neonatal sprague - dawley rats ( seven days old ) together with their maternal rats were obtained from a commercial breeder ( orient co. , seoul , korea ) .
the experimental procedures were performed in accordance with the animal care guidelines of the national institutes of health ( nih ) and the korean academy of medical sciences .
each animal was housed under controlled temperature ( 232 ) and lighting ( 08:00 - 20:00 ) conditions with free feeding .
the neonatal rats were randomly divided into five groups ( n=15 in each group ) : a control group , and four cataract - induction groups , treated with either 0 , 50 , 100 , or 200 mg / kg catechin .
the rats in the catechin - treatment groups received catechin ( sigma chemical co. , st .
louis , mo , usa ) orally once a day for ten consecutive days at the respective doses , starting five days after cataract - induction .
the rats in the control group and in the cataract - induction groups received an equal amount of distilled water for the same duration .
cataracts were induced using a previously described procedure . in brief , at ten days of postnatal age , the neonatal rats received 100 mg / kg n - methyl - n - nitrosourea ( mnu , sigma chemical co. ) intraperitoneally . just before use , mnu was dissolved in physiological saline containing 0.05% acetic acid .
slit - lamp biomicroscopic examination was performed on each eye to provide a morphological assessment of the degree of opacification at 15 days after cataract induction .
prior to the examination , mydriasis was achieved using a topical ophthalmic solution containing tropicamide with phenylephrine hydrochloride ( santen pharmaceutical , osaka , japan ) .
one drop of the solution was instilled in each eye every 30 minutes for 2 hours , while the animals were in a dark room .
after 2 hours , the eyes were examined by slit - lamp biomicroscopy at 12magnification .
the rats were sacrificed immediately after determination of cataract formation with slit - lamp biomicroscopy ( 15 days after cataract induction ) .
the animals were anesthetized using zoletil 50 ( 10 mg / kg , i.p . ; vibac laboratories , carros , france ) . at necropsy ,
both lenses were quickly removed under a surgical microscope gl-99b - v7 ( davis , california , ca , usa ) ; a complete necropsy was performed on all animals .
the lenses were fixed in 4% paraformaldehyde , dehydrated in graded ethanol , treated in xylene , and infiltrated and embedded in paraffin .
coronal sections of 5 m thickness were made using a paraffin microtome ( leica , nussloch , germany ) and were mounted on coated slides , then dried at 37 overnight on a hot plate .
the lenses were collected and immediately frozen at -70. the tissues were homogenized with lysis buffer containing 50 mm tris - hcl ( ph 8.0 ) , 150 mm nacl , 10% glycerol , 1% triton x-100 , 1.5 mm mgcl26h2o , 1 mm egta , 1 mm pmsf , 1 mm na2vo4 , and 100 mm naf , and then ultracentrifuged at 50,000 rpm for 1 hours .
protein content were measured using a bio - rad colorimetric protein assay kit ( bio - rad , hercules , ca , usa ) .
forty micrograms of protein was separated on sds - polyacrylamide gels and were transferred onto a nitrocellulose membrane .
mouse antibodies against actin ( 1:2000 ; santa cruz biotech , santa cruz , ca , usa ) , bax ( 1:1000 ; santa cruz biotech ) , and bcl-2 ( 1:1000 ; santa cruz biotech ) were used as primary antibodies .
horseradish peroxidase - conjugated anti - mouse antibodies for bax and bcl-2 ( 1:2000 ; amersham pharmacia biothech gmbh , freiburg , germany ) were used as secondary antibodies .
the experiment was performed in normal lab conditions at room temperature , with the exception of membrane transfer .
membrane transfer was performed at 4 with a cold pack and a pre - chilled buffer .
band detection was performed using an enhanced chemiluminescence ( ecl ) detection kit ( santa cruz biotech ) .
detected bands were calculated densitometrically using molecular analyst version 1.4.1 ( bio - rad ) , in order to compare the relative expressions of proteins .
tunel staining was performed using an in situ cell death detection kit ( roche , mannheim , germany ) according to the manufacturer 's protocol , in order to visualize dna fragmentation , a marker of apoptotic cell death .
the epithelial cells were suspended in 10 mm tris - hcl buffer , ph 8.0 , containing 1 mm edta , through incubation at 55 for 30 minutes .
sections were then incubated with proteinase k ( 100 g / ml ) , rinsed , incubated in 3% h2o2 , permeabilized with 0.5% triton x-100 , rinsed again , and incubated in tunel reaction mixture .
the sections were rinsed and visualized using converter - pod with 0.03% 3,3'-diaminobenzidine ( dab ) and then mounted onto gelatin - coated slides .
the slides were air dried overnight at room temperature , and coverslips were mounted using permount. to visualize caspase-3 expression , we performed caspase-3 immunohistochemistry using a previously described method .
sections were drawn from each lens and incubated overnight with mouse anti - caspase-3 antibody ( 1:500 ; santa cruz biotech ) and then for another 1 h with biotinylated mouse secondary antibody ( 1:200 ; vector laboratories , burlingame , ca , usa ) . bound secondary antibodies were then amplified with a vector elite abc kit ( 1:100 ; vector laboratories ) .
the antibody - biotin - avidin - peroxidase complexes were visualized using 0.03% dab , and the sections were finally mounted onto gelatin - coated slides .
the slides were air dried overnight at room temperature , and coverslips were mounted using permount. to compare relative expressions of proteins , we examined detected bands densitometrically using molecular analyst version 1.4.1 ( bio - rad ) .
the area of the lens epithelium region in each slice was measured using the image - pro plus computer - assisted image analysis system ( media cybernetics inc .
, silver spring , md , usa ) attached to a light microscope ( olympus , tokyo , japan ) .
the tunel - positive and caspase-3-positive cells within each lens epithelium region were counted through the light microscope .
statistical analysis was performed using one - way anova followed by duncan 's post - hoc test .
neonatal sprague - dawley rats ( seven days old ) together with their maternal rats were obtained from a commercial breeder ( orient co. , seoul , korea ) .
the experimental procedures were performed in accordance with the animal care guidelines of the national institutes of health ( nih ) and the korean academy of medical sciences .
each animal was housed under controlled temperature ( 232 ) and lighting ( 08:00 - 20:00 ) conditions with free feeding .
the neonatal rats were randomly divided into five groups ( n=15 in each group ) : a control group , and four cataract - induction groups , treated with either 0 , 50 , 100 , or 200 mg / kg catechin .
the rats in the catechin - treatment groups received catechin ( sigma chemical co. , st .
louis , mo , usa ) orally once a day for ten consecutive days at the respective doses , starting five days after cataract - induction .
the rats in the control group and in the cataract - induction groups received an equal amount of distilled water for the same duration .
cataracts were induced using a previously described procedure . in brief , at ten days of postnatal age , the neonatal rats received 100 mg / kg n - methyl - n - nitrosourea ( mnu , sigma chemical co. ) intraperitoneally . just before use , mnu was dissolved in physiological saline containing 0.05% acetic acid .
slit - lamp biomicroscopic examination was performed on each eye to provide a morphological assessment of the degree of opacification at 15 days after cataract induction . prior to the examination
, mydriasis was achieved using a topical ophthalmic solution containing tropicamide with phenylephrine hydrochloride ( santen pharmaceutical , osaka , japan ) .
one drop of the solution was instilled in each eye every 30 minutes for 2 hours , while the animals were in a dark room .
after 2 hours , the eyes were examined by slit - lamp biomicroscopy at 12magnification .
the rats were sacrificed immediately after determination of cataract formation with slit - lamp biomicroscopy ( 15 days after cataract induction ) .
the animals were anesthetized using zoletil 50 ( 10 mg / kg , i.p . ; vibac laboratories , carros , france ) . at necropsy ,
both lenses were quickly removed under a surgical microscope gl-99b - v7 ( davis , california , ca , usa ) ; a complete necropsy was performed on all animals .
the lenses were fixed in 4% paraformaldehyde , dehydrated in graded ethanol , treated in xylene , and infiltrated and embedded in paraffin .
coronal sections of 5 m thickness were made using a paraffin microtome ( leica , nussloch , germany ) and were mounted on coated slides , then dried at 37 overnight on a hot plate .
the lenses were collected and immediately frozen at -70. the tissues were homogenized with lysis buffer containing 50 mm tris - hcl ( ph 8.0 ) , 150 mm nacl , 10% glycerol , 1% triton x-100 , 1.5 mm mgcl26h2o , 1 mm egta , 1 mm pmsf , 1 mm na2vo4 , and 100 mm naf , and then ultracentrifuged at 50,000 rpm for 1 hours .
protein content were measured using a bio - rad colorimetric protein assay kit ( bio - rad , hercules , ca , usa ) .
forty micrograms of protein was separated on sds - polyacrylamide gels and were transferred onto a nitrocellulose membrane .
mouse antibodies against actin ( 1:2000 ; santa cruz biotech , santa cruz , ca , usa ) , bax ( 1:1000 ; santa cruz biotech ) , and bcl-2 ( 1:1000 ; santa cruz biotech ) were used as primary antibodies .
horseradish peroxidase - conjugated anti - mouse antibodies for bax and bcl-2 ( 1:2000 ; amersham pharmacia biothech gmbh , freiburg , germany ) were used as secondary antibodies .
the experiment was performed in normal lab conditions at room temperature , with the exception of membrane transfer .
membrane transfer was performed at 4 with a cold pack and a pre - chilled buffer .
band detection was performed using an enhanced chemiluminescence ( ecl ) detection kit ( santa cruz biotech ) .
detected bands were calculated densitometrically using molecular analyst version 1.4.1 ( bio - rad ) , in order to compare the relative expressions of proteins .
tunel staining was performed using an in situ cell death detection kit ( roche , mannheim , germany ) according to the manufacturer 's protocol , in order to visualize dna fragmentation , a marker of apoptotic cell death .
the epithelial cells were suspended in 10 mm tris - hcl buffer , ph 8.0 , containing 1 mm edta , through incubation at 55 for 30 minutes .
sections were then incubated with proteinase k ( 100 g / ml ) , rinsed , incubated in 3% h2o2 , permeabilized with 0.5% triton x-100 , rinsed again , and incubated in tunel reaction mixture .
the sections were rinsed and visualized using converter - pod with 0.03% 3,3'-diaminobenzidine ( dab ) and then mounted onto gelatin - coated slides .
the slides were air dried overnight at room temperature , and coverslips were mounted using permount.
sections were drawn from each lens and incubated overnight with mouse anti - caspase-3 antibody ( 1:500 ; santa cruz biotech ) and then for another 1 h with biotinylated mouse secondary antibody ( 1:200 ; vector laboratories , burlingame , ca , usa ) . bound secondary antibodies were then amplified with a vector elite abc kit ( 1:100 ; vector laboratories ) .
the antibody - biotin - avidin - peroxidase complexes were visualized using 0.03% dab , and the sections were finally mounted onto gelatin - coated slides .
the slides were air dried overnight at room temperature , and coverslips were mounted using permount.
to compare relative expressions of proteins , we examined detected bands densitometrically using molecular analyst version 1.4.1 ( bio - rad ) .
the area of the lens epithelium region in each slice was measured using the image - pro plus computer - assisted image analysis system ( media cybernetics inc . , silver spring , md , usa ) attached to a light microscope ( olympus , tokyo , japan ) .
the tunel - positive and caspase-3-positive cells within each lens epithelium region were counted through the light microscope .
statistical analysis was performed using one - way anova followed by duncan 's post - hoc test .
the degree of lenticular opacification was enhanced in the cataract - induction rats and was reduced by catechin treatment .
2 . the number of tunel - positive cells was 8.302.78/section in the control group , 157.5010.26/section in the cataract - induction group , 143.308.94/mm in the cataract - induction and 50 mg / kg catechin group , 136.4011.36/section in the cataract - induction and 100 mg / kg catechin group , and 97.106.98/section in the cataract - induction and 200 mg / kg catechin group .
these results show that mnu injection enhanced apoptotic cell death in the lens epithelium , and catechin treatment significantly suppressed cataract - induced apoptosis in a dose - dependent manner .
3 . the number of caspase-3-positive cells was 6.202.23/section in the control group , 103.206.78/section in the cataract - induction group , 101.203.67/mm in the cataract - induction and 50 mg / kg catechin group , 103.003.10/section in the cataract - induction and 100 mg / kg catechin group , and 84.202.73/section in the cataract - induction and 200 mg / kg catechin group .
these results show that mnu injection enhanced caspase-3 expression in the lens epithelium , and that catechin ( 200 mg / kg ) treatment significantly suppressed cataract - induced caspase-3 expression . to verify cataract - induced apoptosis
, we ascertained the relative protein expressions of bax and bcl-2 related to apoptosis , and levels of bax and bcl-2 are presented in fig .
the level of bax ( 24 kda , the pro - apoptotic factor ) in the control group was set at 1.00 . in the cataract induction group ,
the levels of bax were 3.110.28 in the 0mg / kg catechin group , 2.790.28 in the 50 mg / kg catechin group , 1.980.18 in the 100 mg / kg catechin group , and 1.970.26 in the 200 mg / kg catechin group .
in contrast , catechin administration reduced the expression of bax in a dose - dependent manner .
specifically , 100 mg / kg and 200 mg / kg catechin significantly suppressed the expression of bax protein .
the level of bcl-2 ( 26~29 kda , the anti - apoptotic factor ) in the control group was set at 1.00 . in the cataract induction groups ,
the levels of bcl-2 were 1.430.04 in the 0mg / kg catechin group , 1.550.03 in the 50 mg / kg catechin group , 1.430.05 in the 100 mg / kg catechin group , and 1.630.03 in the 200 mg / kg catechin group .
cataract formation increased bcl-2 expression , while catechin ( 200 mg / kg ) administration slightly enhanced the expression of bcl-2 .
we calculated the ratio of bax to bcl-2 , one of the crucial factors determining if cells will undergo apoptosis .
the ratio of bax to bcl-2 in the control group was set at 1.00 . in the cataract induction groups ,
the ratios of bax to bcl-2 were 2.170.20 in the 0mg / kg catechin group , 1.800.18 in the 50 mg / kg catechin group , 1.380.13 in the 100 mg / kg catechin group , and 1.210.16 in the 200 mg / kg catechin group .
cataract enhanced the expression of both bax and bcl-2 , but bax expression increased much more than did increased bcl-2 expression . on the other hand , administration of catechin significantly suppressed the expression of bax protein . as a result
, the ratio of bax to bcl-2 was increased by cataract formation , representing ongoing apoptotic cell death in the lens epithelium .
catechin treatment suppressed the ratio of bax to bcl-2 , representing inhibition of apoptotic cell death in the lens epithelium .
the degree of lenticular opacification was enhanced in the cataract - induction rats and was reduced by catechin treatment .
2 . the number of tunel - positive cells was 8.302.78/section in the control group , 157.5010.26/section in the cataract - induction group , 143.308.94/mm in the cataract - induction and 50 mg / kg catechin group , 136.4011.36/section in the cataract - induction and 100 mg / kg catechin group , and 97.106.98/section in the cataract - induction and 200 mg / kg catechin group .
these results show that mnu injection enhanced apoptotic cell death in the lens epithelium , and catechin treatment significantly suppressed cataract - induced apoptosis in a dose - dependent manner .
3 . the number of caspase-3-positive cells was 6.202.23/section in the control group , 103.206.78/section in the cataract - induction group , 101.203.67/mm in the cataract - induction and 50 mg / kg catechin group , 103.003.10/section in the cataract - induction and 100 mg / kg catechin group , and 84.202.73/section in the cataract - induction and 200 mg / kg catechin group .
these results show that mnu injection enhanced caspase-3 expression in the lens epithelium , and that catechin ( 200 mg / kg ) treatment significantly suppressed cataract - induced caspase-3 expression .
to verify cataract - induced apoptosis , we ascertained the relative protein expressions of bax and bcl-2 related to apoptosis , and levels of bax and bcl-2 are presented in fig .
4 . the level of bax ( 24 kda , the pro - apoptotic factor ) in the control group was set at 1.00 . in the cataract induction group ,
the levels of bax were 3.110.28 in the 0mg / kg catechin group , 2.790.28 in the 50 mg / kg catechin group , 1.980.18 in the 100 mg / kg catechin group , and 1.970.26 in the 200 mg / kg catechin group .
in contrast , catechin administration reduced the expression of bax in a dose - dependent manner .
specifically , 100 mg / kg and 200 mg / kg catechin significantly suppressed the expression of bax protein .
the level of bcl-2 ( 26~29 kda , the anti - apoptotic factor ) in the control group was set at 1.00 . in the cataract induction groups ,
the levels of bcl-2 were 1.430.04 in the 0mg / kg catechin group , 1.550.03 in the 50 mg / kg catechin group , 1.430.05 in the 100 mg / kg catechin group , and 1.630.03 in the 200 mg / kg catechin group .
cataract formation increased bcl-2 expression , while catechin ( 200 mg / kg ) administration slightly enhanced the expression of bcl-2 .
we calculated the ratio of bax to bcl-2 , one of the crucial factors determining if cells will undergo apoptosis .
the ratio of bax to bcl-2 in the control group was set at 1.00 . in the cataract induction groups ,
the ratios of bax to bcl-2 were 2.170.20 in the 0mg / kg catechin group , 1.800.18 in the 50 mg / kg catechin group , 1.380.13 in the 100 mg / kg catechin group , and 1.210.16 in the 200 mg / kg catechin group .
cataract enhanced the expression of both bax and bcl-2 , but bax expression increased much more than did increased bcl-2 expression . on the other hand , administration of catechin significantly suppressed the expression of bax protein . as a result
, the ratio of bax to bcl-2 was increased by cataract formation , representing ongoing apoptotic cell death in the lens epithelium .
catechin treatment suppressed the ratio of bax to bcl-2 , representing inhibition of apoptotic cell death in the lens epithelium .
several animal species experience spontaneously occurring cataract of known inheritance and offer valuable model for studying human cataract .
various chemicals are known to contribute to the development of cataract in animals . among these chemicals ,
mnu , a direct - acting alkylating agent that does not require metabolic activation , is known as a cataractogenic agent in rats .
in addition , young animals are reported to be more susceptible to mnu than are adult animals .
therefore , in this study , a cataract model was constructed using a single intraperitoneal injection of nmu in rats at postnatal day 10 .
lens epithelial cells play a vital role in the metabolic homeostasis and maintenance of transparency in the lens , and damage to lens epithelial cells potently contributes to cataractogenesis .
moreover , apoptosis of lens epithelial cells has been reported to be the earliest event in the experimental formation of cataracts , such as those inducted by hydrogen peroxide and mnu . in human studies ,
the number of tunel - positive cells is above 50% in the lens epithelium after cataract surgery .
in addition , caspase-3 is up - regulated and activated in the early stages of apoptosis following cataractogenesis .
we found that the numbers of tunel - positive and caspase 3-positive cells in the lens epithelium were significantly higher following cataract induction .
these findings indicate that mnu injection - induced cataracts increased apoptosis in the lens epithelium . the bcl-2 family of proteins - including bcl-2 and bcl - xl - plays an important role in the regulation of apoptosis in the nervous system .
however , bcl-2 and bcl - xl form heterodimers with the main pro - apoptotic member bax , which can incapacitate their protective functions .
the bcl-2/bax balance is one of the crucial factors determining if cells undergo apoptosis , and the balance can change during cataract formation .
however , cataract formation increased bax expression much more than it increased bcl-2 expression . in the lens epithelium of the cataract ,
lens cell death occurs by apoptosis , and inhibition of apoptosis can delay cataract formation .
it has been reported that catechins can modulate apoptosis by altering the expressions of anti - apoptotic and pro - apoptotic genes . among the constituents of catechin
, egcg is known to protect against oxidative stress - induced and chronic glutamate - induced apoptosis in several human cells .
yao et al . reported that catechin protects against mitochondria - mediated apoptosis induced by h2o2 in human lens epithelial cells through the modulation of caspases and the mapk and akt pathways .
we observed that catechin significantly suppressed both cataract - induced increases in dna fragmentation and caspase-3 expression in the lens epithelium in dose - dependent manners .
many studies have shown that egcg , the main component of catechin , inhibits the expression of pro - apoptotic genes such as bax , bad , and mdm2 , and that egcg increases the expression of anti - apoptotic genes such as bcl-2 , bcl - w , and bcl - xl . in the present study ,
however , cataracts induced apoptosis by increasing the bax expression much more than it did the bcl-2 expression .
administration of catechin suppressed the expression of bax protein in a dose - dependent manner , but only slightly enhanced bcl-2 expression .
the ratio of bax to bcl-2 was increased by cataract formation . in contrast , catechin suppressed the ratio of bax to bcl-2 , showing that apoptosis was inhibited by catechin treatment . taken together , our results demonstrate that catechin alleviated cataract - induced apoptosis in lens epithelial cells .
catechin could potentially be used to delay cataractogenesis through the suppression of apoptotic cell death in the lens epithelium .
 purposeto investigate the effect of catechin on apoptotic cell death in the lens epithelium of rats with cataract.methodscataract was induced by intraperitoneal injection of 100 mg / kg n - methyl - n - nitrosourea ( mnu ) to ten day - old sprague - dawley rats . 
 the neonatal rats were randomly divided into five groups ( n=15 in each group ) : a control group , and four cataract - induction groups , treated with either 0 , 50 , 100 , 200 mg / kg catechin . 
 we performed slit - lamp biomicroscopic analysis , terminal deoxynucleotidyl transferase - mediated dutp nick end labeling ( tunel ) assay , western - blot for bcl-2 and bax , and immunohistochemistry for caspase-3.resultsapoptotic cell death in lens epithelial cells that increased following cataract formation in rats was suppressed by cathechin.conclusionscatechin inhibited cataract - induced apoptotic cell death in the lens epithelium and may prove useful for the prevention of cataract progression . 