| Cataract is a kind of worldwide eye diseases leading to blind, occupying the largest proportion in all ophthalmological diseases leading to blind. The current treatment of cataract is operation merely, but the high cost of operation and post-operation complications are currently the most troubled problems. Therefore, to develop the prevention and treatment of cataract has become the current research hotspot.Selenite cataract, an experimental animal model for simulating human senile cataract, because of its quick and convenient molding, is widely used in investigating various mechanisms of cataract formation, as well as screening for anti-cataract drugs. Interestingly, selenite cataract appears, only when sodium selenite injection is before eyelid opening of the rats; after this time, even increasing the dose of sodium selenite, selenite cataract can not appear. With regard to this age specific phenomenon in the molding process, people do not know the reason, but the beneficial research on this issue will further reveal the deep reason of selenite cataract formation, and expand the application of senelite cataract animal model in screening anti-cataract drugs.In this dissertation, animal model of selenite cataract and human lens epithelial (hLE) cells SRA01/04were used as the experimental objects, to investigate the relationship between development of blood-retina barrier and selenite cataract formation, also that between selenium and apoptosis pathway of lens epithelial cell, and so on. The main results are as followed:1. Effect of blood-retinal barrier development on formation of selenite nuclear cataract in ratAccording to the method in literatures, injection of sodium selenite to eyelid unopening Wistar rats subcutaneously produced nuclear cataract successfully. Initially, we detected mRNA expression of GPxl, MsrA, MsrB1and activity of GPx in rat lens of different ages, the results show that mRNA expression of these enzymes and enzyme activity of GPx were the highest before eyelid opening, and then progressively decreased with age. Further, we used lanthanum hydroxide tracer method to detect development status of blood-retina barrier at different ages, the results show that distribution of lanthanum hydroxide in retinal pigment epithelial layer of19-day-old rats was significantly less than10-day-old rats. Injecting sodium selenite to8-day-old rats, lanthanum hydroxide increases obviously and extends to the inner layers of the retina after48h, and the retinal pigment epithelial layer was damaged seriously; while injecting sodium selenite to17-day-old rats with the same dose, number of lanthanum hydroxide decreased significantly and does not extend to the inner layer after48h. Correspondingly, in these groups of rats, selenium content in the eyeballs and MDA level in the lens are in agreement with the change of lanthanum hydroxide distribution. Injecting sodium selenite before eyelid unopening, the damage degree of epithelial cells was significantly greater than injecting after that. These results indicate that antioxidant capacity in the eyelid unopening rats is not the main reason for easy cataract formation, but the real reason is that blood-retina barrier development is not mature in the eyelid unopening rats, and then a lot of sodium selenite gets into the eyes, resulting in oxidative damage to eye lens and formation of cataract.2. Apoptosis of lens epithelial cells induced by selenium and its pathwayIn order to study the mechanism of cataract formation induced by sodium selenite, we investigated apoptosis of hLE cells SRA01/04induced by selenium and its pathway. Cell viability was determined by MTT method. The data displayed that, the survival rate of hLE cell showed a concentration-dependent decrease to the increasing concentration of sodium selenite. When the concentration of Na2SeO3is6μM and8μM, the cell survival rates are about50%. Annexin V-FITC/PI double staining and flow cytometry analysis showed that, as the increasing concentration of sodium selenite, apoptosis rate of hLE cell increased gradually:it was23.8%at6μM, however it has reached74.6%at8μM. This result is coincident with the qualitative result of Hoechst33258staining. We further measured the activity change of Caspase-3in hLE cells; the results showed that after treatment with7μM sodium selenite for24h, Caspase-3activity increased by49.4%. Correspondingly, level of ROS increased by73.1%, content of MDA increased by80.5%, and the mitochondrial membrane potential is greatly decreased, mitochondrial depolarization is very obvious. Thus, apoptosis of hLE cells SRA01/04induced by sodium selenite, is closely related to mitochondrial apoptotic pathway mediated by generated reactive oxygen species.3. Influence of selenite cataract on redox balance in liver, kidney and brain of Wistar ratIn the animal model of selenite cataract, there is little report on the effect of sodium selenite on the redox status of major metabolic organ, so we measured the changes of antioxidant status of some tissues in selenite cataract rat model, such as liver, kidney, etc. The results show that, compared with the control group, injecting sodium selenite to rats resulting in mRNA expression level of GPxl decreased by25%,20%in liver, brain, but that in kidney increased by43.5%; the change trend of GPx activity is also like this. Compared to GPxl, mRNA expression of MsrA also presented similar change trend; however, mRNA expression levels of MsrB1increased by86%,62%and71%in rat liver, kidney and brain after injection of sodium selenite. It is shown by further detection, significant oxidative stress appeared in liver tissue of selenite cataract model animal; compared with the control group, after injection of sodium selenite, MDA content in rat liver increased by78%, but reduced by29%in kidney, and remained unchanged in brain. These results indicate that, the balance of redox state in liver and kidney of selenite cataract rat model has been broken, so this change should be taken into account when screening anti-cataract drugs by this model animal. |
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