Researches On The Apoptosis-inducing Effects Of Gatifloxacin And Rifampicin On The Human Corneal Epithelial Cells

Human corneal epithelium (HCEP), located at the anterior end of the cornea, isthe first defence barrier against external pathogens invasion. Therefore, HCEP issusceptible to infection, which results in keratitis thus impairing visual clarity.Gatifloxacin and rifampicin, as two antibacterial drugs, are currently used inophthalmologic clinic for keratitis. With the wide application of this kind of medicine,the concentration and dose of antibacterial agents have been proposed to reduce bymore and more reserches. Whether gatifloxacin and rifampicin have toxic effects havedrawed increasing attention. Unfortunately, detailed reports on toxic effects ofgatifloxacin and rifampicin to HCEP cells have not been reported. In order to explorethe cytotoxicity of gatifloxacin and rifamipicin as ophthalmic drugs to HCEP cells, anHCEP cell line without tumorigenicity builded in my laboratory was used as the studysystem. And the apoptosis-inducing effects of gatifloxacin and rifampicin to HCEPcells had been deeply investigated in this study.In order to test and verify apoptosis inducing effect of gatifloxacin to HCEP cells,the cultured HCEP cells were treated with0.1875~3g/L (3g/L is clinicalconcentration) gradient concentration of gatifloxacin respectively. The results showedthat HCEP cells treated with gatifloxacin at concentration above0.75g/L showedsome changes such as cell shrinkage, detachment and quantity decreasing under lightmicroscope. The results indicated that gatifloxacin had significant cytotoxicity toHCEP cells at the concentration of0.75~3g/L and the cytotoxicity was dose-andtime-dependence. Aridine orange/ethidium bromide (AO/EB) double-fluorescentstaining of0.1875~3g/L treated cells showed gatifloxacin could induce membranepermeability increase at a concentration above0.75g/L. The results showed that thetoxic effect of gatifloxacin to HCEP cells varied in a dose-and time-dependentmanner, suggesting that the toxic effects may be apoptosis. Agarose gelelectrophoresis of DNA samples of HCEP cells treated with1.5g/L gatifloxacin for28h and32h showed a remarkable DNA ladder, illustrating that gatifloxcin couldlead to DNA fragmentation in HCEP cells. Transmission electron microscope (TEM)of gatifloxacin treated HCEP cells showed some characteristic features of apoptosis,including cell structure disorder, plasma membrane blebbing, chromatin condensation,and presence of apoptotic bodies, indicating that HCEP cells treated with gatifloxacinhad ultrastructural characteristics of apoptosis. HCEP cells treated with1.5g/L for4h were in the state of early apoptosis by (flow cytomytry) FCM, Most cells treated for24h were in the state of late apoptosis. Meanwhile, the mitochondrial membranepotential of HCEP cells treated by gatifloxacin was detected to find the mitochondrialmembrane potential of HCEP cells depolarized. It revealed that gatifloxacin couldinduce the apoptosis of HCEP cells through mitochondrial.In order to test and verify apoptosis inducing effect of rifampicin on HCEP cells，cultured HCEP cells were treated with31.25mg~0.5g/L gradient concentration ofrifampicin respectively. The results showed that HCEP cells treated with rifampicin atconcentration above0.125g/L showed the change of shape, obvious plasmamembrane blebbing, and the detachment under light microscope, the results indicatedthat rifampicin had significant cell toxicity to HCEP cells at a concentration above0.125g/L and the cell toxicity was dose-and time-dependence. AO/EBdouble-fluorescent staining of gatifloxcin treated cells showed obvious improvementof cell membrane permeability, suggesting that the toxic effects of rifampicin may beapoptosis. By means of agarose gel electrophoresis, DNA ladder was observed afterHCEP cells were treated with0.5g/L rifampicin for32h, which illustrats thatrifampicin at a clinical concentration could lead to DNA fragmentation in HCEP cells.TEM of HCEP cells treated with rifampicin showed some characteristic features ofapoptosis, including cell structure, chromatin condensation, and the plasma membraneblebbing, indicating that the cytotoxicity of rifampicin was apoptosis. HCEP cellstreated with rifampicin for6h were in the state of early apoptosis by FCM. And mostcells treated for24h were in the state of late apoptosis. The test results of themitochondrial membrane potential proved that the mitochondrial membrane potentialof HCEP cells treated by rifampicin was depolarized and the apoptosis was relatedwith mitochondria.The effects of gatifloxcin and rifampicin to HCEP cells as an ideal study systemin vitro had been investigated in this study. The results showed that gatifloxacin andrifampicin at clinical concentration of4-fold dilution indeed had apoptosis-inducingeffect to HCEP cells. According to the results, it was helpful to avoid the harm of thecornea for repeated use of the drugs. And the clinical dosage of gatifloxacin andrifampicin should be used. The research results could be used to guide for the clinicaluse of ophthalmologic antibacterial drugs.