An Investigation Of Inhibition Of Tetrandrine On Rabbit Corneal Stroma Cells And Rabbit Corneal Epithelial Cells In Vitro

Objective To investigate the effect of tetrandrine on inhibition of RCSCs and RCECs and to explore the mechanism. To try hard to search out a strong effect and weak side effect drug which can replace corticosteroid to treat haze after refractive surgery.Methods 1. The primary cultured and subcultured RCSCs were exposed to different concentrations of tetrandrine(1~10μg/ml) and fluorometholone(5~90μg/ml) in experimental groups and to blank culture media in contrast group. The primary cultured and subcultured RCECs were exposed to different concentrations of tetrandrine(1~10μg/ml) and fluorometholone(5~130μg/ml) in experimental groups and to blank culture media in contrast group. Then RCSCs and RCECs were cultivated for 24 h,48 h,72h. The inhibitive characteristics to the cells'proliferation were measured with methyl thiazolyl tetrazolium (MTT) assay. The antiproliferative rate of cells and the IC50 were calculated and compared. 2. After the effect of tetrandrine on RCSCs and RCECs for 48h, the changes of cells cycles and the apoptosis rate were observed with flow cytometer (FCM). 3. After the effect of tetrandrine on RCSCs and RCECs for 48h, the changes of the expression of P21 and E2F1 were measured by immunocytochemistry.Results 1. After the treatment of tetrandrine , RCSCs showed the marked inhibition in 24h,48h,72h and showed marked time-dosage -effect dependence. Its IC50 of three time-point were 4.14,3.42 and 2.84μg/ml in RCSCs. After the treatment of tetrandrine , RCECs showed the inhibition in 24h,48h,72h and showed marked time-dosage -effect dependence. Its IC50 of three time-point were 7.38,4.14 and 3.37μg/ml in RCECs. The IC50 of tetrandrine at each time point in RCSCs was lower than that in RCECs. After the treatment of fluorometholone, RCSCs and RCECs showed the inhibition in 24h,48h,72h and showed marked time-dosage -effect dependence. The IC50 of fluorometholone at each time point in RCSCs and RCECs were 52.81,36.27,33.16μg/ml and 69.19,42.01,35.83μg/ml. The IC50 of tetrandrine at each time point in RCSCs and RCECs were lower than that of fluorometholone. 2. After the treatment of tetrandrine, FCM result showed that the percentage of RCSCs and RCECs in G0/G1 phase increased obviously and the apoptosis cusp and rate also increased. 3. Immunocytochemistry showed increased expression of P21 and decreased expression of E2F1 in RCSCs and RCECs after the treatment of tetrandrine. Conclusion 1. Tetrandrine has inhibitory effect on the proliferation of RCSCs and RCECs. The effect is more dominant in comparison with fluorometholone. The inhibitory effect of tetrandrine on RCSCs is stronger than that on RCECs . 2. Tetrandrine may exert its antiproliferative effect on RCSCs and RCECs by ceasing cell cycle with P21 and E2F1 and by inducing cell apotosis.