Researches On The Apoptosis-inducing Effects Of Ofloxacin And Oxybuprocaine Hydrochloride On The Human Corneal Epithelial Cells

Along with the development of medicine technology, ophthalmic drugs are more andmore widely used. Anesthetic and antibacterial medicines provide clinical treatment withmore opportunities and choices, but the toxic effects of these ophthalmic drugs have attractedour much more focus on safe use of these medicine and subsequently become an importantresearch subject in the fields of medcine. Most ophthalmic drugs are applied on ocular surface,it potentially cause toxic and side effects on cornea, or even lead to blindness. Corneaepithelium, as the outer tissue of cornea, serves as physical barrier to defend against outsidepathogenic factors. Its integrity is a decisive factor for maintaining the normal structure andfunction of cornea. Therefore, it is necessary to research the effects of ophthalmic drugs onhuman corneal epithelial (HCEP) cells. Until now, the effects of oxybuprocaine and ofloxacinto HCEP cells have not been reported yet. In this study, HCEP cells obtained from theuntransfected human corneal epithelial cell line was used as experimental material toinvestigate the effects of oxybuprocaine and ofloxacin to HCEP cells.The HCEP cells from HCEP cell line were treated with oxybuprocaine and ofloxacin atdifferent gradient concentrations on HCEP cells in the light of their clinical concentration.Then the growth situation and morphology of in vitro cultured HCEP cells were checkedunder an inverted light microscope, and MTT assay, respectively．The results indicated thatoxybuprocaine and ofloxacin had significant cell toxicity to HCEP cells at a concentrationabove0.5g/L and1.5g/L respectively. And the cell cycle phase experiment observed throughflow cytometry which showed the cells treated with oxybuprocaine and ofloxacin arrested inS phase.Therefore the cellular toxicity mechanisms of oxybuprocaine and ofloxacin werestudied through membrane permeability, Annexin V/PI, DNA fragmentations andultrastructure of HCEP cells, by employing the method of acridine orange/ethidium bromide(AO/EB) double－fluorescent staining, DNA agarose gel electrophoresis, transmissionelectron microscopy(TEM) and flow cytometry respectively. The results showed that bothoxybuprocaine and ofloxacin lead HCEP cells to apoptosis with the phenomena of increased rate of apoptosis cells, nuclear fragmentation (DNA ladder), cell structure disorder, plasmamembrane blebbing, chromatin condensation, and the presence of apoptotic bodies.Furthermore, to explores the pathways of cell apoptosis induced by the two drugs,mitochondrial membrane potential and the expression of caspase-2,-3,-9were detected. Theresults showed that the decrease of mitochondrial membrane potential and stress response ofmitochondria in HCEP cells after treated with the two drugs. Meanwhile the concentration ofcaspase-2and caspase-9increase within a certain time, this activates expression of caspase-3,thus causes the cell apoptosis. Thus, this research assume that oxybuprocaine and ofloxacincould induce HCEP cells apoptosis through mitochondrial pathway, but further tests areneeded to prove this assumption.With the untransfected human corneal endothelial cell line as an ideal study system invitro, the effects of oxybuprocaine and ofloxacin on HCEP cells had been investigated in thisstudy. The results showed that oxybuprocaine and ofloxacin had apoptosis-inducing effect onHCEP cells indeed, and apoptosis could be induced through mitochondrial pathway. Inaddition, these findings using the cornea epithelial cell line cells provided importantexperimental bases for the research and development of innocuous novel ophthalmologydrugs.