title: Protective effect of recombinant hirudin variant III against galactose-mediated rat lens epithelial cell damage.
abstract: OBJECTIVE
  In our previous studies, the protective activity of recombinant hirudin variant III (rHV3) against galactose-mediated lens epithelial cells damage was shown using human lens epithelial cell line. In this study, we applied similar methodology to primary rat lens epithelial cells (rLECs) to see whether the same effect and mechanism remain.
  
  
  METHODS
  The rLECs were cultured in D/F(12)-10% fetal bovine serum (FBS) medium containing 50 mM d-galactose with or without rHV3. Cell viability was assessed by methylthiazol tetrazolium (MTT) assay. Reactive oxygen species (ROS) were detected with 2',7'-dichlorofluorescein (DCF). Levels of malondialdehyde (MDA) and free glutathione (GSH), activities of glutathione s-transferase (GST), catalase, superoxide dismutase (SOD) and glutathione peroxidase (GSHPx) were measured using commercial quantification kits. Cell apoptosis was evaluated by DNA fragmentation assay and flow cytometric analysis. The gene expression of Bcl-2 and Bax was analyzed using reverse transcription-PCR (RT-PCR) and western blot.
  
  
  RESULTS
  Cell viability, ROS, MDA and GSH levels, antioxidant enzymes were significantly altered when rHV3 was administrated. rHV3 also showed prevention of apoptosis of rLECs. In addition, rHV3 treatment significantly increased galactose-induced Bcl-2 downregulation, and decreased Bax upregulation.
  
  
  CONCLUSIONS
  The protective effects of rHV3 may be due to effective recovery of the antioxidative defense system, resulting in antiapoptosis. In addition, rHV3 may be able to inhibit apoptosis of lens epithelial cells through the mitochondrial pathways of apoptosis by regulating Bcl-2 and Bax expression.
authors.affiliation: School of Life Science and Technology, China Pharmaceutical University, Nanjing, China. ouyu2008@126.com
authors.forename: Yu
authors.initials: Y
authors.lastname: Ou
authors.forename: Gaoyong
authors.initials: G
authors.lastname: Liao
authors.forename: Zhijun
authors.initials: Z
authors.lastname: Yuan
authors.forename: Wutong
authors.initials: W
authors.lastname: Wu
mesh_terms: D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western; D002470:Cell Survival; D002478:Cells, Cultured; D053938:DNA Fragmentation; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D005343:Fibrinolytic Agents / Q000494:pharmacology*; D005434:Flow Cytometry; D005690:Galactose / Q000633:toxicity*; D005978:Glutathione / Q000378:metabolism; D006629:Hirudins / Q000494:pharmacology*; D007908:Lens, Crystalline / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D008297:Male; D008315:Malondialdehyde / Q000378:metabolism; D010088:Oxidoreductases / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism; D011994:Recombinant Proteins / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013778:Tetrazolium Salts / Q000378:metabolism; D013844:Thiazoles / Q000378:metabolism; D051028:bcl-2-Associated X Protein / Q000235:genetics
chemical_list: D005343:Fibrinolytic Agents; D006629:Hirudins; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D011994:Recombinant Proteins; D013778:Tetrazolium Salts; D013844:Thiazoles; D051028:bcl-2-Associated X Protein; C072300:hirudin HV3; D008315:Malondialdehyde; D010088:Oxidoreductases; C022616:thiazolyl blue; D005978:Glutathione; D005690:Galactose