMGECs, especially in THP-1 cells treated with the Ganoderma
species (GL and GT) and LO. Because most medicinal mushrooms
contain polysaccharides (e.g. β-glucans) and steroids/triterpenoids
(e.g. ergosterol and ganoderic acid), they are known not only for
their potent antioxidant activity but also for their antiproliferative
and proapoptotic effects. A number of studies have demonstrated
such effects of Ganoderma species (Cheng et al., 2007;Gao et al.,
2004;Ren et al., 2021). In addition, triterpenoids isolated from LO
have demonstrated cytotoxicity against various cancer cell types
(Feng and Yang, 2015;Han et al., 2020). In contrast, IO showed no
cytotoxicity in HCE-T, iHMGEC, and THP-1 cells which is in line
with previous studies (Chen et al., 2009;Shnyreva et al., 2018).
The ability of the studied fungal extracts to scavenge free radicals
was concentration-dependent for all extracts and particularly
pronounced for GT, GO, and IO. Similarly, GT, GO, and IO
were observed to have strong effects in both UVB-exposed HCE-
T cells and iHMGECs.
Among the tested extracts, GO exhibited the highest free radical
scavenging ability. The characterization of the significant
antioxidant activity of GO is based on a rich phenolic profile.
Fumaric acid and protocatechuic acid were identified as the
major constituents (Tel-Çayan, 2019). Furthermore, antioxidant
and radical scavenging properties of polysaccharides of GT
extracts are also known (Mau et al., 2005;Tseng et al., 2008). In
in vivo studies on UV-induced oxidative stress, IO was shown to
possess cytoprotective properties by reducing ROS and lipid
peroxide levels (Yun et al., 2011). It was also found to have
genoprotective properties by upregulating DNA repair genes (Eid
FIGURE 4
Impact of fungal extracts on the IL-6 (A), IL-8 (B) and TNF-a (C) cytokine secretion of HCE-T cells. HCE T cells were exposed to UV light for 24 h and
treated with DMSO (vehicle) or the fungal extracts. After incubation with the extracts, the supernatants were collected to quantify the secreted mediators
using LEGENDplex™. As an inhibition control, the cells were treated with 10 µM dexamethasone (DEX) prior to UVB exposure. Relative cytokine secretions
were normalised to the UVB-stimulated control (Stim.) and presented as mean ± standard deviation. n = 3; *p <0.05, **p <0.01, ***p <0.001,
****p <0.0001.
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et al., 2021). IO grows on birch trees, where it acquires a large
quantity of betulin and betulinic acid (Ryvarden and Gilbertson,
1993). Both of which proved themselves through their potent
antioxidant activity (Bai et al., 2012;Drenkhan et al., 2022;Géry
et al., 2018).
Activation of NF-κB is crucial to initiate a wide range of
transcription processes during inflammation. Numerous mushroom
species contain secondary metabolites and polysaccharides that are
known to reduce inflammatory mediators, including nitric oxide (NO),
a variety of pro-inflammatory cytokines, and NF-κB(Amirullah et al.,
2024;Taofiq et al., 2016). In this study, NF-κB expression in THP-1 cells
was concentration-dependently reduced in all extracts. Only treatment
with the IO extract had no effect on NF-κB expression. However, other
studies showed a reduction in NF-κB level in murine macrophage cells
(RAW 264.7 cells) after treatment with IO extract (Ma et al., 2013).
Further studies are needed to investigate the impact of IO on other
monocytic/macrophage cells, such as primary cells, to confirm
this aspect.
FIGURE 5
Impact of fungal extracts on the viability (A),NF-κB expression (B),IP10(C), TNF-a (D),IL-6(E), and IL-8 (F) cytokine secretion of THP-1 cells. (A)
THP-1 cells were treated with DMSO (vehicle) or the fungal extracts for 24 h. Their viability was determined using the colorimetric WST-1 assay. Triton-X
100 (Triton-X; 1% v/v) was used as a control. The data were normalised to the negative control (Unstim) and expressed as mean ± standard deviation. n =
3; *p <0.05, **p <0.01, ****p <0.0001. (B) Retroviral transduced NF-κB-eGFP-THP-1 human monocytic cells were stimulated with LPS (100 ng/mL)
and treated with the mushroom extracts for 24 h. Dexamethasone (Dex) was used as an inhibitor control. NF-κB expression was analysed by flow
cytometry. Relative NF-κB expression was normalised to the control (Stim) and presented as the mean ± standard deviation. n = 3. *p <0.05, **p <0.01,
****p <0.0001. (C, D, E, F) THP-1 cells were stimulated with LPS (100 ng/mL) and treated with the fungal extracts for 48 h. Dexamethasone (Dex) was used
as an inhibitor control. The supernatants were collected and analysed using LEGENDplexTM. Relative cytokine concentration was normalised to the
control (Stim.) and presented as mean ± standard deviation. n = 3; *p <0.05, **p <0.01, ***p <0.001, ****p <0.0001.
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Decreased NF-κB expression results in a reduction of the
expression of the transcription factor and consequently in a
decreased expression of various pro-inflammatory cytokines such
as TNF-α, IL-1β, IL-6 and cyclooxygenase-2 (Liu et al., 2017). The
inhibition of cytokine levels in THP-1 cells after treatment with the
extracts observed in this study reflects this relationship, aside from
the IO extracts. However, in addition to the NF-κB pathway, other
factors also mediate the expression of pro-inflammatory factors,
such as the nuclear factor of activated T cells (NFAT) or activator
protein 1 (AP-1) (Lorentz et al., 2003).
Oxidative stress and eye irritation lead to cytokine-mediated
(TNF-α, IL-6, and IL-8) inflammation. To determine the potential as
a DED treatment, the extracts were therefore examined for their
inhibitory properties against these cytokines. In HCE-T cells, GO
showed the strongest inhibitory effects on cytokine levels at a
concentration of 30 μg/mL. However, good effects were also
achieved with 3 and 10 μg/mL GL. Furthermore, stronger effects
and lower standard deviation errors were observed in HCE-T cells
when testing lower concentrations than when testing high
concentrations. Cytotoxic stress could likely occur during
FIGURE 6
Effects of mushroom extracts on IHMGEC viability (A), intracellular ROS level (B), lipid droplet/cell ratio (C), lipid dro plet size (D), cell number (E) and
cell size (F).(A) Confluent IHMGECs were incubated with DMSO (vehicle) or the mushroom extracts in 4% differentiation medium for 24 h before cell
viability was measured using WST-1. Triton-X 100 (Triton-X; 1% v/v) was used as a control. (B) IHMGECs were treated with H
2
DCFDA (10 µM) for 30 min
and incubated with the fungal extracts for 10 min before exposure to a UV lamp. The fluorescence intensity was measured and the values normalised
to the control (NC) and presented as mean ± standard deviation. n = 3; *p <0.05, **p <0.01, ***p <0.001. (C,D,E, F) IHMGECs were incubated for 24 min
4% FCS differentiation medium with or without fungal extracts. Cells incubated in CM, 0% FCS or 10% FCS were used as controls. A cocktail of LipidTOX
and DAPI was used to evaluate the lipid droplets using a fluorescence microscope and Fiji software. Data were normalised to 4% FCS and presented as
mean ± standard deviation. Replication = Rep; n = 3; **p <0.01, ***p <0.001, ****p <0.0001.
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treatment with high concentrations (30 μg/mL) and affect
cytokine secretion.
In comparison to HCE-T cells, the secretion of all the cytokines
examined was significantly inhibited in THP-1 cells. The strongest
effects using non-toxic concentrations (3 or 10 μg/mL) were
observed for GT, followed by GL, IO, and GO. One study has
shown that GT has bidirectional immunomodulatory effects on the
secretion of cytokines by THP-1 cells, by downregulating TNF-α
and IL-1 αwhen exposed to a high dose of LPS and phorbol
myristate acetate (PMA) and the opposite occurs when exposed
to a low dose of the stimulants (Gao et al., 2000). For IO, significantly
higher TNF-αlevels and IL-8 levels were detected at the highest
concentration (30 μg/mL) compared to the positive control
group. Another study also describes an induction of pro-
inflammatory factors (TNF-α, IFN-γ, IL-1β, and IL-2) in human
peripheral blood mononuclear cells (PBMCs) treated with IO
extract, possibly due to the composition of heteropolysaccharides
such as glucose, galactose and mannose (Xu et al., 2014).
The extracts of GO and GL showed significant inhibitory effects
on the secretion of the observed pro-inflammatory cytokines in both
HCE-T cells and THP-1 cells. In the present study, GO exhibited the
strongest cytokine-inhibiting effect among the extracts tested.
However, the anti-inflammatory properties of GO in cells have
rarely been described in the literature to date. Hence, further studies
are needed to explore its therapeutic properties in more detail.
GL, on the other hand, is one of the oldest and best-known
traditional Chinese medicines, that has been demonstrated to have
immunomodulating effects (Lin and Zhang, 2004) and to inhibit
inflammatory mediators such as IL-6, IL-8, IL-10, and TNF-αin
various cell types (Barbieri et al., 2017;Habijanic et al., 2015;Wu
et al., 2019). Likewise, GL lowers IFN-γ, IL-1β, and TNF-αlevels in
THP-1 cells (Rathor et al., 2014) and reduces oxidative damage in
corneal epithelial cells (Tsai et al., 2021). Thus, both GL and GO
demonstrated significant anti-inflammatory potential.
In order to determine the impact of the extracts on the cell-tight
junction, the TEER value was determined. Both GL and GO at a
concentration of 30 μg/mL decreased the TEER values after 48 h of
incubation. Since epithelial cell viability correlates with high TEER
readings (Konsoula and Barile, 2005;Reichl, 2008), cytotoxicity
caused by long-term exposure to the extracts could explain the
results. On the other hand, LO, which was found to be the most
cytotoxic extract in this study, has shown higher TEER values than
the negative control with low permeability of the sodium fluorescein.
Since the wound healing experiment showed no stimulatory effect of
the extracts on cell proliferation, this is unlikely to be the cause.
Thus, the morphology of the membrane proteins such as ZO-1 and
occludin or the cell size could be affected by LO. Further studies are
needed to confirm this assumption.
The three primary ingredients of the tear film layer are lipids,
water, and mucin. Meibomian gland cells release lipids that coat the
outermost layer of the cornea, shielding it from the environment and
preventing evaporation of the tear fluid. It has been demonstrated
that meibomian gland dysfunction is a common factor of DED
development, where reduced lipid secretion disrupts the tear film
homeostasis. This results in ocular irritation, hyperosmolarity and
inflammation (Baudouin et al., 2016). It has been discovered that
monounsaturated oleic fatty acid is present at reduced
concentrations in patients with meibomian dysfunction (Shine
and McCulley, 2000). Stearoyl-CoA desaturase and fatty acid
synthase are some the important lipid synthesis markers that
have been found to increase meibomian lipid production when
expressed (Liu et al., 2011). Our test samples may lack essential
compounds to upregulate the aforementioned related lipid genes to
stimulate lipid production. On the other hand, a study has shown
that ergosterol peroxide, a steroid commonly found in mushrooms
and extracted from GL inhibited lipid droplet production,
triglycerides, and differentiation of 3T3-L1 adipocytes (Jeong and
Park, 2020). Ergosterol peroxide has also been found in FF, GO and
IO (Kahlos, 1996;Ma et al., 2013;Rösecke and König, 2000) but was
not detected in the herein investigated extracts. Moreover, in this
study, no impacts on the amount of secreted lipid droplets were
detected in treated IHMGECs, an increase in the size of secreted
lipid droplets was observed when treated with GT extract.
Finally, the influence of the extracts on the tight junction
connections of HCE-T cells was investigated. Both GL and GO
disrupted the tight junctions of HCE-T cells after 24 h of treatment,
while LO strengthened the cells’tight junctions and consequently
the barrier function of the cells, which was also confirmed by the low
paracellular permeability of the sodium fluorescein tracer in the
basolateral chamber of the transwell. The main group of constituents
of LO consists of triterpenoids of the lanostane type (Grienke et al.,
2014). These triterpenoids have been shown to induce
neuromuscular blockade, which causes botox-like effects (Santana
et al., 2011). Botox has been proposed for the treatment of DED
because of its ability to reduce lacrimal drainage (Sahlin and Chen,
1996). However, in practice, no noticeable improvement has been
observed in DED patients treated with botox (Horwath-Winter
et al., 2003).
5 Conclusion
In this study, we investigated the mycochemical compositions and
therapeutic potential of six ethanolic fungal extracts (FF, GL, GT, GO,
IO, and LO) for the treatment of DED using a set of in vitro assays.
The mycochemical analysis revealed that except for FF, all extracts
contain lanostane triterpenes as main secondary metabolites.
Interestingly, among the triterpene containing extracts a broad
chemical diversity of this structure class was observed. This is
reflected by modifications such as oxygenation and esterification.
This might explain the different biological properties observed. In
general, the extracts were shown to be effective in reducing oxidative
stress, one of the relevant factors in the production of ROS.
Pronounced antioxidant activities in corneal cells were observed
for GT, GO, and IO. Cytokine secretion by THP-1 cells was
significantly inhibited in a concentration-dependent manner by all
extracts except for IO. Nevertheless, the anti-inflammatory
investigations of the extracts revealed that in all HCE-T, iHMGEC,
and THP-1 cells, GO and GL had significant effects in reducing the
aetiology associated with DED. Fungal extracts in particular those of
GO and GL warrant further investigations to clarify their specific
therapeutic benefits and mechanisms of action and to identify their
bioactive components in relation to DED. They showed to possess the
potential for controlling inflammatory response and oxidative stress,
which paves the way for their application in modern therapeutic
contexts, such as in managing dry eye disease (DED).
Frontiers in Pharmacology frontiersin.org13
Areesanan et al. 10.3389/fphar.2025.1557359
Data availability statement
The raw data supporting the conclusions of this article will be
made available by the authors, without undue reservation.
Author contributions
AA: Data curation, Formal Analysis, Investigation, Methodology,
Writing–original draft, Writing–review and editing. AW: Formal
Analysis, Investigation, Methodology, Writing–original draft,
Writing–review and editing. SN: Formal Analysis, Investigation,
Methodology, Writing–review and editing. UG: Conceptualization,
Funding acquisition, Writing–review and editing. AZ-K:
Writing–original draft, Writing–review and editing. JR:
Conceptualization, Funding acquisition, Supervision,
Writing–review and editing. CG: Conceptualization, Funding
acquisition, Writing–review and editing.
Funding
The author(s) declare that financial support was received for the
research, authorship, and/or publication of this article. AA, AZ-K,
and CG are financially supported by a PRIAM-based consortium of
multiple funders. This research was funded in part by the Austrian
Science Fund (FWF) (Grant-DOI: 10.55776/P34028).
Acknowledgments
During the preparation of this work the authors made use of
assistance of the DeepL Translator in order to improve language.
After using this tool/service, the authors reviewed and edited the
content as needed and take full responsibility for the content of the
publication.
Conflict of interest
The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be
construed as a potential conflict of interest.
Generative AI statement
The author(s) declare that no Generative AI was used in the
creation of this manuscript.
Publisher’s note
All claims expressed in this article are solely those of the authors
and do not necessarily represent those of their affiliated
organizations, or those of the publisher, the editors and the
reviewers. Any product that may be evaluated in this article, or
claim that may be made by its manufacturer, is not guaranteed or
endorsed by the publisher.
Supplementary material
The Supplementary Material for this article can be found online
at: https://www.frontiersin.org/articles/10.3389/fphar.2025.1557359/
full#supplementary-material
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