ABSTRACT
Background Doxycycline is a broad spectrum antibiotic,and it is frequently used in the treatment of ocular surface diseases.Objective The purpose of the present study was to investigate the effect of doxycycline on the inhibition of cell proliferation in bovine corneal myofibroblasts in vitro and assess its contribution to ocular surface repairing mechanism.Methods Six fresh bovine corneas were collected.The corneal stromal layer was isolated by two-step method of 1.0 g/L and 2.0 g/L collegenase-1.Isolated cells were plated at mantaryay culture flask in 10％ FBS of RPMI-1640.Vimentin and alpha-smooth muscle actin (α-SMA) organization were evaluated by immunocytochemistry,and the cells with influoresccence staining for vimentin and α-SMA were identified as the corneal myofibroblasts.Doxycycline at the concentrations of 10,20,40,60,80 mg/L was added to the medium,respectively,in different concentrations of doxycycline groups.Dexamethasone (120 mg/L)was used in the same way in the positive control group,and no drug was used in the negative control group.Cell proliferation was evaluated by MTT and the cell cycle was analyzed by BD FACScan flow cytometer assay 24 hours and 48 hours after addition of any drug.Results The cells grew well and showed the positive response for vimentin and α-SMA.MTT assay showed that the A570values of bovine corneal myofibroblasts were gradually declined with the increase of the concentration of doxycycline and lapse of active time,showing statistically significant difference (Fconcentration =1233.778,P＜0.001 ; Ftime =227.564,P ＜ 0.001).And the difference between the two factors was also statistically significant (Ftime*concentration =51.656,P＜0.001).Flow cytometry cell cycle analysis showed that 24 hours after 10,20,40,60,80 mg/L doxycycline treated,the perentage of of corneal myofibroblast cell in G0-G1 phase was 82.85％,84.36％,85.18％,87.12 ％ and 89.31％,showing significant increase in comparison with 63.89％ of the negative control group (all P＜0.05),and that of 40 mg/L doxycycline group was near the positive control group.Forty-eight hours after 10,20,40,60,80 mg/L doxycycline treated,the perentage of of corneal myofibroblast cell in G0-G1 phase was 82.78％,86.15％,88.23％,89.57％,93.00％,with significant increase in comparison with 70.17％ of the negative control group (all P ＜ 0.01),and that of 40 mg/L doxycycline group was near the positive control group.Conclusions The growth of the bovine corneal myofibroblasts is inhibited by doxycycline in time-and dosedependent manner in the range from 10 mg/L to 80 mg/L,and 40 mg/L of doxycycline has an obviously inhibitory action as 120 mg/L dexamethasone.