title: Cytotoxicity of pilocarpine to human corneal stromal cells and its underlying cytotoxic mechanisms.
abstract: OBJECTIVE
  To examine the cytotoxic effect of pilocarpine, an anti-glaucoma drug, on human corneal stromal (HCS) cells and its underlying cytotoxic mechanisms using an in vitro model of non-transfected HCS cells.
  
  
  METHODS
  After HCS cells were treated with pilocarpine at a concentration from 0.15625 g/L to 20.0 g/L, their morphology and viability were detected by light microscopy and MTT assay. The membrane permeability, DNA fragmentation and ultrastructure were examined by acridine orange (AO)/ethidium bromide (EB) double-staining. DNA electrophoresis and transmission electron microscopy (TEM), cell cycle, phosphatidylserine (PS) orientation and mitochondrial transmembrane potential (MTP) were assayed by flow cytometry (FCM). And the activation of caspases was checked by ELISA.
  
  
  RESULTS
  Morphology observations and viability assay showed that pilocarpine at concentrations above 0.625 g/L induced dose- and time-dependent morphological abnormality and viability decline of HCS cells. AO/EB double-staining, DNA electrophoresis and TEM noted that pilocarpine at concentrations above 0.625 g/L induced dose- and/or time-dependent membrane permeability elevation, DNA fragmentation, and apoptotic body formation of the cells. Moreover, FCM and ELISA assays revealed that 2.5 g/L pilocarpine also induced S phase arrest, PS externalization, MTP disruption, and caspase-8, -9 and -3 activation of the cells.
  
  
  CONCLUSIONS
  Pilocarpine at concentrations above 0.625 g/L (1/32 of its clinical therapeutic dosage) has a dose- and time-dependent cytotoxicity to HCS cells by inducing apoptosis in these cells, which is most probably regulated by a death receptor-mediated mitochondrion-dependent signaling pathway.
authors.affiliation: Laboratory for Corneal Tissue Engineering, College of Marine Life Sciences, Ocean University of China, Qingdao 266003, Shandong Province, China.
authors.forename: Xiao-Long
authors.initials: XL
authors.lastname: Yuan
authors.affiliation: Laboratory for Corneal Tissue Engineering, College of Marine Life Sciences, Ocean University of China, Qingdao 266003, Shandong Province, China.
authors.forename: Qian
authors.initials: Q
authors.lastname: Wen
authors.affiliation: Laboratory for Corneal Tissue Engineering, College of Marine Life Sciences, Ocean University of China, Qingdao 266003, Shandong Province, China.
authors.forename: Meng-Yu
authors.initials: MY
authors.lastname: Zhang
authors.affiliation: Laboratory for Corneal Tissue Engineering, College of Marine Life Sciences, Ocean University of China, Qingdao 266003, Shandong Province, China.
authors.forename: Ting-Jun
authors.initials: TJ
authors.lastname: Fan
keywords: apoptosis; cytotoxicity; human corneal stromal cells; mitochondrion; pilocarpine