Transcript Effect of Topical Epigallocatechin Gallate to Treat Dry

The Effect of Topical Epigallocatechin Gallate
(EGCG) Treatment on Murine Dry Eye
Hyun Soo Lee, Andre Okanobo, Nambi Nallasamy,
Sunil K. Chauhan, M. Reza Dana
Schepens Eye Research Institute Boston, Massachusetts
Department of Ophalmology, Harvard Medical School and Massachusetts
Eye and Ear Infirmary, Boston, Massachusetts
The authors have no financial interest in the subject matter of this poster.
Introduction
Dry eye is the most common cause for clinical disease in
ophthalmology and it is widely recognized that dry eye
disease (DED) is associated with inflammation of the ocular
surface.
Epigallocatechin gallate (EGCG), one of main extracts in
green tea, has an inhibitory effect on inflammation by
reducing the expression of IL-1, IL-6, MCP-1 and TNF-α
through inhibition of NF-kB activation.
Hypothesis
Topical EGCG administration may have a therapeutic effect
for the treatment of DED by inhibiting proinflammatory
cytokines through the modulation of NF-kB.
Here, we aimed to investigate the efficacy of topical EGCG
on a murine model of dry eye.
Materials and Methods
Female 7-8 week old C57BL/6 mice were housed in the
controlled environment chamber (relative humidity < 30%,
constant temperature of 21°C to 23°C, and airflow of 15 L/min
for all day) to induce DED. Subcutaneous administration of
scopolamine (tid) was used to maximize ocular dryness.
Forty-eight hours after induction of dry eye, mice were randomly
divided into the following 4 different groups: 1) untreated group,
2) topical vehicle treatment, 3) topical 0.01% EGCG, and 4)
topical 0.1% EGCG.
Materials and Methods
Corneal fluorescein staining (using the National Eye
Institute grading scheme) at baseline (day 0), 48 hrs, and
day 4 and day 9 after induction of DED
Immunohistochemical staining (day 9)
1) corneal CD11b+ cells, 2) NF-kB p65
Real-time PCR (day 9) : corneal cytokines IL-1β, TNF-α,
monocyte chemotactic protein (MCP)-1
Corneal TUNEL assay (day 9)
Results: corneal fluorescein staining score
Topical 0.1% EGCG treatment produced a significant decrease in the corneal fluorescein
staining score compared with the vehicle and untreated groups at days 9. Asterisk
indicates P<.0001 vs untreated, P=.001 vs vehicle, P=.006 vs 1.0% EGCG. But there was
no significant difference between 0.01% EGCG and vehicle. Data are presented as mean
and SEM (error bars).
Results: CD11b+ cells in dry eye cornea
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Center
Periphery
Both 0.01% and 0.1% EGCG treatment decrease the number of CD11b+ cells in the center
and periphery of dry eye corneas, compared with the untreated and vehicle groups.
Asterisk indicates P < .05 ; dagger, P<.01 ; and double dagger, P<.001. Data are
presented as mean and SEM (error bars).
Results: real-time PCR
A
B
IL-1b
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TNF-α
C
MCP-1
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Real-time polymerase chain reaction results showing that corneas treated with
0.1% and 0.01% EGCG have significantly decreased relative expression of IL-1β (A)
and monocyte chemoattractant protein (MCP)-1(C) transcripts in dry eye cornea.
Data were normalized to GAPDH mRNA and values are expressed as fold change
over normal control corneas. Data from three independent experiments are
presented as mean and SEM (error bars)
Results : Immunohistochemistry of NF-kB p65
) Normal control
) Normal control
) Untreated
) Untreated
) Vehicle
) Vehicle
) 0.1% EGCG
) 0.1% EGCG
The p65 protein was localized in the
cytoplasm of the corneal epithelial
cells of normal controls (A).
Significant nuclear translocation for
p65 protein was observed in the
epithelium of the untreated DED
cornea (C). In contrast, 0.1% EGCG
(G) decreased the translocation of
NF-kB p65 compated to the vehicletreated group (E). Left column:
Immunohistochemistry staining of
NF-kB p65. Right column: DAPI (4’, 6
diamidino-2-phenylindole) staining of
nucleus.
Results : Tunel assay
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The number of apoptotic cells was significantly increased in dry eye corneal epithelial
layer compared with normal controls (P<.0001 vs normal control). Although 0.01%
and 0.1% EGCG showed decrease in the number of apoptotic cells compared to the
untreated group (P=.009 and P=.003, respectively), But topical 1.0% EGCG
significantly increased the corneal epithelial apoptosis compared with 0.01% and
0.1% EGCG groups (P=.013 and P=.007, respectively)
Conclusions
Topical EGCG treatment is able to reduce the clinical signs
and inflammatory changes in experimental dry eyes
through suppressing the inflammatory cytokine expression
and infiltration of CD11b+ cells in the cornea.
Our
study
suggests
pharmacologically
active
that
EGCG,
agent
available
a
potential
for
topical
administration, could be used therapeutically for the
treatment of dry eye.