Apoptosis Of Cattle Lens Epithelial Cells Induced By C2-ceramide

Background and ObjectiveCataract is the main reason for the blinding of the worldwide at present, and its pathogenesis is a question that domestic and foreign scholars have concerned. There are studies which have found that the apoptosis of lens epithelial cells(LECs) is an important reason for lens opacification and cataract formation.Sphingomyelin is an important component of cell membrane structure. Ceramide, a product of Sphingomyelin with Sphingomyelinase acted on, plays a decisive role in inducing the signal transduction process of apoptosis and is one of the characteristic signs of measuring apoptosis.Ceramide is not only related closely to the development of the tumor in blood system, nervous system and digestive system, but also to non-neoplastic diseases. Sphingomyelin is a major lipid composition of lens membrane, and there has been report about sphingomyelinase in the lens epithelial, cortical and nuclear regions. Moreover, some scholars have reported that the concentration of ceramide in lens of cataract patients is four times as the normal lens in the same age. However, the action of ceramide in normal LECs and the development of cataract is not entirely clear.Caspase-3 is an apoptotic protease, located in the downstream of apoptotic cascade "waterfall", plays a pivotal role in the end of the process of apoptosis which was started by many factors. It has been reported that ceramide can induce caspase-dependent and caspase-independent apoptosis.This study is to investigate the effects of exogenous C2-ceramide on the survival and apoptosis of cattle LECs and observe the variation of caspase-3 activity.Methods1. Preparation of C2-ceramideC2-ceramide powder was prepared with anhydrous ethanol to 400mmol/L solutions,—20℃preserved. Diluted anhydrous ethanol to the end of the volume≤0.1% with DMEM culture medium when it was used.2. Cultivation of cattle LECsA fresh cattle eye was obtained from the slaughter house in Zhengzhou. Removed the lens under sterile conditions. Teared the anterior capsule of lens carefully, and put it into DMEM culture medium contained with 20% FBS. Then, the capsule was cut to the size of 1mm×1mm and planted into 25ml culture bottle uniformly. Replaced the culture medium every 2 to 3 days. When primary cultured cells reached confluence, subcultures were prepared using 2.5g/L trypsin by the ratio of 1:2. Replaced the culture medium every 3 days.3. Determination of the proliferation of cattle LECs with C2-ceramide treatedCattle LECs, the concentration of which was 1×10⁵/ml, was treated by different concentrations (50μmol/L, 100μmol/L, 200μmol/L, 400μmol/L) of C2-ceramide for 24h. Measured the OD value of 570nm wavelength, and calculated inhibition ratio. Inhibition ratio=(OD value of control group—OD value of C2-ceramide Group/OD value of control group)×100%.4. Apoptosis of Cattle LECs was determined by TUNEL stainingCattle LECs, the concentration of which was 1×10⁵/ml, was treated by different concentrations (25μmol/L, 50μmol/L, 100μmol/L) of C2-ceramide for 12h and operated according to the instructions of one-step TUNEL apoptosis detection kit. Selected 5 fields of vision under the fluorescent microscope of 100 times and acquire the images. Then, analyzed them with Image-Pro Plus 5.0 and calculated the integral optical density, which reflected the situation of apoptosis.5. Determination of caspase-3 activityDiluted pNA (10mM) to 200μmol/L, which was the standard substance. Cattle LECs was treated by different concentrations (25μmol/L, 50μmol/L, 100μmol/L) of C2-ceramide. Operated according to the instructions of activity of caspase-3 detection kit, measured the OD value of 405nm wavelength, and calculated the rate of activity of caspase-3. Rate of activity of caspase-3 = (OD value of C2-ceramide Group and control group/OD value of positive control group)×100%.6. Statistical analysisStatistical analysis was performed with SPSS10.0 software. Numerical data were performed by X|—±S . Several groups were compared by simple factor analysis of variance, and every two groups were compared by q test. The size of test was a = 0.05.Results1. Morphological observation of cattle LECsIt could be seen that primary cattle LECs grew from the edge of the capsule membranes during 24h to 48h. The original cells growing from the tissue, which were transparent, were in the shapes of polygon and long-spindle and in different size. With the continuous cell division, cells grew in monolayer and reached confluence after 15-18 days.Subcultured cells were round after trypsin digestion, and proliferated rapidly after complete adherence. The cells reached confluence after 6-8 days. There was no significant difference between the original and Subcultured cells.2. Result of the determination of cells inhibition ratioIC₅₀ was in the concentration interval of 50μmol/L～100μmol/L, so chose 100μmol/L for the upper limit of concentration in subsequent experiments. Inhibition ratio was signification declined when action time was 12h, so chose 12h for the upper limit of time in subsequent experiments3. Result of the determination of cells apoptosisAfter TUNEL staining, the apoptotic cells were marked with green fluorescence. Integral optical density of apoptotic cells increased along with the enlargement of concentrations of C2-ceramide(P<0.01), C2-ceramide could induce the apoptosis of cattle LECs in a dose-dependent manner.4. Result of the determination of caspase-3 activityCaspase-3 activity increased along with the enlargement of concentrations of C2-ceramide(P<0.01).Conclusions1. C2-ceramide could inhibit the growth of cattle LECs.2. C2-ceramide could induce the apoptosis of cattle LECs, which was in a dose-dependent manner.3. The expression of caspase-3 activity existed in this apoptotic process.