Researches On The Apoptosis-reducing Effects Of Levofloxacin And Rifampicin On The Human Corneal Endothelial Cells

Being widely used as antibiotics and antiseptics clinically, levofloxacin andrifampicin as eye drops have gained attention for their potential toxicity on corneals.Human cornea are composed of endothelial cells, keraticytes, epithelial cells and theirderivatives, within which endothelial cells in mammals such as human beingpostnatally lose the ability of proliferation completely, thus once the endothelial cellsare injured, the cell layer cannot repair itself my cell renewal. Levofloxacin are afluoroquinolone antimicrobial agent, is an optically active isomer of its racemate,ofloxacin. Levofloxacin exerts a bactericidal effect by inhibiting bacterial DNAsynthesis through interference with the enzymes DNA grase and topoisomerase IV.Rifampicin is one of the most potent and broad spectrum antibiotics against bacterialpathogens and is a key component of anti-tuberculosis therapy, stemming from itsinhibition of the bacterial RNA polymerase. Skelnik reseached that levofloxacin hadboth time-and concentration-related toxic effects on both human corneal stromalkeratocyte and endothelial cells by using a calcein AM fluorescent bioassay(Skelnik,2003). Rifampicin eye drops have a severe allergic reaction, like papule. In this study,HCEC obtained from the untransfected human corneal endothelial cell line was usedas experimental material to investigate the effects of levofloxacin and rifampicin toHCEC. In addition, cats were used in the further study of effects to cornealendothelial cell caused by levofloxacin and rifampicin eye drops. According to theresults of in vitro and in vivo experiments, the accurate evaluation of the two kinds oftopical antibiotic eye drops effect on HCEC and the experimental basis for clinicaluse is given.Under light microscope, the cultured HCEC treated with levofloxacin at2.44g/L~4.88g/L showed cell shrinkage, cell junction disappeared, detachment andquantity decrease. AO/EB double-fluorescent staining of2.44g/L~4.88g/Llevofloxacin treated cells showed the death rate varies in a dose-and time-dependentmanner. Agarose gel electrophoresis of DNA samples of HCEC treated with2.44g/L~4.88g/L levofloxacin showed DNA ladder, illustrating that levofloxacin couldlead to DNA fragmentations in HCEC. Transmission electron microscope (TEM) of4.88g/L alcaine treated HCEC showed some characteristic features of apoptosis,including cell structure disorder, plasma membrane blebbing, chromatin condensation,and the presence of apoptotic bodies, indicating that HCEC treated with levofloxacin had ultrastructural characteristics of apoptosis. Summarize all the above studies, itwas proved that levofloxacin at2.44g/L~4.88g/L could induce HCEC apoptosis.After treated with0.25g/L~0.5g/L rifampicin respectively, cultured HCECshowed cell shrinkage, cell junction disappeared, detachment and quantity decreaseobserved by light microscope when treated with rifampicin at a concentration above0.25g/L. AO/EB double-fluorescent staining showed the death rate varies in a dose-and time-dependent manner. Remarkable DNA ladder was found at a concentration of0.5g/L, illustrating that rifampicin could lead to DNA fragmentations in HCEC. TEMof0.5g/L rifampicin treated HCEC showed some characteristic features of apoptosis,including cell structure disorder, plasma membrane blebbing, chromatin condensation,and the presence of apoptotic bodies. Based on the results above, it was demonstratedthat rifampicin could induce HCEC apoptosis.In order to further prove that the two kinds of topical antibiotic eye drops haveapoptosis-inducing effect on HCEC, cats were used as experimental animals in studies.Levofloxacin and rifampicin eye drops were droped under subconjunctival four timesa day, duration of treatment was20d. The endothelial specular microscopeobservation was taken every5d. The result showed that, the endothelial cell densityreduced, average cell area increased, The cats’ corneas were identified in vitro.According to alizarin red staining, pleomorphic cell number and cell area of theexperimental eyes increased obviously. SEM showed a small number of apoptoticcells in the corneal endothelium of experimental eyes. According to the studies above,levofloxacin and rifampicin in clinical used concentration could lead to cornealendothelial cell apoptosis was proved.The results showed that levofloxacin and rifampicin had apoptosis-inducingeffect on HCEC and had toxic side effects on CCEC. Consequently, in clinicalapplications, the use of levofloxacin and rifampicin should be used carefully. Inaddition, these findings using the cornea endothelial cell line cells provided importantexperimental bases for the research and development of innocuous topical antibioticeye drops.