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Fermented Cordyceps cicadae Mycelia Extracts Ameliorate Dry Eye Symptoms through Reduction of Cornea Epithelial Cell Apoptosis and Maintenance of Conjunctival Goblet Cells in a Mouse Dry Eye Model

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Journal of Food and Nutrition Research, 2017, Vol. 5, No. 5, 320-330
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Fermented Cordyceps cicadae Mycelia Extracts
Ameliorate Dry Eye Symptoms through Reduction of
Cornea Epithelial Cell Apoptosis and Maintenance of
Conjunctival Goblet Cells in a Mouse Dry Eye Model
Tung-Yu Lin1,#, Han-Hsin Chang2,#, Yu-Jun Tang3, Chin-Chu Chen4,5, Li-Ya Lee6, David Pei-Cheng Lin1,7,*
1Department of Medical Laboratory and Biotechnology, Chung Shan Medical University, Taichung City, Taiwan
2Department of Nutrition, Chung Shan Medical University, Taichung City, Taiwan
3Institute of Biochemistry, Microbiology, and Immunology, Chung Shan Medical University, Taichung City, Taiwan
4Institute of Food Science and Technology, National Taiwan University, Taiwan
5Department of Food Science, Nutrition, and Nutraceutical Biotechnology, Shih Chien University, Taiwan
6Department of Veterinary Medicine, National Chung Hsin University, Taiwan
7Department of Ophthalmology, Chung Shan Medical University Hospital, Taichung City, Taiwan
#These are first authors.
*Corresponding author:
Abstract Cordyceps cicadae (Cc), a traditional Chinese medicine, has been shown to possess immunomodulatory
and anti-inflammatory activities, and is regarded as having effects in vision improvement, but with no reported
evidence. This study investigated the effects of Cordyceps cicadae fermented mycelia extracts (Cc extracts) in a
benzalkonium chloride (BAC)-induced mouse dry eye model. Female ICR mice aged 6 weeks were randomly
divided into four groups: blank, BAC-damaged without Cc extracts, BAC-damaged with 10 mg/kg bodyweight of
Cc extracts, BAC-damaged with 100 mg/kg bodyweight of Cc extracts. The results showed that tear volume, tear
film breakup time, and cornea surface indexes, including smoothness, opacity, topography, and the extent of
lissamine green staining, were all improved with intake of Cc extracts intake, when compared to the status of the
BAC-damaged group without Cc extracts. Immunohistochemical assays showed moderate change of Ki-67+ and
Np63+ epithelial cell populations, while apoptotic epithelial cells, as detected by TUNEL assay, were decreased.
PAS stain showed that the conjunctival goblet cell number and total cell area were decreased in the BAC-damaged
group with Cc extracts at 10 mg/kg bodyweight. This study demonstrated that Cc extracts effectively ameliorate
BAC-induced dry eye symptoms through enhancement of cornea resilience against BAC-induced damages and
maintenance of conjunctival goblet cells.
Keywords: Cordyceps cicadae, mycelia extracts, amelioration, dry eye symptoms, cornea epithelial cell apoptosis,
conjunctival goblet cells
Cite This Article: Tung-Yu Lin, Han-Hsin Chang, Yu-Jun Tang, Chin-Chu Chen, Li-Ya Lee, and David
Pei-Cheng Lin, Fermented Cordyceps cicadae Mycelia Extracts Ameliorate Dry Eye Symptoms through
Reduction of Cornea Epithelial Cell Apoptosis and Maintenance of Conjunctival Goblet Cells in a Mouse Dry
Eye Model.” Journal of Food and Nutrition Research, vol. 5, no. 5 (2017): 320-330. doi: 10.12691/jfnr-5-5-6.
1. Introduction
Dry eye disease (DED) prevails between 7.4% and
33.7% among human populations [1] and it was estimated
that 25% of patients who visited ophthalmic clinics due to
symptoms of dry eye [2]. The risk factors of DED include
age, gender, autoimmune disease, long-term contact lens
wearing, refractive laser surgery, excessive visual task
performance, intake of certain systemic medications,
smoking, and low humidity environments [2,3]. These risk
factors characterize DED as a chronic ocular surface
disorder that affects significantly quality of life through
enduring discomfort and visual disturbances. Due to such
extended impact, DED has attracted much attention and
many efforts have been exerted in finding the ways to
alleviate its symptoms.
DED is a multifactorial disease that is manifested in
many aspects. Previous studies indicated that inflammation,
such as seen in systemic autoimmune diseases including
Sjogren’s syndrome and systemic lupus erythematosus, is
a common manifestation of dry eye symptoms [4,5]. Some
researchers have thus proposed to use topical or systemic
immunomodulatory therapy in severe dry eye conditions
[6,7]. Increase of pro-inflammatory markers with
excessive oxidants in the tear is also a hallmark of DED
[8,9]. Thus, anti-inflammation by use of anti-oxidants,
Journal of Food and Nutrition Research 321
such as the use of dietary alpha-lipoic acid or topic
administration of CoQ10, has been experimentally applied
for dry eye symptom alleviation [10,11,12].
Cordyceps cicadae (Cc), a traditional Chinese health
food, was shown to possess immunomodulatory activities
[13] and anti-inflammatory effects [14]. These effects, if
exerted in the eye, may help to relieve dry eye symptoms.
Nevertheless, whether intake of Cc extracts may
ameliorate dry eye symptoms is not known. This study
investigated this potential effect in benzalkonium chloride
(BAC)-induced mouse dry eye model [15].
2. Results
2.1. Dietary Cc Extracts Ameliorate Ocular
Surface Damages Induced by
Benzalkonium Chloride
The ocular surface damages were assessed by semi-
quantitative grading systems according to smoothness,
topography, opacity, and the extent of corneal staining by
lissamine green. The results showed that dietary intake of
Cc extracts helped to alleviate ocular surface damages in
all of the four grading systems, except for opacity, while
those without intake of Cc extracts showed significant
damages on the ocular surface (Figure 1). The effective
dose was 100 mg/kg of body weight and this effective
dose was consistent for mitigation of the smoothness,
topography, and corneal staining grading.
2.2. Tear Production under the Influence of
Cc Extracts
Dietary intake of Cc extracts helped to increase the
aqueous tear production after ocular surface damage by
BAC (Figure 2). At day 7 after the BAC-induced damage,
Cc extracts given at 100 mg/Kg of body weight lead to
significant increase of tear production as compared to that
of the BAC-damaged group. At day 10 after the damage,
both groups given at 10 mg/Kg and 100 mg/Kg of body
weight of Cc extracts showed significant increase of tear
production. Notably, starting from day 4, with intake of
Cc extracts at 10 mg/Kg or 100 mg/Kg, the tear
production was higher than either the control or the BAC-
damaged group.
In A, less irregularity of reflective light was observed with intake of intake of Cc extracts. Negative images were also shown for the extent of lissamine
green staining. In B, semi-quantitative analysis showed that the effective dose was at 100 g/Kg of bodyweight. BAC: benzalkonium chloride-damaged
group without intake of CC extracts. BAC-[C] 10 mg/kg and BAC-[C] 100 mg/kg: benzalkonium chloride-damaged groups with intake of CC extracts
at 10 mg/kg and 100 mg/Kg, respectively.
Figure 1. Ocular surface grading for corneal smoothness, opacity, topography, and extent of lissamine green staining at day 10 of the experiment
322 Journal of Food and Nutrition Research
BAC: benzalkonium chloride-damaged group without intake of CC extracts. 10 mg/kg and 100 mg/kg: benzalkonium chloride-damaged groups with
intake of Cordyceps cicadae mycelia fermented extracts at 10 mg/kg and 100 mg/Kg, respectively.
Figure 2. Aqueous tear production (in millimeter) assessed with a 1-mm-width strip cut off from a pH test paper at days 1, 4, 7, and 10 of the
BAC: benzalkonium chloride-damaged group without intake of Cc extracts. 10 mg/kg and 100 mg/kg: benzalkonium chloride-damaged groups with
intake of CC extracts at 10 mg/kg and 100 mg/Kg, respectively.
Figure 3. Tear film breakup time (in seconds) assessed after fluorescein staining and recording at days 1, 4, 7, and 10 of the experiment
Journal of Food and Nutrition Research 323
2.3. Increased Tear Film Breakup Time with
Cc Extracts
Since the tear volume was increased with intake of Cc
extracts, we further assessed the tear quality as reflected
by tear film breakup time (TBUT). The results showed that
BAC caused significant reduction of TBUT at day 1 after
the damage (Figure 3) and this detrimental effect aggravated
more severely at days 4, 7, and further to day 10. TBUT was
significantly increased at day 4 with intake of Cc extracts
in both 10 mg/Kg and 100 mg/Kg dose groups, as compared
to that of the BAC-damaged group. However, the significance
of increase did not persist to day 7. At day 10, the TBUT
increase regained its significance. Furthermore, the TBUTs
of 100 mg/Kg dose group was significantly longer than
that of the 10 mg/Kg dose group at both day 4 and day 10.
2.4. Cc Extracts Help to Maintain Central
Cornea Epithelium Thickness after BAC-
induced Damage
Histological analysis showed that BAC administration
generally caused cornea damage on the surface (Figure 4A),
but did not reduce the total cornea thickness (Figure 4B).
On the contrary, BAC caused significant increase of total
cornea thickness in the peripheral cornea area (Figure 4B).
The main reduction of thickness caused by BAC was on
the central cornea epithelium (Figure 4C). This reduction,
however, did not extend to the peripheral cornea. There
was even a slight increase of epithelial thickness in the
peripheral cornea (Figure 4C).
BAC administration caused increase of cornea stroma
thickness in the central and peripheral cornea, but not in
the limbus when compared with that of the control group
(Figure 4D). With intake of Cc extracts, the thickness of
stroma in the central cornea and peripheral cornea was
even more increased, particularly in the central cornea
where dietary intake of Cc extracts at 100 mg/Kg dose
lead to the most significant increase. On the contrary, in
the limbus, intake of Cc extracts did not increase stromal
thickness (Figure 4D). At 10 mg/Kg dose, the stroma
thickness in the limbus was even significantly reduced as
compared to that of the BAC-damaged group.
2.5. Moderate Mobilization of Cornea Ki-67+
and Np63+ Epithelial Cells with Cc
Extracts Intake
To further characterize the damages caused by BAC
administration, we performed immunohistochemistry for
Ki-67+ and Np63+ cells and calculated the cell numbers in
the cornea central, peripheral, and limbus regions at day
10 of the experiment. The results showed that Ki-67+ cells
in all of the 3 cornea regions were fewer in the blank
group than in the BAC-damaged group in all of the three
cornea regions (Figure 5A). Interestingly, dietary intake of
Cc extracts did not appear to promote further increase of
Ki-67+ cells, adding to the mobilizing effect occurred after
BAC damage. Instead, the number of Ki-67+ cells became
less as compared to that of the BAC-damaged group,
particularly in the peripheral cornea and limbus areas
when 100 mg/Kg dose was administered (Figure 5B).
CC: central cornea. PC: peripheral cornea. L: limbus. BAC: benzalkonium chloride-damaged group without intake of Cc extracts. 10
mg/kg and 100 mg/kg: benzalkonium chloride-damaged groups with intake of CC extracts at 10 mg/kg and 100 mg/Kg, respectively.
Figure 4. Histological analysis and thickness measurement of total cornea, cornea epithelium, and cornea stroma at day 10 of the experiment after
Hematoxylin-Eosin staining
324 Journal of Food and Nutrition Research
CC: central cornea. PC: peripheral cornea. L: limbus. BAC: benzalkonium chloride-damaged group without intake of CC extracts. 10 mg/kg
and 100 mg/kg: benzalkonium chloride-damaged groups with intake of Cordyceps cicadae mycelia fermented extracts at 10 mg/kg and 100
mg/Kg, respectively
Figure 5. Immunohistochemical staining and quantitative analysis of Ki-67+ cells at day 10 of the experiment
Immunohistochemical detection of Np63+ cells at day 10 of the experiment. CC: central cornea. PC: peripheral cornea. L: limbus. BAC:
benzalkonium chloride-damaged group without intake of Cc extracts. 10 mg/kg and 100 mg/kg: benzalkonium chloride-damaged groups with
intake of CC extracts at 10 mg/kg and 100 mg/Kg, respectively
Figure 6. Immunohistochemical detection of Np63+ cells at day 10 of the experiment
In contrast to the Ki-67+ cell population, the Np63+
cells did not appear to be mobilized to the same extent in
the peripheral cornea and limbus areas (Figure 6). When
compared with that of the BAC-damaged group, the
Np63+ cell number with dietary intake of Cc extracts
showed no significant difference in either the 10 mg/Kg
group or in the 100 mg/Kg group (data not shown).
2.6. Cc Extracts Helps to Reduce
BAC-induced Apoptosis in
the Cornea Epithelium
Since our data indicated that dietary intake of Cc
extracts can effectively ameliorate ocular surface damages
induced by BAC administration, the cornea epithelium
Journal of Food and Nutrition Research 325
must either be protected from the damages or they will die
and be replaced though mobilization of limbal stem cells
or basal progenitor cells [17]. Therefore, we performed
TUNEL assay to examine the apoptotic status of the corneal
epithelial cell populations. The results showed extensive
apoptotic cells in all 3 cornea areas following BAC
administration (Figure 7A). Quantitatively, the peripheral
cornea area contained more apoptotic epithelial cells than
the central cornea and the limbus areas following the
damages induced by BAC (Figure 7B). With dietary
intake of Cc extracts, the numbers of apoptotic cornea
epithelial cells were generally reduced in both 10 mg/Kg
and 100 mg/Kg study groups and the 100 mg/Kg study
group apparently reduced more apoptotic epithelial cells.
2.7. Maintenance of Conjunctival Goblet Cell
with Cc Extracts Intake
Since the tear quality was improved with intake of Cc
extracts, as indicated by increased tear film breakup
time, we performed PAS stain to assess the conjunctival
goblet cell status. The results showed that PAS+
conjunctival goblet cells became fewer after the damage
caused by BAC treatment (Figure 8A). With intake of
CC extracts at 10 mg/Kg, the number of PAS+
conjunctival goblet cells was higher than that in the
damaged group (Figure 8B). However, less conjunctival
goblet cells were observed with intake of Cc extracts at
100 mg/Kg dose.
In A, the upper panel showed an example of combination of PI (in red) and FITC (in green) images to yield cells with yellow signals (indicated by
arrows) that were regarded as positive for TUNEL assay. In B, quantitative analysis showed evident reduction of apoptotic cornea epithelial cells with
intake of Cc extracts. CC: central cornea. PC: peripheral cornea. L: limbus. BAC: benzalkonium chloride-damaged group without intake of CC extracts.
10 mg/kg and 100 mg/kg: benzalkonium chloride-damaged groups with intake of CC extracts at 10 mg/kg and 100 mg/Kg, respectively.
Figure 7. In situ terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay at day 10 of the experiment
326 Journal of Food and Nutrition Research
In A, the morphology and number of goblet cells were shown in the inferior conjunctiva. In B, the PAS+ conjunctival
goblet cells were quantified. The results showed that more goblet cells were maintained with intake of Cc extracts.
BAC: benzalkonium chloride-damaged group without intake of CC extracts. 10 mg/kg and 100 mg/kg: benzalkonium
chloride-damaged groups with intake of CC extracts at 10 mg/kg and 100 mg/Kg, respectively.
Figure 8. Periodic acid Schiff (PAS) staining at day 10 of the experiment
3. Discussion
Dry eye disease (DED) is a multifactorial disorder of
the tear film and ocular surface [3,5]. For many years,
researchers have been trying to unveil the core mechanism
of DED, which is reflected by the number of publications
directly related to dry eye syndromes. A recent review
paper [18] was published by using the key words “dry eye,
inflammation” on the PubMed and Web of Science
databases for scientific articles published in English
between January 1, 1900 and August 30, 2013. Based on
the literature survey, the authors clearly demonstrated that
inflammation is the core mechanism and plays a key role
in the pathogenesis of DED. The authors concluded that
immune dysregulation leads to a cycle of continued
inflammation and eventually leads to DED. Thus,
functional foods that possess both anti-inflammatory and
immunomodulatory activities may be used as candidates
for alleviation of dry eye symptoms.
Cordyceps cicadae (Cc), a caterpillar-shaped medicinal
mushroom that derives its nutrients from larvae of Cicada
flammata Dist., has been used as a dietary supplement in
Chinese for hundreds of years. Previous studies have
shown that the ergosterol peroxide from C. cicadae
inhibits the activation and proliferation signals in
primary human T lymphocytes [19] and ameliorates
TGF-β1-induced activation of kidney fibroblasts [14].
Another earlier study [13] indicated that C. cicadae
extracts possess immunomodulatory functions. We
therefore hypothesized that C. cicadae may help to relieve
dry eye symptoms and evaluated its fermented mycelia
extracts in a benzalkonium chloride (BAC)-induced
mouse dry eye model. The results demonstrated that
dietary intake of Cc extracts, particularly at 100 mg/Kg
dose, helped to ameliorate BAC-induced dry eye
symptoms through multiple aspects, including improvement
of ocular surface grading of smoothness, topography, and
lissamine green staining, increasing tear volume and
TBUT period, prevention against the extensive epithelial
layer apoptosis in the central, peripheral, and limbal areas
of the cornea, and maintenance of conjunctival goblet cell
number and area at 10 mg/Kg dose.
Multi-fold mechanisms may be unveiled underlying the
efficacy after intake of Cc extracts. The amelioration of
BAC-induced damages to the ocular surface may be
through accelerated repair by mobilization of limbal stem
cells or corneal progenitor cells or both [17,20].
Alternatively, intake of Cc extracts may render the corneal
epithelial cells more resilient to the damages caused by
BAC. We conducted immunohistochemistry of Ki-67 and
Np63 cellular proliferation markers to investigate the
changes of corneal epithelial cells following damages
induced by BAC and compare the status with or without
intake of Cc extracts. Ki-67 is a marker for proliferating
but poorly differentiated corneal epithelial cells [21] and
unspecified ΔNp63 isoforms are detectable in the basal
layer of the corneal epithelium at all times [21]. The
results demonstrated more Ki-67+ cells in all of the three
cornea areas following BAC damage, in contrast to the
steady status in the blank control group. Evidently, BAC
Journal of Food and Nutrition Research 327
damage caused more mobilization of proliferative cornea
epithelial cells, either from the limbus or form the local
progenitor pool.
If dietary intake of Cc extracts ameliorated the damages
caused by BAC through accelerated cell mobilization, it
would have promoted even more increase of Ki-67+ cells
as compared to that of the BAC damage group without Cc
extracts intake. Instead, Ki-67+ cell number became
significantly less (p<0.05 at 100 mg/Kg dose) as compared to
that of the BAC-damaged group, particularly in the
peripheral cornea and limbus areas. The result suggests
that intake of Cc extracts did not help to ameliorate
BAC-induced dry eye symptoms mainly through promotion
for faster corneal epithelium replacement. Instead, a moderate
condition of mobilization was more likely to occur. The
result that Np63+ cells did not appear to be evidently
mobilized in the peripheral cornea and limbus areas also
indicates this moderate condition of mobilization.
If the corneal stem cells or progenitor cells were only
moderately mobilized under the effects of BAC damage,
ocular surface improvement with intake of Cc extracts
would be resulted from more resilience to the damages.
The results of TUNEL assay confirmed that fewer
apoptotic cells were found as Cc extracts were given,
particularly in the peripheral and limbus areas when the
Cc extracts were given at 100 mg/Kg dose.
Notably, as Cc extracts were given to the mice, the
effective dose at 100 mg/Kg correlates in almost all
aspects of dry eye symptom amelioration, except for the
cell number and area of conjunctival goblet cells.
Significant amelioration was observed at 100 mg/Kg dose
for 3 out of the 4 ocular surface grading criteria. The
results of tear production and TBUT were also better with
100 mg/Kg dose. Another result that is also noteworthy is
the significant increase of stromal thickness in the central
cornea, in contrast to the non-significance in the
peripheral cornea and the limbus. This data is conversely
correlated with the non-significant change of Ki-67+ cell
number in the central cornea and the significant change in
the peripheral cornea and in the limbus. Interestingly,
fewer apoptotic cornea epithelial cells were detected in the
central cornea in the BAC-damaged group as well as in
the groups given Cc extracts. The reduction of central
cornea epithelial apoptotic activity is likely due to the
more increase of stromal thickness in the central cornea
where differential corneal epithelial-keratocyte cell
interactions may occur in this area, leading to differential
release of protective factors from the keratocytes. For
example it has been known that paracrine mediators such
as hepatocyte growth factor (HGF) and keratinocyte
growth factor (KGF) are produced by the keratocytes to
regulate proliferation, motility, differentiation, and
possibly other functions, of epithelial cells [22,23]. If the
differential epithelial-keratocyte cell interaction occurred,
since the epithelial cells in the central cornea were more
protected, apoptotic activities were relatively low and the
loss of central cornea epithelial cells would be less. The
demand for repair would be less, as reflected by the
non-significant change of Ki-67+ cell number in the
central cornea. Arguably, however, increase of the central
stroma thickness may be an indication of edema caused by
inflammatory activities therein. Besides, the effects to
maintain conjunctival goblet cell were better with Cc
extracts at 10 mg/Kg than at 100 mg/Kg dose.
Complicated local factors may be involved, resulting in
impedance of goblet cell generation with Cc extracts given
at higher dose. Obviously, this entire scenario demands
further investigations.
The underlying mechanisms between maintenance of
healthy cornea epithelial layers and improvement of tear
production and quality are multifold. Particularly, since
Cc extracts possess both immunomodulatory and
anti-inflammatory activities, the ameliorating effects
found in the present study are of complexity and warrant
further investigation. Also, the clinical significance
between protecting corneal epithelium from extensive
apoptosis rather than acceleration of stem cell
mobilization and replacement of damaged cells is different.
To the aged corneas or corneas with limbal stem cell
deficiency, as their cornea stemness capacity is diminished,
stimulation to accelerate cornea repair is less likely to
ameliorate dry eye symptoms. Under these conditions,
dietary Cc extracts are potentially more helpful.
4. Materials and methods
4.1. Mice and Study Groups
A total of 24 six-week-old female ICR mice were
purchased from BioLASCO Taiwan Co., Ltd, Taipei,
Taiwan. The mice were fed ad libitum and kept under
standard conditions with a 12-h light/dark cycle. The mice
were acclimatized and habituated to the laboratory for at
least one week before experiments. All mice were
examined with a slit lamp (Model 99 BQ; Haag-Streit,
Bern, Switzerland). Only mice without anomalies of the
anterior segment of the eye (cornea, anterior chamber, iris,
or lens) were included in the experiments. The mice were
randomly split into 4 groups: blank with 0.9 % NaCl as
vehicle control, BAC-damaged without CC extracts,
BAC-damaged with 10 mg/kg bodyweight of Cc extracts,
BAC-damaged with 100 mg/kg bodyweight of Cc extracts.
Each group contained 6 mice. All experiment protocols
were reviewed and approved by the Animal Care and Use
Committee of Chung Shan Medical University, Taichung,
Taiwan and were performed in agreement with the
Association for Research in Vision and Ophthalmology
(ARVO) Resolution on the Use of Animals in Research.
4.2. Cornea Surface Damage by
Benzalkonium Chloride
The BAC damages were performed from day 1 to day
10, with the mice anaesthetized and their ocular surfaces
exposed to 5 μL of 0.2% BAC administered topically
twice daily (9 AM and 9 PM). Care was taken to ensure
that BAC covered the entire mouse ocular surface for at
least 1 minute during each BAC administration.
4.3. Preparation and Feeding of Cc Extracts
Cordyceps cicadae was harvested from mountain areas
of New Taipei City, Taiwan. The mycelium was isolated
and cultured on potato dextrose agar and genotyped to
328 Journal of Food and Nutrition Research
confirmed its identity. The colony has been stored (stock
number MU30106) in Bioresource Collection and
Research Centre (BCRC), Hsinchu, Taiwan. The
mycelium was primarily cultured at 25°C in 2% glucose,
1% yeast extract, 1% soy bean powder at pH 6.0, followed
by expanded culture and fermentation. The crude
fermented fluids were concentrated, cold-dried into
powder, and stored at 4°C before feeding. The Cc extracts
were given by daily tube feeding at 10 AM starting from 3
days prior to the BAC damage and continuing until day 10.
All mice were sacrificed on day 10 for tissue collection.
4.4. Grading of Corneal Surface Damages
Corneal surface damages were graded according to
smoothness, topography, opacity, and the extent of
lissamine green staining, following a previous publication
[10]. Briefly, one eye of each mouse was randomly
selected for assessment of corneal smoothness and
topography. The other eye was then assessed for corneal
opacity. Images of the cornea surface were taken with a
stereoscopic zoom microscope equipped with ring
illuminator (SMZ 1500; Nikon). The digital images were
then used to score corneal smoothness on a 5-point scale
according to the number of distorted quadrants in the
reflected ring: 0, no distortion; 1, distortion in one
quadrant of the ring (3 clock hours); 2, distortion in two
quadrants (6 clock hours); 3, distortion in three quadrants
(9 clock hours); 4, distortion in all four quadrants (12
clock hours); and 5, severe distortion, in which no ring
could be recognized. The scoring of corneal topography
was the same as that of corneal smoothness, but covered
much more area of the corneal surface. The other set of
images on the other eye was used for corneal opacity
scoring (0, normal cornea; 0.5, mild haze seen only under
dissection microscope; 1, mild haze; 2, moderate haze
with visible iris; 3, severe haze with invisible iris; 4,
severe haze with corneal ulceration). For lissamine green
staining, both corneas from each mouse were stained with
2% lissamine green B (Sigma-Aldrich, cat. no. 199583) in
0.9% NaCl. Images were taken and scored according to
the areas of stain. Briefly, the total area of punctuate
staining was designated as grade 0; grade 1, less than 25%
of cornea stained with scattered punctuate staining; grade
2, 25% to 50% of cornea stained with diffuse punctate
staining; grade 3, 50% to 75% of cornea stained with
punctuate staining and apparent epithelial defects; grade 4,
more than 75% of cornea stained with abundant punctuate
staining and large epithelial defects. All scorings were
performed by two observers without prior knowledge of
the experiment and study groups.
4.5. Tear Production and Tear Film Breakup
Time Assessment
Aqueous tear production was assessed with phenol
red-impregnated cotton threads (Zone-Quick; Oasis,
Glendora, CA, USA) at days 1, 4, 7, and 10. The animals
were anesthetized and rest for a fixed period of 20 seconds.
A 1-mm-width strip was held with forceps under a
dissection microscope and placed in the lateral cantus of
the conjunctival fornix of the eye for 20 seconds. The tear
distance (in millimeters) was read under a microscope.
Tear film breakup time was assessed by in vivo staining
with 0.1% liquid sodium fluorescein [10,16]. The mice
were anesthetized with intraperitoneal tribromoethanol
injection (0.3 ml at 20 mg/ml). One or two microliter of
fluorescein stain was dropped into the conjunctival sac.
After 3 blinks, tear film fluorescein signals were recorded
under an Olympus BX51 fluorescence microscope
(Olympus, Tokyo, Japan). The tear film breakup time was
read in seconds by two observers without prior knowledge
of the experiment and study groups.
4.6. Histology and Immunohistochemistry
After mouse sacrifice by cervical dislocation, the
eyes were extracted, processed, and stained with
Hematoxylin-Eosin for histopathologic analysis following
conventional procedures as previously described [10,16].
For immunohistochemistry, the tissue sections were boiled
in citrate buffer (pH 6.0) for 20 minutes for antigen
retrieval and then incubated, respectively, with either
mouse anti-Np63 (1/50, cat. no. sc-8431; Santa Cruz
Biotechnology, Santa Cruz, CA) or rabbit anti-Ki-67
antibody (1/100, cat. no. NB11089719; Novus Biologicals),
followed by incubation with a horseradish peroxidase
conjugated secondary antibody (1/200), either anti-mouse
or anti-rabbit IgG (Jackson ImmunoResearch Laboratories,
Inc., West Grove, PA), and followed by washes and incubation
in diaminobenzidine tetrahydrochloride solution for color
detection, and counterstained with hematoxylin.
4.7. Demarcation of Central, Peripheral, and
Limbal Cornea Areas and Thickness
To describe differential cell distribution and corneal
thickness in different areas, each cornea was demarcated
into central, peripheral, and limbal areas. The eye was
sectioned on sagittal plane and the cornea length, spanning
from the upper to the lower limbus, was split into 3 equal
parts. The upper one third and the lower one third were
regarded as the peripheral corneas, while the middle one
third as the central cornea. Following Hematoxylin-Eosin
stain, the tissue sections with the longest corneal length
were selected and measured for total, epithelial, and
stromal thickness under a Nikon E 100 microscope (Nikon,
Tokyo, Japan), equipped with a digital camera linked to a
desktop computer. Each thickness measurement was
determined by 2 observers without prior information of
the experiments and study groups. All measurements were
performed on ImageJ2 (National Institutes of Health,
Bethesda, USA) software program.
4.8. In Situ Terminal Deoxynucleotidyl
Transferase dUTP Nick End
Labeling (TUNEL) Assay
In situ TUNEL labeling was performed using the
DeadEnd™ Fluorometric TUNEL System (G3250;
Promega, Madison, WI) according to manufacturer's
instructions. Tissue sections were fixed in acetone at 4°C,
rinsed with phosphate-buffer saline (PBS), permeabilized
Journal of Food and Nutrition Research 329
by 0.2% Triton X100, and incubated in equilibration
buffer for 10 min. The sections were then incubated with
TdT reaction mix for 60 min, followed by immersion in
standard saline citrate to stop reaction. For positive
controls, tissue sections were incubated in DNase I prior
to addition of equilibration buffer. For negative controls,
DDW was added instead of TdT reaction mix. All
preparations were rinsed with PBS several times,
counterstained with propidium iodide to locate the cells
(P3566; ThermoFisher), and mounted for photography.
The photo images were taken with an Olympus BX51
fluorescence microscope (Olympus, Tokyo, Japan),
followed by digital combination of the fluorescein
isothiocyanate (FITC) (green) with the propidium iodide
(red) color to produce yellow signals. Only cells with
yellow signals were regarded as positive for TUNEL assay.
4.9. PAS Stain for Conjunctival Goblet Cell
Number and Total Cell Areas
Periodic acid Schiff (PAS) staining was performed to
evaluate conjunctival epithelial morphology, and the
number of goblet cells and total cell areas in the inferior
conjunctiva was counted under a microscope (ECLIPSE
E100; Nikon, Melville, NY) with a ×20 objective and
ImageJ2 software program. For each eye, 5 different
sections were randomly selected for counting, and an average
was calculated. All scorings were performed by two
observers without prior knowledge of the study groups.
4.10. Statistics
All data were obtained from repeats (n > 6). The data
are presented as the means ± standard error of the means
(SEMs) and were compared among groups. The corneal
smoothness, opacity, and fluorescein staining scores were
compared with the KruskalWallis test. The corneal total,
epithelial, and stromal thickness and the number of cells
positive in immunohistochemistry and TUNEL assay were
analyzed with the MannWhitney test. All statistical
analyses were performed by using the SPSS program
(SPSS, Inc., Chicago, IL).
5. Conclusion
We demonstrated that C. cicadae fermented mycelia
extracts help to ameliorate dry eye symptoms after ocular
surface damages caused by benzalkonium chloride
administration. The efficacy was shown in multiple aspects,
including tear quality and quantity as well as ocular surface
assessments. Furthermore, we elucidated that C. cicadae
fermented mycelia extracts help to ameliorate the dry eye
symptoms through enhancement of cornea resilience and
maintenance of conjunctival goblet cells after benzalkonium
chloride administration. Results of this study support that
C. cicadae fermented mycelia extracts may be used as a
functional food ingredient for vision protection.
Conflicts of Interests
The authors declare no conflict of interest.
Author Contributions
D.P.-C.L. and H.-H.C conceived and designed
experiments and wrote the paper. T.-Y.L. and Y.-J.T.
performed experiments. C. –C.C. and L.-Y.L. provided
materials and reagents and analyzed the data.
This study was supported by research grants: MOST
102-2320-B-040-013 from Ministry of Science and
Technology, Taiwan; COA 104-20-26 and COA 105-
A005-C from Council of Agriculture, Taiwan.
[1] Lin, P. Y.; Tsai, S. Y.; Cheng, C. Y.; Liu, J. H.; Chou, P.; Hsu, W.
M. Prevalence of dry eye among an elderly Chinese population in
Taiwan: the Shihpai Eye Study. Ophthalmology. 2003; Volume
110, pp. 1096-1101.
[2] O’Brien, P. D.; Collum, L. M. Dry eye: diagnosis and current
treatment strategies. Curr. Allergy Asthma. Rep. 2004; Volume 4,
pp. 314-319.
[3] Dry Eye WorkShop. Research in dry eye: report of the Research
Subcommittee of the International Dry Eye Workshop. Ocul. Surf.
2007; Volume 5, pp. 179-193.
[4] Arevalo, J. F.; Lowder, C. Y.; Muci-Mendoza, R. Ocular
manifestations of systemic lupus erythematosus. Curr. Opin.
Ophthalmol. 2002; Volume 13, pp. 404-410.
[5] Yen, J. C.; Hsu, C. A.; Li, Y. C.; Hsu, M. H. The Prevalence of
dry eye syndrome's and the likelihood to develop Sjögren's
syndrome in Taiwan: A Population-Based Study. Int. J. Environ.
Res. Public Health. 2015; Volume 12, pp. 7647-7655.
[6] Cordero-Coma, M.; Anzaar, F.; Sobrin, L.; Foster, C. S. Systemic
immunomodulatory therapy in severe dry eye secondary
to inflammation. Ocul. Immunol. Inflamm. 2007; Volume 15,
pp. 99-104.
[7] Huang, J. F.; Yafawi, R.; Zhang, M.; McDowell, M.; Rittenhouse,
K. D.; Sace, F.; Liew, S. H.; Cooper, S. R.; Pickering, E. H.
Immunomodulatory effect of the topical ophthalmic Janus kinase
inhibitor tofacitinib (CP-690,550) in patients with dry eye disease.
Ophthalmology. 2012; Volume 119, pp. e43-50.
[8] Zheng, Q.; Ren, Y.; Reinach, P. S.; Xiao, B.; Lu, H.; Zhu, Y.; Qu,
J.; Chen, W. Reactive oxygen species activated NLRP3
inflammasomes initiate inflammation in hyperosmolarity stressed
human corneal epithelial cells and environment-induced dry eye
patients. Exp. Eye Res. 2015; Volume 34, pp. 133-140.
[9] Deng, R.; Hua, X.; Li, J.; Chi, W.; Zhang, Z.; Lu, F.; Zhang, L.;
Pflugfelder, S. C.; Li, D. Q. Oxidative stress markers induced by
hyperosmolarity in primary human corneal epithelial cells. PLoS
One. 2015; pp. e0126561.
[10] Chen, B. Y.; Lin, D. P.; Chang, L. S.; Huang, T. P.; Liu, H. J.; Luk,
C. P.; Lo, Y. L.; Chang, H. H. Dietary α-lipoic acid prevents
UVB-induced corneal and conjunctival degeneration through
multiple effects. Invest. Ophthalmol. Vis. Sci. 2013; Volume 54,
pp. 6757-6766.
[11] Andrade, A. S.; Salomon, T. B.; Behling, C. S.; Mahl, C.D.;
Hackenhaar, F. S.; Putti, J.; Benfato, M. S. Alpha-lipoic acid
restores tear production in an animal model of dry eye. Exp. Eye
Res.2014; Volume 120, pp. 1-9.
[12] Mencucci, R.; Favuzza, E.; Boccalini, C.; Lapucci, A.; Felici, R.;
Resta, F.; Chiarugi, A.; Cavone, L. CoQ10-containing eye drops
prevent UVB-induced cornea cell damage and increase cornea
wound healing by preserving mitochondrial function. Invest.
Ophthalmol. Vis. Sci. 2014; Volume 55, pp. 7266-7271.
[13] Weng, S. C.; Chou, C. J.; Lin, L. C; Tsai, W. J.; Kuo, Y. C.
Immunomodulatory functions of extracts from the Chinese
medicinal fungus Cordyceps cicadae. J. Ethnopharmacol. 2002;
Volume 83, pp. 79-85.
[14] Zhu, R.; Zheng, R.; Deng, Y.; Chen, Y.; Zhang, S. Ergosterol
peroxide from Cordyceps cicadae ameliorates TGF-β1-induced
330 Journal of Food and Nutrition Research
activation of kidney fibroblasts. Phytomedicine. 2014; Volume 21,
pp. 372-378.
[15] Lin, Z.; Liu, X.; Zhou, T.; Wang, Y.; Bai, L.; He, H.; Liu Z. A
mouse dry eye model induced by topical administration of
benzalkonium chloride. Mol. Vis. 2011; Volume 17, pp. 257-264.
[16] Lin, D. P.; Chang, H. H.; Yang, L. C.; Huang, T. P.; Liu, H. J.;
Chang, L. S.; Lin, C. H.; Chen, B. Y. Assessment of ultraviolet
B-blocking effects of weekly disposable contact lenses on corneal
surface in a mouse model. Mol. Vis. 2013; Volume 19, pp. 1158-1168.
[17] Majo, F.; Rochat, A.; Nicolas, M.; Abou Jaoudé, G.; Barrandon, Y.
Oligopotent stem cells are distributed throughout the mammalian
ocular surface. Nature. 2008; Volume 456, pp. 250-254.
[18] Wei, Y.; Asbell, P. A. The core mechanism of dry eye disease
(DED) is inflammation. Eye Contact Lens. 2014; Volume 40,
pp. 248-256.
[19] Kuo, Y. C.; Weng, S. C.; Chou, C. J.; Chang, T. T.; Tsai, W. J.
Activation and proliferation signals in primary human T
lymphocytes inhibited by ergosterol peroxide isolated from
Cordyceps cicadae. Br. J. Pharmacol. 2003; Volume 140,
pp. 895-906.
[20] Yoon, J. J.; Ismail, S.; Sherwin, T. Limbal stem cells: central
concepts of corneal epithelial homeostasis. World J. Stem Cells.
2014; Volume 6, pp. 391-403.
[21] Pearton, D. J.; Yang, Y.; Dhouailly, D. Transdifferentiation of
corneal epithelium into epidermis occurs by means of a multistep
process triggered by dermal developmental signals. Proc. Natl.
Acad. Sci. USA. 2005; Volume 102, pp. 3714-3719.
[22] Wilson, S. E.; Liu, J. J.; Mohan, R. R. Stromal-epithelial
interactions in the cornea. Prog. Retin. Eye Res. 1999; Volume 18,
pp. 293-309.
[23] Ramaesh, T.; Ramaesh, K.; Martin Collinson, J.; Chanas, S. A.;
Dhillon, B.; West, J. D. Developmental and cellular factors
underlying corneal epithelial dysgenesis in the Pax6+/- mouse
model of aniridia. Exp. Eye Res. 2005; Volume 81, pp. 224-35.
... The experimental groups were administered CCM-DW (50 mg/kg/bw/ day) and CCM-EtOH (50 mg/kg/bw/day) by gastric gavage. Doses with no toxic effects were selected based on previous studies [26,27]. IOP in the right eyes of the rats was then measured weekly. ...
... Although other factors have been proposed as playing roles in glaucoma, high IOP remains the primary documented risk factor for glaucomatous optic nerve damage [29]. For the treatment of eye disease, following the administration of benzalkonium chloride, fermented mycelia extracts of C. cicadae reportedly enhanced corneal resilience and the maintenance of conjunctival goblet cells, thus causing an improvement in dry-eye symptoms [27]. In the present study, we first evaluated the IOP-lowering effects of C. cicadae mycelia extract in a steroid-induced glaucoma rat model for glaucoma disease treatment. ...
Full-text available
Glaucoma is a leading cause of irreversible blindness worldwide. This study evaluates the reduction of intraocular pressure (IOP) induced by C. cicadae mycelia extract in a steroid-induced rat model of glaucoma. Cordyceps cicadae mycelia is a well-known and valued traditional Chinese herbal medicine. C. cicadae mycelia were cultured using a liquid fermentation technique. The harvested C. cicadae mycelia were then lyophilized and extracted with two solvents, water and ethanol. The aqueous extract (CCM-DW) and ethanolic extract (CCM-EtOH) of the mycelia were obtained through lyophilization. Sprague Dawley rats were randomly divided into four groups (n = 6 in each group): a normal group, a control group, and experimental groups treated with CCM-DW, or CCM-EtOH (both at 50 mg/kg/body weight). Except for those in the normal group, all rats received a subconjunctival injection of betamethasone to induce high IOP. The rats in the experimental groups received a daily administration of CCM by oral gavage for four consecutive weeks. IOP reduction is the known treatment for glaucoma. The results revealed that steroid treatment caused a significant increase in the animals' IOP (control group). Elevated IOP decreased significantly after treatment with CCM-DW and CCM-EtOH (p < 0.01), and CCM-DW was more effective than CCM-EtOH. CCM-DW and CCM-EtOH were capable of causing significant decreases in high IOP-induced lesions in pathological studies in which it was shown that the efficacy of CCM-DW surpassed that of CCM-EtOH. After CCM-DW administration for 28 days, there were significant decreases in malondialdehyde and lactate dehydrogenase levels and significant increases in catalase, superoxide dismutase, and glutathione peroxidase levels. In summary, C. cicadae mycelia may be beneficial for preventing or treating glaucoma due to its significant IOP-lowering and antioxidant activities.
... In a previous study, we explored the ameliorative effects of C. cicadae mycelial extracts on dry eye disease using a benzalkonium chloride (BAC)-induced mouse model [28]. Furthermore, we discovered that C. cicadae exhibited an alleviating effect on dry eye symptoms in humans [29]. ...
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Cataracts, a prevalent age-related eye condition, pose a significant global health concern, with rising rates due to an aging population and increased digital device usage. In Taiwan, cataract prevalence is particularly high, reaching up to 90% among individuals aged 70 and above. The lens of the eye absorbs short-wave light, which can lead to oxidative stress in lens epithelial cells and contribute to cataract formation. Exposure to ultraviolet (UV) light further exacerbates the risk of cataracts by generating reactive oxygen species. Heat-shock proteins (HSPs), involved in protein maintenance and repair, have been linked to cataract development. Cordyceps cicadae (C. cicadae), a traditional Chinese medicine, has a long history of use and is known for its pharmacological effects. N6-(2-hydroxyethyl) adenosine (HEA), a bioactive compound found in C. cicadae, exhibits anti-inflammatory, immunomodulatory, and neuroprotective properties. Previous studies have shown that C. cicadae mycelial extracts improve dry eye disease and reduce intraocular pressure in animal models. Additionally, C. cicadae possesses antioxidant properties, which are beneficial for combating cataract formation. In this study, we aim to evaluate the preventive efficacy of C. cicadae mycelial extracts in UV-induced cataract development. By investigating the ameliorative effects of C. cicadae on eye diseases and its potential role in ocular health improvement, we hope to uncover new options for cataract prevention and provide insights into the mechanisms of action. The findings of this research could provide a novel approach for nutritional supplements targeting cataract prevention, offering potential benefits in the field of ocular health.
... C. cicadae-fermented mycelia extract in a benzalkonium chloride (BAC)-induced dry eye model has been evaluated in mice. C. cicadae extract effectively ameliorated BAC-induced dry eye symptoms via enhancement of cornea resilience against BAC (10 mg/kg)-induced damage and maintenance of conjunctival goblet cells [20]. In a steroid-induced high IOP rat model of glaucoma, elevated IOP levels decreased significantly after four weeks of oral treatment with water or ethanol extracts of C. cicadae. ...
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Cordyceps cicadae (CC), an entomogenous fungus that has been reported to have therapeutic glaucoma, is a major cause of blindness worldwide and is characterized by progressive retinal ganglion cell (RGC) death, mostly due to elevated intraocular pressure (IOP). Here, an ethanolic extract of C. cicadae mycelium (CCME), a traditional medicinal mushroom, was studied for its potential in lowering IOP in rat and rabbit models. Data showed that CCME could significantly (60.5%) reduce the IOP induced by microbead occlusion after 56 days of oral administration. The apoptosis of retinal ganglion cells (RGCs) in rats decreased by 77.2%. CCME was also shown to lower the IOP of normal and dextrose-infusion-induced rabbits within 60 min after oral feeding. There were dose effects, and the effect was repeatable. The active ingredient, N6-(2-hydroxyethyl)-adenosine (HEA), was also shown to alleviate 29.6% IOP at 0.2 mg/kg body weight in this rabbit model. CCME was confirmed with only minor inhibition in the phosphorylated myosin light chain 2 (pMLC2) pathway.
... Our in vivo manifestations of fine PM effects are consistent with the clinical characteristic of dry eye [32,33], the diagnosis of which in clinical practice is widely based on the TBUT, the tear secretion test and goblet cell counting. In consistent with other studies that presented TBUT in normal mouse less than 10 s [34,35], which is the minimum human normal value, we found that TBUT of mice was 4.59 ± 0.22 s at 3 rd and 4.49 ± 0.14 s at 6 th month. The lower value of mice may be attributed to materials, animals, handling techniques and protocols. ...
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Exposure to ambient fine particulate matter (fine PM) pollution has been previously associated with ocular surface diseases. But, to the best of our knowledge, the in vivo long-term effects of fine PM on the ocular surface have not been investigated. We aimed to evaluate the effects of fine PM on cultured human corneal epithelial (HCE) cells and on the ocular surfaces of mice, with standard reference material of fine PM(SRM 2786). We applied fine PM suspension to the eyes of C57BL/6 mice for up to 6 months. In vivo examinations, including tear secretion, tear film break-up time (TBUT) and corneal fluorescein staining, were performed in the 3rd and 6th month. At the end of the in vivo study, the corneal histological changes and conjunctival goblet cells were examined by staining, and cytokines in tissue were also detected. In addition, HCE cells were treated with fine PM for 12 h and 24 h. Then, cell apoptosis and reactive oxygen species (ROS) formation was detected. We found that fine PM damages the mouse eye in a dose- and time-dependent manner. In mice, the tear secretion and tear film break-up time were significantly reduced, along with the development of corneal epithelial damage, apoptosis of conjunctival epithelial cells and hypoplasia of conjunctival goblet cells. In addition, IL-18, IL-22, IL-23 and MCP-1 were increased in both conjunctiva and cornea of the fine PM-treated animals. Furthermore, increased apoptosis and ROS production were observed in time- and dose-dependent manner in HCE cells after fine PM exposure for 12 h and 24 h. Our results indicate that fine PM is cytotoxic to both HCE cells and the ocular surface. Long-term topical application of fine PM suspension in mice results in ocular surface changes that are similar to those observed with dry eye.
Dry eye disease (DED), a multifactorial inflammatory ocular surface disorder, affects up to 50% of individuals over 50 years old worldwide and is one of the most common reasons for seeking ophthalmologic care. Generally, topical eye drops or oral drugs are administered to treat DED; however, the use of preservatives in eye drops or the adverse effects of oral drugs are disadvantageous for long-term therapy. Cordyceps cicadae, a traditional Chinese medicinal fungus, possesses anti-inflammatory effects without evident toxicity and is obtainable at low price. Our previous study demonstrated that C. cicadae mycelium effectively ameliorates dry eye symptoms in the benzalkonium chloride (BAC)-induced mouse dry eye model by increasing tear volume and tear film breakup time (TBUT). However, the effects of C. cicadae mycelium for human dry eye amelioration remains unknown. Thus, the present study investigated the mitigation of dry eye conditions and related discomforts through oral supplementation of fermented C. cicadae mycelium. A total of 70 healthy individuals were recruited and randomly allocated to receive a daily oral dose of 1,050 mg preparation in sachet containing either freeze-dried C. cicadae mycelium powder with 0.3 mg of adenosine and 1.5 mg of HEA per gram or placebo for 90 days. The participants were subjected to anthropometric measurements, dry eye questionnaires (DEQ), Schirmer's tests, intraocular pressure (IOP) measurements, tear film breakup time (TBUT) tests, tear osmolality measurements, and tear electrolyte analysis prior to and right after completion of the study. The results showed a significantly increased TBUT as well as a significant decrease in tear osmolarity, in parallel with the decrease of tear electrolytes, especially Na+ and Cl ions. Although significant increase of tear volume was not observed, the increased TBUT suggests mitigation of dry eye through improvement of tear quality. Therefore, C. cicadae mycelium supplementation may be used for dry eye alleviation as a novel therapeutic intervention.
Cordyceps cicadae mycelium (CCM) reduces high intraocular pressure (IOP) in animal models. This clinical study determined the decrease in IOP after one CCM dose in humans. Forty-six patients with IOP ≥ 21 mmHg in either eye were enrolled and their pulse, blood pressure (BP), and IOP were assessed at 0 and 90 min after CCM administration. CCM did not affect the pulse, but it lowered systolic and diastolic BP overall at 90 min, particularly in men. CCM significantly reduced the IOP in patients IOP ≥ 21 mmHg in left and right eyes in overall, and women had a significant reduction compared with men. The IOP decrease in patients ≥ 60 years old showed a significant decrease in the left eye overall and in women, but the right eye showed only a minor decrease at 90 min. This is the first report indicating that CCM can quickly and significantly reduce high IOP in humans.
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Dry eye and dry mouth are the principal sources of morbidity for patients with Sjögren's syndrome (SS). There are few effective treatments, particularly systemic ones. Targeting aquaprin-5 (AQP5)-mediated tear secretion has been tested as a novel ancillary strategy and has proved promising. Patients have a great interest in using complementary medicine, including nutraceuticals and bioactive compounds to alleviate their symptoms. Potential mechanisms by which phytocompounds and bioactive compounds may benefit SS ocular and mouth symptoms through modulation of AQP5 activity are presented within this review. Supplementation with prebiotics (such as polyphenols with high bioavailability) in SS patients with lower Firmicutes/Bacteroides (F/B) community ratio phenotype, through administration of butyrate-producing diets, is proposed as ancillary strategy for dry eye and mouth. The potential use of natural bioactive compounds to treat dry eye could also apply to dry mouth occurring in the context of aging and SS. This novel hypothesis could have implications with respect to planning a successful dietary regimen for achieving and maintaining a normal gut microbiota in SS patients. This regimen would include augmenting butyrate-producing foodstuffs and/or polyphenol-rich syrups, and high amounts of some specific probiotic-rich foodstuffs such as yogurt, soy yogurt, or as probiotic supplements. There are applications for pharmaceutical and nutraceutical products aiming to relieve dry eye and mouth.
The salient rise of chronic disease from the mid-twentieth century threatens to overwhelm public health systems in an increasing number of countries and is now considered an epidemic. Dry eye disease is an underappreciated disorder that bears all the hallmarks of chronic disease. Preventative health care seeks improved and sustainable patient engagement in the self-management of health to limit the progress and extent of chronic disease. Anthropogenic environments engendering lifestyles and behaviours that can be detrimental to human health, can be considered as direct or indirect threats to successful preventative health strategies. Chronic disease can be viewed as the result of physiological responses of the human body to the modern environment. The quest for an increasingly convenient, global, and disease-free lifestyle is ironically threatening to undo the gains in health and quality of life made over the last one hundred years. Considering dry eye disease as an anthropogenic chronic disease, contributions of diet (food and beverages consumed) and nutrition (extending to relationships with self, community, and nature) to development of dry eye disease are explored in this review. Evidence of environmental and behavioural instigators of chronic disease with an emphasis on production, disbursement, and preservation of food, is presented. Furthermore, evidence of traditional food practices that offer resistance to the development of chronic systemic inflammatory disorders are reviewed as an exemplar of potential strategies that can be put into practice by individuals and communities to reinstate a balanced life, community and planet.
Objective: Cordyceps cicadae, a medicinal fungus, is assessed as having many functions: anti-cancer, anti-fatigue, anti-aging, immune-boosting, renal and liver protection. Since the industrial production of C. cicadae mycelium consistently manufactures bioactive compounds superior to wild fruiting bodies, there is a need to confirm the toxicity of liquid fermented C. cicadae mycelium. Studies showed the toxicity evaluation of C. cicadae mycelium in animal models, but safety reports in clinical studies are scarce. As such, a safety assessment of oral N6-(2-hydroxyethyl) adenosine (HEA-enriched) C. cicadae mycelium in humans is provided here. Method: After 49 participants ingested granules of 1.05 g of freeze-dried C. cicadae mycelium once a day for 3 months, their blood samples were collected at the beginning and end of the experiment for analysis. Results: There were no significant differences between the initial and final measurements in renal and liver function. Also, there was no influence on blood electrolytes as well as blood lipid levels. In clinical observation, there were also no side effects or adverse feelings mentioned by participants. Conclusion: These results suggested that HEA-enriched C. cicadae mycelium produced by liquid fermentation is safe and can be developed as a functional health food.
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Background: Dry eye syndrome (DES) is one of the key clinical features and possibly an early clinical presentation of Sjögren's syndrome (SS). We explore DES prevalence and assess the likelihood of DES patients to develop SS in Taiwan through the National Health Insurance Research Database (NHIRD). Methods: Through a cohort comparison study, longitudinal data from the NHIRD (2000 to 2008) in Taiwan was used to probe the prevalence of DES and the odds that DES patients would later develop SS. Results: The prevalence of DES in the present study is 4.87%. The incidence rates of developing SS were 4.8% for the DES group and 1.5% for comparison group. The median age and interquartile range of DES and comparison patients was 49.8 (10) and 48.7 (15) years old, respectively. The crude hazard ratio (with 95% confidence interval) for DES patients to develop SS was 3.13 (3.10-3.50) for the DES group, and the adjusted hazard ratio (with 95% confidence interval) was 3.64 (3.43-3.87). The observation period and interquartile range for DES and comparison patients to develop SS later were 1418 (781-2316) versus 1641 (971-2512) days respectively. Conclusions: DES patients carried a higher risk for developing SS (hazard ratio 3.13) and presented for SS 3.88 years earlier than comparison group patients in this study.
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Oxidative stress has been known to be involved in pathogenesis of dry eye disease. However, few studies have comprehensively investigated the relationship between hyperosmolarity and oxidative damage in human ocular surface. This study was to explore whether and how hyperosmolarity induces oxidative stress markers in primary human corneal epithelial cells (HCECs). Primary HCECs were established from donor limbal explants. The hyperosmolarity model was made in HCECs cultured in isosmolar (312 mOsM) or hyperosmotic (350, 400, 450 mOsM) media. Production of reactive oxygen species (ROS), oxidative damage markers, oxygenases and anti-oxidative enzymes were analyzed by DCFDA kit, RT-qPCR, immunofluorescent and immunohistochemical staining and Western blotting. Compared to isosmolar medium, ROS production significantly increased at time- and osmolaritydependent manner in HCECs exposed to media with increasing osmolarities (350-450 mOsM). Hyperosmolarity significantly induced oxidative damage markers in cell membrane with increased toxic products of lipid peroxidation, 4-hydroxynonenal (4-HNE) and malondialdehyde (MDA), and in nuclear and mitochondria DNA with increased aconitase-2 and 8-OHdG. Hyperosmotic stress also increased the mRNA expression and protein production of heme oxygenase-1 (HMOX1) and cyclooxygenase-2(COX2), but reduced the levels of antioxidant enzymes, superoxide dismutase-1(SOD1), and glutathione peroxidase-1 (GPX1). In conclusion, our comprehensive findings demonstrate that hyperosmolarity induces oxidative stress in HCECs by stimulating ROS production and disrupting the balance of oxygenases and antioxidant enzymes, which in turn cause cell damage with increased oxidative markers in membrane lipid peroxidation and mitochondrial DNA damage.
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A strong cohort of evidence exists that supports the localisation of corneal stem cells at the limbus. The distinguishing characteristics of limbal cells as stem cells include slow cycling properties, high proliferative potential when required, clonogenicity, absence of differentiation marker expression coupled with positive expression of progenitor markers, multipotency, centripetal migration, requirement for a distinct niche environment and the ability of transplanted limbal cells to regenerate the entire corneal epithelium. The existence of limbal stem cells supports the prevailing theory of corneal homeostasis, known as the XYZ hypothesis where X represents proliferation and stratification of limbal basal cells, Y centripetal migration of basal cells and Z desquamation of superficial cells. To maintain the mass of cornea, the sum of X and Y must equal Z and very elegant cell tracking experiments provide strong evidence in support of this theory. However, several recent studies have suggested the existence of oligopotent stem cells capable of corneal maintenance outside of the limbus. This review presents a summary of data which led to the current concepts of corneal epithelial homeostasis and discusses areas of controversy surrounding the existence of a secondary stem cell reservoir on the corneal surface.
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Purpose: The purpose of this article is to review the evidence for the hypothesis that the core mechanism of dry eye disease (DED) is inflammation, including evidence from recent basic, clinical, and translational research involving human patients, animal models, and cell cultures. Methods: Using the key words "dry eye + inflammation," the authors conducted a comprehensive search of the PubMed and Web of Science databases for scientific articles published in English between January 1, 1900 and August 30, 2013 on the role of inflammation in DED in cell cultures, animal models, and humans. The resulting articles were then categorized and reviewed. Results: The literature search revealed a total of 458 publications, almost all published after 1992. The percentages of original studies and review articles are 77.29% (354) and 22.71% (104), respectively. Among the original studies, the number of reports on human DED is 200 (43.7%), on animal models is 115 (25.1%), and cell cultures is 39 (8.5%). A yearly distributing plot revealed that 76% were published from 2003 to 2011, 53% from 2008 to 2012, and 11% during the first 9 months of 2013. This distribution signifies a rapidly growing awareness of the importance of inflammation in DED pathogenesis. Conclusions: Inflammation plays a key role in the pathogenesis of DED as evidenced by research using tissue culture, animal models, and subjects with DED. Developing biomarkers for inflammation of the ocular surface will provide improved understanding of the mechanisms leading to DED, classification of the severity of DED, and objective metrics for outcome measures of treatment. The chronicity of the disease suggests that dysregulation of immune mechanisms leads to a cycle of continued inflammation, accompanied by alterations in both innate and adaptive immune responses. Given the underlying mechanism for DED, developing effective and safe anti-inflammatory treatments is likely to be beneficial for patients with DED.
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Weekly disposable soft contact lenses have been widely used recently, but their shield effects against ultraviolet (UV) irradiation remain to be evaluated. This study investigated the bioprotective effects of several weekly soft contact lenses against UVB irradiation on the corneal surface in a mouse model. FIFTY ICR MICE WERE RANDOMLY DIVIDED INTO FIVE GROUPS: (1) blank control, (2) exposed to UVB without contact lens protection, (3) exposed to UVB and protected with Vifilcon A contact lenses, (4) exposed to UVB and protected with Etafilcon A contact lenses, and (5) exposed to UVB and protected with HEMA+MA contact lenses. The exposure to UVB irradiation was performed at 0.72 J/cm(2)/day after anesthesia for a 7-day period, followed by cornea surface assessment for smoothness, opacity, and grading of lissamine green staining. Tissue sections were prepared for hematoxylin and eosin staining and immunohistochemical detection by using antibodies against myeloperoxidase, cytokeratin-5, P63, Ki-67, nuclear factor-kappa B (p65), cyclooxygenase-2, Fas L, and Fas. The results showed impaired corneal surface with myeloperoxidase(+) polymorphonuclear leukocyte infiltration into the stroma after UVB exposure, in contrast to the intact status of the blank controls. The corneas with Etafilcon A and HEMA+MA contact lenses maintained more cells positive for cytokeratin-5, P63, and Ki-67 compared to those with Vifilcon A or without contact lens protection. Furthermore, less proinflammatory factors, including nuclear factor-kappa (p65), cyclooxygenase-2, Fas L, and Fas, were induced in the corneas protected by Etafilcon A and HEMA+MA. This study demonstrated various protective effects of weekly disposable contact lenses against UVB irradiation. The mouse model used in the present study may be used extensively for in vivo assessment of UV shield efficacy.
In studies on dry eye (DE) disease, an association has been identified between tear film hyperosmolarity and inflammation severity elicited through receptor-induced increases in proinflammatory cytokine and chemokine release. These immune reactions might be mediated by inflammasomes, macromolecular complexes mounted around the NLRP3 protein and can be activated by reactive oxygen species (ROS) over-generation. Hence in this study we determine whether: a) ROS activated NLRP3 inflammasomes mediate hyperosmotic stress-induced inflammation in human corneal epithelial cells (HCECs); b) the ROS-NLRP3-IL-1β axis activation is associated with environment-induced DE. Immortalized HCECs were exposed to 500 mOsm medium in the presence and absence of a ROS inhibitor, N-acetyl-l-cysteine (NAC). HCECs transfected with NLRP3 siRNA or a negative control (NC) siRNA. Intracellular ROS was measured by fluorometric analysis using the probe 2',7'-dichlorofluorescin diacetate (DCFH-DA). Real-time PCR evaluated NLRP3, ASC, pro-caspase-1 and pro-IL-1β mRNA levels. Western blot analysis assessed NLRP3 protein expression whereas caspase-1 activity was determined with a fluorometric assay. Bioactive IL-1β release was assessed by ELISA. ROS production, NLRP3 inflammasome and pro-IL-1β gene expression as well as IL-1β secretion were also evaluated in the conjunctival epithelial cells and tear fluid samples of environment-induced DE patients and normal subjects. NAC suppressed hyperosmolarity-induced rises in ROS levels, NLRP3 inflammasome formation and activation, caspase-1 activity and IL-1β release. On the other hand, NLRP3 siRNA knockdown inhibited hyperosmotic stress-induced NLRP3 activation, which led to ASC, pro-caspase-1 and pro-IL-1β mRNA dowregulation followed by suppression of associated caspase-1 activity and IL-1β secretion. In addition, in ocular surface samples of environment-induced DE patients, ROS generation, NLRP3, ASC, pro-caspase-1 and pro-IL-1β gene expression as well as IL-1β secretion were upregulated. Taken together, NLRP3 mediated innate immune responses triggered by rises in ROS generation induce inflammation in hyperosmotic stressed HCECs. ROS-NLRP3-IL-1β signaling pathway might play a priming role in environment-induced DE development. Copyright © 2015. Published by Elsevier Ltd.
Purpose: We evaluated the potential protective effects of Coenzyme Q10 (CoQ10) on human corneal cells and rabbit eyes after ultraviolet B (UVB) exposure and a model of wound healing in rabbit eyes after corneal epithelium removal. Methods: Human corneal epithelium cells (HCE) were exposed to a source of UVB radiation (312 nM) in the presence of different CoQ10 concentrations or vehicle. The mitochondrial function and cell survival were evaluated by means of 3-(4,5-dimethylthiazole-2-yl)2,5-diphenyl-tetrazolium (MTT) reduction and lactic dehydrogenase (LDH) release. Furthermore, quantitation of oxygen consumption and mitochondrial membrane potential were conducted. In vivo rabbit models were adopted to evaluate the effect of CoQ10 on UVB-induced conjunctival vessel hyperemia and corneal recovery after ethanol induced corneal lesion. Results: In UVB-exposed HCE cells, CoQ10 addition led to an increased survival rate and mitochondrial function. Furthermore, oxygen consumption was maintained at control levels and adenosine triphosphate (ATP) decline was completely prevented in the CoQ10-treated cells. Interestingly, in an in vivo model, CoQ10 was able dose-dependently to reduce UVB-induced vessel hyperemia. Finally, in a model of corneal epithelium removal, 12 hours from surgery, animals treated with CoQ10 showed a reduction of damaged area in respect to vehicle controls, which lasted until 48 hours. Conclusions: We demonstrated that CoQ10 reduces corneal damages after UVB exposure in vivo and in vitro by preserving mitochondrial function. Also, for the first time to our knowledge we showed that the administration of CoQ10 after corneal epithelium removal promotes corneal wound healing.
Chronic kidney disease is a growing public health problem with an urgent need for new pharmacological agents. Ergosterol peroxide (EP) is the major sterol produced by Cordyceps cicadae Shing (C. cicadae), a widely used traditional Chinese medicine. C. cicadae has been used to treat many kinds of diseases and has a potential benefit on renoprotection. This study aimed to investigate the anti-fibrotic effects of EP as well as the underlying mechanisms. A normal rat kidney fibroblast cell line (NRK-49F) was stimulated to undergo fibroblast activation by transforming growth factor-β1 (TGF-β1) and EP treatment was applied to explore its potential anti-fibrotic effects. Cell proliferation was investigated using MTT analysis. Fibrosis-associated protein expression was analyzed using immunohistochemistry and/or Western blotting. EP treatment attenuated TGF-β1-induced renal fibroblast proliferation, expression of cytoskeleton protein and CTGF, as well as ECM production. Additionally, EP blocked TGF-β1-stimulated phosphorylation of ERK1/2, p38 and JNK pathway. Moreover, the TGF-β1-induced expression of fibronectin was attenuated by either inhibition of MAPKs or by EP treatment. In conclusion, our findings demonstrate that EP is able to suppress TGF-β1-induced fibroblasts activation in NRK-49F. This new information provides a line of theoretical evidence supporting the use of C. cicadae in the intervention of kidney disease and suggests that EP has the potential to be developed as a therapeutic agent to prevent renal fibrosis.
Purpose: This study investigated the effects of dietary α-lipoic acid (α-LA) against ultraviolet B (UVB)-induced corneal and conjunctival degeneration in a mouse model. Methods: Female CBA mice were randomly divided into five study groups, including blank control, UVB without α-LA, and UVB with dietary α-LA at 1, 10, and 100 mg/kg body weight. Following UVB exposure, corneal surfaces were assessed along with immunohistochemistry for nuclear factor-κB (NF-κB), cyclooxygenase-2 (COX-2), malondialdehyde (MDA) accumulation, and P63⁺ basal cell distribution. Matrix metalloproteinase (MMP)-2 and MMP-9 activities were determined by gelatin zymography. ELISA assay was performed to confirm the findings of immunohistochemistry for NF-κB, COX-2, and MDA, along with the levels of TNF-α and IL-6. Tear production and goblet cell density were determined after tear strip assay and periodic acid Schiff staining, respectively. Results: The results showed that UVB irradiation caused corneal surface damage, polymorphonuclear leukocyte infiltration, and loss of P63⁺ basal cells. Dietary α-LA ameliorated the UVB-induced corneal damage while simultaneously reducing MDA accumulation and maintaining P63⁺ basal cell survival. NF-κB-p65, COX-2, TNF-α, IL-6, and MMP-9 activity were all reduced by dietary α-LA. In addition, α-LA helped to reverse aqueous tear reduction, conjunctival squamous epithelium metaplasia, and goblet cell loss after UVB exposure. Conclusions: Dietary α-LA can prevent UVB-induced corneal damage and can be used as a prophylactic agent prior to excessive UVB exposure.