Isolation And Identification Of The Primary Targets Of Dexamethasone-attacked Proteins In Rat Lens

Glucocorticoid (GC) has been widely and effectively used in the clinic. However, the cataract may be induced with the long-term or the higher dose of useage. Steroid-induced cataract is often involved with both eyes marked with the opacity of posterior subcapsular. And it is usually complicated with other type cataract (such as the age-related cataract).It remains unclear of the pathogenesis of steroid-induced cataract. At present, many hypothesises have been proposed, such as protein adduct formation, oxidative damage, disequilibrium of osmotic pressure. Many studies revealed that, in the early stage, anomalous connection, degeneration and the decrease of transparency occurred in the lens. However, what is the volunable targetted proteins in the lens by glucocorticoids? Identification of these proteins would facilitate the study of its character and function, which will be helpful to understand the development of steroid-induced cataract starting from the primary stage of its formation.These will foundamentally benefit to the research of pathogenesis and medication of steroid-induced cataract. ObjectiveThe purpose of this study is to isolate and identify the lens proteins which are attacked by dexamethasone at the early stage. With the isolation and identification of these volunable proteins, we could reveal the modification of lens crystallin by dexamethasone and help to the further study of its character and function.MethodsForty-two fresh lenses (female Sprague-Dawley rat, 2 months) were divided into 3 groups (5μM dexamethasone group; 10μM dexamethasone group and control group). After incubation with fluorescence dexamethasone (5μM, 10μM) or culture fluid without dexamethasone, the transparency of the lenses was observed daily. The lenses were also cut into 10μm frozen sections and the fluorescence intensities were detected by a fluorescence microscope for the selection of the optimal time and the concentration of incubation.One hundred and fifty fresh lenses of rats (2 months) were again divided into 3 groups, and then incubated with fluorescence dexamethasone(10μM) for 3 and 5 days, while incubated with culture fluid without dexamethasone was treated as the controls under the same condition. After the homogenizing of the incubated lenses, the water-soluble proteins were separated by size-exclusion chromatography (Sephacry1 S-300HR) at a flow rate of 50 ml/h with 5ml of each fraction collection. Absorbance of elutions at 280 nm was read from a spectrophotometer and the fluorescence intensity (INT) value was monitored by a fluorophotometer. The elution profile of proteins and the curve of INT value were drawn. Then the SDS polyacrylamide gel electrophoresis (SDS-PAGE) was performed to identity the fraction with the highest INT value. This portion was further analyzed by mass spectrometry to identify the primary target proteins by dexamethasone.Results1. The opacity of the lenses occurred at the incubation time of 3 d and 2 d in 5μM and 10μM dexamethasone groups, respectively. The fluorescence was not detected in 5μM dexamethasone group lenses at different incubation time. However, the different fluorescence intensity was detected gradually at 3 d and the maximum fluorescence intensity was observed at 7 d.2. Chromatogram of lens protein showed that the occurance of crystallin peaks were ahead of that of the controls at different time in 5μM and 10μM groups. A high INT value corresponding to alpha-crystallin absorption peak was oberved, while a very low INT value corresponding to beta-crystallin and gamma-crystallin peaks were noticed after 3d and 5d.3. SDS-PAGE results showed that the highest INT value fraction were mainly approx 20 KD bands. Compared with the controls, the dexamethasone groups had more protein bands and distributed around 20 KD, and even up to 40 KD and 60 KD. Mass spectrometry demonstrated that the 20 KD bands were mostly matched with the masses of alpha A-crystallin of rat lens. However, the peptid conjugated with dexamethasone could not be identified.Conclusions1. As an indicator, the dihydroxyfluorane can reflect the incubation state of the rat lens. The distribution of dexamethasone can be decteced directly at the early stage by fluorescence microscope, and the fluorescence intensity corresponding to the content of dexamethasone can be also dynamically measured in each fraction.2. Alpha A-crystallin is the primary target of lens protein by dexamethasone. The modification of alpha-crystallin and further decreased molecular chaperone function induced by a steroid may contribute to the steroid-induced cataract formation.