Researches On The Apoptosis Inducing Effects Of Diclofenac Sodium On Three Types Of Human Corneal Cells

Diclofenac sodium (DFS) is an important class of nonsteroidalanti-inflammatory drugs (NSAID) topically applied in Ophthalmology. It can inhibitthe synthesis and release of prostaglandins (PG) and leukotriene (LT) and otherinflammatory factors through suppressing the activity of cyclooxygenase (COX) andlipoxygenase (LOX). It functions potently in anti-inflammatory, reducing pain andalso has the function to protect the blood-ocular barrier. Resently, DFS is widelyapplied in Ophthalmology. It is now reported in the literature that DFS can inhibitfibroblast and rabbit corneal epithelial cell proliferation, suggesting that highconcentrations and prolonged medication may damage normal cells. The research onthe effect of ophthalmic drugs on corneal cells is mainly through clinical trials andanimal experiments, limit the development process of the experiment in large. Thecultured cell lines in vitro as models for drug toxicity studies with uniformity, fastresponse, economical, convenient and many other advantages, making the cells morewidely applied in toxicology. This paper used the human corneal epithelium (HCEP)cell line, human corneal stromal (HCS) cell line and human corneal endothelium(HCE) cell line built in our laboratory as experimental materials, studied the effect ofDFS on vitro HCEP, HCS and HCEC and on domestic cat corneal in vivo.In order to detect the effect of DFS at different concentrations on human cornealcells and to determine the effective concentration and processing time, this paperdilute the pharmaceutical concentration to0.03125g/l on the basis of existing clinicalconcentration (1g/l). Under light microscopy, the cultured HCEP, HCS and HCECtreated with1g/l,0.5g/l and0.25g/l DFS showed morphology changes: cell shrinkage,plasma membrane blebbing, detachment and death.For the purpose of determining the effect of DFS at different concentrations afterdifferent times on human corneal cells, the experiment of acridine orange/ethidiumbromide (AO/EB) fluorescence double staining showed that the effect on HCEP, HCSand HCEC was significantly positively correlated with the concentration and processing time of DFS. The apoptotic rate of HCEPC separately treated with1g/lDFS for16h and0.5g/l DFS for24h were both100%; the mortality of HCEPseparately treated with0.25g/l,0.125g/l and0.0625g/l DFS for28h were27.77%,25.00%and19.76%separately. The apoptotic rate of HCSC separately treated with1g/l DFS for8h and0.5g/l DFS for16h both reached100%; the mortality of HCEPseparately treated with0.25g/l,0.125g/l and0.0625g/l DFS for28h were75.21%,58.76%and26.43%separately.When HCEC were treated with1g/l DFS for8h,0.5g/l DFS for20h or0.25g/l DFS for28h, The apoptotic rate were all100%; themortality of HCE separately treated with0.125g/l and0.0625g/l DFS for24h were21.59%and19.64%. Summarize all the above studies, it was proved that DFS at aconcentration above0.0625g/l could increase the plasma membrane permeability,induce cell apoptosis.In order to further determine the induction of apoptosis of DFS on humancorneal cells, we conduct the DNA agarose gel electrophoresis and transmissionelectron microscopy (TEM). The DNA extracted from HCEPC treated with0.5g/l and0.25g/l DFS respectively after16h and28h, HCSC treated with0.5g/l and0.25g/lDFS separately after8h and24h, HCEC treated with1g/l,0.5g/l and0.25g/l DFSrespectively after4h,12h and20h, agarose gel electrophoresis showed the ladderbands which mean DNA appeared fragments. Then HCEPC treated with0.5g/l DFSfor12h, HCSC treated with0.25g/l DFS after20h and HCEC treated with0.5g/lDFS for12h, were fixed slices. Under transmission electron microscopy, the slicesappeared chromatin condensation and marginalized distribution in the nucleus,cytoplasmic vacuoles and apoptotic bodies. This mean DFS which is higher than0.25g/l can indeed induce the cell apoptosis of three human cornea cells.Finally, in order to verify the apoptosis-inducing effect of DFS on corneal cellsin vivo, cats were used as the experimental animals. In accordance with the clinicalconcentrations, DFS eye drops was applied on cats for2weeks. Using the cornealendothelial microscopy, we determine the domestic cat corneal endothelial celldensity, the average area of the corneal endothelial cells, the ratio of hexagonal cells,cell area variation rate (CV) and so on. The results found corneal endothelial celldensity of cats was decreasing, corneal endothelial cell area was increasing,differenced in control eyes significantly (P <0.01), the CV values increased, thehexagonal cell ratio decreased, but no significant difference with the control eyes (P>0.05) in20days. After40days, the cat corneal drawn by Alizarin red staining, frozen sections and HE staining, scanning electron microscopy and transmission electronmicroscopy observation. Alizarin red staining results showed that the domestic catexperimental corneal endothelial cell density and hexagonal cell ratio decreasedsignificantly, part of the cell area increased. HE staining showed the domestic catexperimental corneal stromal collagen fiber structure loosened, this may has relationwith DFS induction of MMPs over expression. Scanning electron microscopy resultsshowed that a small amount of endothelial cells of the experimental cornea of thedomestic cat appeared apoptosis, transmission electron microscopy results showedthat the connection between the corneal epithelial cells was destroyed. Theexperimental conclusions above verify the DFS apoptosis-inducing effect on cornealcells.This paper used non-transfected the HCEP cell lines, HCS cell lines and HCEcell lines as research system in vivo, identified the apoptosis-inducing effect of DFSon HCEPC, HCSC, HCEC and verified the apoptosis-inducing effect on cat’s corneacells through domestic cats experiment in vivo. The research results confirmed thatDFS has great toxic effects on cornea cells and it can induce the cell apoptosis. DFSshould be avoided in clinical applications, use other non-steroidal anti-inflammatorydrugs without toxic side effects for substitute and develop the new greenanti-inflammatory drugs without toxic side effects to avoid or reduce the humancornea toxicity damage caused by the eye drugs as far as possible.