Expression Of Chloride Channel Protein-3and Aquaporin-1in Glucocorticoid-induced Cataract Lens In Rats

Objective:Glucocorticoid (GC) has been widely and effectively used in the clinic.Long term systemic or topical application of glucocorticoids benefit theanti-inflammatory and immune systems. At the same time, it also brings manyside-effects. Glucocorticoid Induced Cataract (GIC) is one of most commoncomplications. The pathogenesis of GIC is complicated and remains unclear. In ourexperiment, the Glucocorticoid Induced Cataract rats’ lenses were cultured in vitro.Following incubation, we used a surgical microscope to evaluated the degree ofopacity lenses daily. The expression of chloride channel protein-3(CLC-3) andaquaporin-1(AQP-1) gene in the cultured lenses of rats were investigated to determinewhether CLC-3mRNA and AQP-1mRNA expression were related to the occurrence andprogression of GIC.Methods: One hundred and forty-four fresh lenses(female Sprague-Dawley rat,3months old) were divided into3groups (Control group;5mmol/L Dexamethasonegroup and10mmol/L Dexamethasone group).12lenses were evaluated using surgicalmicroscope at day1,3,5,7.Expression of CLC-3mRNA and AQP-1mRNA in the lensesduring different cataract phases were determined by real-time quantitative PCR.Results:The Glucocorticoid-Induced Cataract of cultured rats’ lenses model wassuccessfully established and carried on correspondingly divided stages and groups.2.The evaluation of the lenses(according to Mathur’s method of grading culturedlens): The stage1opacity of the lenses occurred at the incubation time of3d (11/12) in5mmol/L dexamethasone group; the stage2opacity of the lenses occurred at theincubation time of5d(10/12) and3d(10/12) in5mmol/L and10mmol/L dexamethasonegroups respectively; the stage3opacity of the lenses occurred at the incubation time of7d(10/12) and5d(9/12) in5mmol/L and10mmol/L dexamethasone groups respectively;the stage4opacity of the lenses occurred at the incubation time of5d (2/12) and 7d(8/12) in10mmol/L dexamethasone group; the stage5opacity of the lenses occurredat the incubation time of7d(4/12) in10mmol/Ldexamethasone group. There weresignificant differences between the control group compared to5mmol/L and10mmol/Ldexamethasone groups respectively from day3(P0.01). And there were significantdifferences between the5mmol/L and10mmol/L dexamethasone groups from day3(P0.01).3.The expression of CLC-3mRNA was determined by real-time quantitative PCR:3.1CLC-3mRNA was expressed in the lenses of control group,5mmol/Ldexamethasone group and10mmol/L dexamethasone group.3.2During our experiment, the expression of CLC-3mRNA was found to have nosignificant differences in the lenses of control group(P>0.05).3.3The expression of CLC-3mRNA in the lenses of5mmol/L dexamethasonegroup began to increase at day3and reached6-fold that of normal level at day7. And itwas found to have significant differences compared to control group(P0.01).3.4The expression of CLC-3mRNA in the lenses of10mmol/L dexamethasonegroup began to increase at day3and reached6-fold that of normal level at day5. Thenit decreased at day7and was less than the normal level for50%. It was found to havesignificant differences compared to control group(P0.01).3.5CLC-3mRNA in the lenses of10mmol/L dexamethasone group are more thanthose in the5mmol/L dexamethasone group at days1,3and5. And they are less thanthose in the5mmol/L dexamethasone group at day7. There are significant differencesbetween these two groups(P0.01).3.6CLC-3mRNA in lenses increased at stages1,2and3, and it reached6-fold thatof normal level. It was found to have significant differences compared to the mRNA inlenses at stage0(P0.01). Then it decreased at stage4and stage5, and it was less thanthe normal level for45%(P0.01).4.The expression of AQP-1mRNA was determined by real-time quantitative PCR:4.1AQP-1mRNA was expressed in the lenses of control group,5mmol/Ldexamethasone group and10mmol/L dexamethasone group.4.2During our experiment, the expression of AQP-1mRNA was found to have nosignificant difference in the lenses of control group(P>0.05).4.3The expression of AQP-1mRNA in the lenses of5mmol/L dexamethasonegroup began to decrease gradually from day3and reached55%that of normal level atday7. And it was found to have significant differences compared to control group(P0.01).4.4The expression of AQP-1mRNA in the lenses of10mmol/L dexamethasonegroup began to decrease gradually from day3and reached35%that of normal level atday7. It was found to have significant differences compared to control group(P0.01).4.5AQP-1mRNA in the lenses of10mmol/L dexamethasone group are more thanthose in the5mmol/L dexamethasone group at days1,3,5and7. There are significantdifferences between these two groups(P0.01).4.6AQP-1mRNA in lenses decreased at stages1,2,3and4. It was found to havesignificant differences compared to the mRNA in lenses at stage0(P0.01).Conclusion:The lens would exhibit opacity when we used dexamethasone widelyin local application. The degree and speed of opacity have a positive correlation withthe dexamethasone’s concentration and duration of action.1.In our experiment, the expression of CLC-3mRNA increased gradually at firstand suddenly dropped after it reached its peak and this may play an important role inGlucocorticoid Induced Cataract (GIC).3.There is a down-expression of AQP-1mRNA in our cultured lenses.The degree of the down-expression of AQP-1mRNA have a positive correlationwith the dexamethasone’s concentration and duration of action.The dexamethasone may inhibit the expression of AQP-1in lens and this may playan important role in Glucocorticoid Induced Cataract (GIC).The innovation of our experiment:In our experiment of Glucocorticoid Induced Cataract, for the first time we use themethod of real-time quantitative PCR for the expression of CLC-3mRNA andAQP-1mRNA.