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Glutathione determination The content of GSH in each lens was determined with 55 dithiobis(2nitrobenzoic acid) following centrifugation (3500r/min) in 2mL of 100g/L trichloroacetic acid for 10 minutes and measured using the colorimetric method at 25℃ and 412nm.
Assay of Na, KATPase activity Na,KATPase activity was measured by adding the clear supernatant of homogenate to a buffer containing 100mmol/L NaCl, 20mmol/L KCl, 5mmol/L MgCl2, 3mmol/L ATP and 50mmol/L Tris, pH 7.4. After 15 minutes preincubation at 37℃, ATP was used as the substrate and the liberated inorganic phosphate was estimated by spectrophotometric estimation. Ouabain was used as a specific blocker of Na, KATPase activity and the ouabain sensitive ATPase activity was estimated and expressed as micromoles of inorganic phosphate released per mg protein per hour.
Assay of catalase activity Catalase (CAT) activity was determined by the method of Beers et al with modification by spectrophotometric recording of the cleavage of H2O2 at 240nm. The activity of catalase was expressed as U/mg protein.
Assay of glutathione reductase activity Glutathione reductase(GR) activity was measured according to the procedure of Bergmeyer. The activity of GR was expressed as U/g protein.
Statistical Analysis Oneway ANOVA was used for testing statistical significance between groups. The median calculation of the lens opacity for each group was analyzed by using the Wilcoxon rank sum test. P<0.05 was considered significant. All the data were dealt by the SPSS 13.0 statistical package.
RESULTS
The Grading of Lenses After seven days, all 6 lenses in the hyperoxiaexposed group exhibited total cortical opacification (Grade 3). In contrast, only few lenses revealed less opacification in the control group and the Tau treated group, and minimal opacification in the NAC treated group.
Figure 1 The watersoluble protein in rabbit lenses（略）
Figure 2 The GSH content in rabbit lenses（略）
Figure 3 The activities of enzymes in rabbit lenses（略）
Protein Determination There was a reduction of the watersoluble protein concentration in the lenses of the hyperoxiaexposed group than that in the control group, about decreased by 10.4%. It increased in the NAC treated group by 9.9% and in the Tau treated group by 5.0%, respectively (Figure 1).
Glutathione determination The level of GSH was significantly lower in the hyperoxiaexposed group than that in the control group, by 37.8% (P<0.05). Surprisingly, NAC enhanced glutathione (GSH) levels about 38.1% (P<0.05, Figure 2).
The activities of enzymes The activity of Na, KATPase was significantly lower in the hyperoxiaexposed group than that in the control group, by 53.5% (P<0.05). It was increased in the NAC treated group and the Tau treated group, by 40.9% (P<0.05) and 5.6%, respectively (Figure 3A). There was a reduction in Cat activities in the lenses exposed to the hyperoxia without NAC treatment when compared to the control lenses, about decreased 21.0%. Compared with the hyperoxiaexposed group, the activities of Cat were increased in the two treated groups, by 13.7% and 6.0%, respectively. However, there were no statistically significant differences in all groups (P>0.05, Figure 3B).
The activity of GR was not decreased dramatically in the hyperoxiaexposed group, and not increased in the two treated groups (P>0.05, Figure 3C).
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