With CCK-8 treatment, expression of iNOS mRNA appeared gradually upregulated during the period of 20 to 40 days of experiment, then downregulated by 60 days (Fig. 5).
Using immunofluorescent staining and Western blotting analysis, we verified that ONOO- was produced during the formation of diabetic cataract. LEC are the most metabolically active region in lens. The restorative inhibition of lens depends on the antioxidant enzymes produced in the LEC.
Using RT-PCR gene array technique, we verified that inducible nitric oxide synthetase (iNOS) might contribute to oxidative stress by helping to produce more powerful oxidants such as ONO-. iNOS is the major enzyme involved in the production of NO, which is a signalling molecule in several pathways.17 Under pathological conditions in the lenses of humans, rats and rabbits,18 upregulation of iNOS mRNA in LEC leads to overproduction of iNOS and NO as well as increasing O2. Increased NO and O2 produce ONOO- a strong oxidant. The changes in NO, iNOS and ONOO- during diabetic cataract formation were not clear. Our studies found that NT increased in the LEC of diabetic rats. Peroxynitrite generation has been implicated in the induction of apoptosis seen in diseases such as diabetes.19
Recent studies also reported that high glucose and peroxynitrite are associated with tyrosine nitration, inactivation of prostacyclin synthase, thromboxane/prostaglandin H2 receptor mediated apoptosis and adhesion molecule expression in cultured human aortic endothelial cells.20 We found that CCK-8 in LEC21 and other tissues reduced the oxidation induced by ONOO- and/or strengthened the antioxidant system.22 CCK-8 could have also inhibited the expression of iNOS mRNA thus decreasing the formation of ONOO-,23 Expression of a small amount of NT in the control provides physiological evidence for the existence of ONOO-. When exogenous NO and ONOO- reacted with ox pulmonary endothelium, only ONOO- could induce cell apoptosis.24 ONOO- affects the cell’s oxidising and repair systems, ionic channels, proteinase, nitroproteins and inhibits respiration in mitochondria, leading to cell apoptosis and apoptosis mediated by mitochondria.25,26 During retinal ischaemia and reperfusion, NO and ONOO- are produced in eyes.27 We also found that ONOO- induced apoptosis in LEC.5 Therefore, iNOS induced overproduction of NO, later combined with O2 to form ONOO- as well as other oxidants.28
CCK-8, which has many physiological functions, is distributed in the stomach, intestine and central nervous system.29-34 Kuntz E and his colleagues35 reported that CCK-8 could improve blood glucose concentrations in type 1 diabetic rats, which correlated with an increase in beta cell mass. This study found that the damaging affect of ONOO and iNOS could be inhibited by CCK-8 which may make it a useful therapeutic agent for diabetic cataracts.36-37 We found that up to 40 days, the effect of CCK-8 was not apparent, while after 40 days its effect was dramatic. The reason for this may be related to time required for the concentration of CCK-8 to reach a critical level.
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