An Experimental Investigation Of Four Anti-Fibrosis Drugs Including Tetrandrine On Inhibition Of Rabbit Corneal Stroma Cells In Vitro

Objective To screen out the best drug by the antiproliferative rate ofrabbit corneal stroma cells (RCSCs) in vitro from four anti-fibrosis drugsand to explore it's mechanism. To try hard to search out a drug which canreplace corticosteroid to treat haze after refractive surgery. Methods 1. The primary culture and subculture of RCSCs wereexposed to different concentrations of tetrandrine(1~10μg/ml), anti-TGF-β(0.5~16μg/ml),α-2b IFN(100~2500 /ml), verapamil(20~100 μg/ml)and FML(16.67~83.33μg/ml) in the experimental groups and to blankculture media in the contrast group, then it were cultivated for 24h,48h or72h. The inhibitive characteristics to the cells' proliferation were measuredwith methyl thiazolyl tetrazolium (MTT) assay. The best drug was screenedout by the antiproliferative rate of RCSCs. 2. After the treatment of the bestdrug, the changes of cells cycles and the apoptosis rate were observed withflow cytometer (FCM). The ultrastructure of cell was observed withtransmission electron microscope. And the changes of the apoptosisindexes including bax and bcl-2 were measured by immunocytochemistry.3. The contents of fibronectin (FN) were detected by immunocytochemistryand enzyme- linked immuno-sorbent assay (ELISA). Results 1. After the treatment of tetrandrine,RCSCs showed themarked inhibition whenever in 24h,48h or 72h and showed time-dosage-effect dependence. Its IC50 of three time-point were 3.71,3.16 and2.82μg/ml. Anti-TGF-β could inhibit the proliferation of RCSCs distinctlyin each experimental group and its IC50 of three time-point were 6.55,3.31and 3.86μg/ml. α- 2b IFN could inhibit the proliferation of RCSCs weakly,while verapamil could not. FML could inhibit the proliferation of RCSCs ineach experimental group too and its IC50 of three time-point were 33.19,37.74 and 42.05μg/ml. Tetrandrine was screened out as the best drug in theend. 2. After the treatment of tetrandrine, Apoptosis of RCSCs wasdetected by transmission electron microscope. FCM result showed that thepercentage of cells in G0/G1 phase increased obviously and the apoptosiscusp and rate also increased. Immunocytochemistry showed bax and bcl-2increased while bax increased more than bcl-2 in the experimental group. 3.Immunocytochemistry showed FN declined, while ELISA showed FNincreased in the experimental group. Conclusion 1. Tetrandrine had the strongest inhibitive effect onRCSCs and showed time-dosage-effect relation. It's inhibitive effect wasstronger than fluorometholone. The inhibitive effect of anti-TGF-β was justweaker than tetrandrine. α- 2b IFN could inhibit the proliferation ofRCSCs weakly, while verapamil could not. Tetrandrine was screened out asthe best drug in the end. 2. Tetrandrine might exert it's antiproliferativeeffect by inducing apotosis and ceasing cells cycles. 3. Tetrandrine coulddecrease FN to deposit between the RCSCs.