Inhibiting apoptosis of lens epithelial cells by vitamin C

Wenjuan LUO; Yunxiao WANG; Honglu YAN; Chuanfu WANG

ABSTRACT

BACKGROUND:

Oxidation injury can lead to the apoptosis of lens epithelial cells. Some researches have found that body supplement of vitamin C is beneficial to inhibit oxidation injury by increasing the level of vitamin C in aqueous humor.

OBJECTIVE:

To observe the inhibitive effect of vitamin C on H2O2 induced apoptosis of lens epithelial cell.

DESIGN:

Randomized controlled trial taking animal's lens as subject.

SETTING:

Department of Ophthalmology, Medical College of Qingdao University.MATERIALS 120 adult New Zealand rabbits of both genders, weighing 3-5 kg, were selected. Vitamin C and 300 g/L H2O2 were purchased from Shanghai Guangda Chemical Reagent Factory.

METHODS:

The experiment was carried out in the Central Laboratory, the Affiliated Hospital of Medical College of Qingdao University from January 2002 to January 2004. ①Culture of lens 240 lenses were isolat ed from 120 rabbits after being killed. Among them, 192 clear lenses were selected and divided randomly to three groups with 64 lenses in each group control group, H2O2 group and H2O2+vitamin C group. Lenses in the control group were incubated in non-serum and non- phenolsulfonphthalein MEM medium, those in the H2O2 group incubated in non serum and non- phenolsulfonphthalein MEM medium containing 1 mmol/L H2O2 (62 μL of 30 g/L H2O2 was added into the medium every 6 hours to keep H2O2 level maintain 1 mmol/L in the medium), and those in the H2O2+vitamin C group incubated in non-serum and non- phenolsulfonphthalein MEM medium containing 1 mmol/L H2O2 and 1 mmol/L vitamin C. Under the same culture condition, the lenses in each group were detected at hours 6, 12, 18, 24, 36, 48, 72 and 108 after culture, respectively. ②Measurement of the opacity of lens Under the white background, two black lines 0.5 mm in width, which were mutually vertical with an interval of 10 mm. The lenses were placed above the crossing to measure the opacity. ③Detecting the apoptosis of lens epithelial cellsThe apoptosis of lens epithelial cells was examined using TUNEL method and DNA fragmentation assay under light microscope to calculate the apoptotic rate of epithelial cells.MAIN OUTCOME

MEASURES:

①Opacity of the lenses in each group;②apoptotic rate of lens epithelial cells in each group.

RESULTS:

①After 108 hours of culture, the opacity of the lenses in the H2O2+vitamin C group was obviously lower than that in the H2O2 group. ②The apoptotic rates in the control group were lower that those in the H2O2group at different time point and those in the H2O2+vitamin C groupat hours 24, 48 and 72 after culture(P＜0.05-0.01). The apoptotic rates in the H2O2+vitamin C group were significantly lower than those in the H2O2group at different time points after culture (P＜0.01). ③The findings of DNA fragment analysis showed that the DNA "ladder" was found in the H2O2 group during 24-72 hours, while no DNA "ladder" but only normal electrophoresis strap were present in the other two groups 24 hours after culture.

CONCLUSION:

Lens can take refuge from the oxidative injury of H2O2with the addition of vitamin C. As a result, the apoptosis rate of lens epithelial cells and further, cataract formation can be restrained.