Intracellular GSH and ROS levels may be related to galactose-mediated human lens epithelial cell apoptosis: role of recombinant hirudin variant III.
Oxidative processes in the lenses are the most commonly found damaging factor for the development of cataracts. Hirudin, a most potent inhibitor of thrombin as an antithrombic drug, also have potential use in cataracts. In order to investigate the mechanisms of hirudin against galactose-induced cataract at the cellular level. We used recombinant hirudin variant III (rHV3) to study the protective effect of hirudin on galactose-mediated human lens epithelial cells injury. The human lens epithelial cells (hLECs) were cultured in D/F(12)-10% FBS medium containing 125 mM D-galactose with or without rHV3. Cell viability was assessed by methylthiazol tetrazolium (MTT) assay and propidium iodide (PI) staining in situ. Cell apoptosis was elevated with comet assay (single cell gel electrophoresis, SCGE), AO/EB double staining and Annexin-V/PI double staining assay. Reactive oxygen species (ROS) were quantified with 2',7'-dichlorofluorescein (DCF), and free glutathione (GSH) levels were measured with a commercial GSH quantification kit. Decreased viability and increased apoptosis of the hLECs were observed when incubated with 125 mM galactose. These hLECs also demonstrated the increased presence of ROS, whereas GSH was reduced. rHV3 blocked the induction of cell death, apoptosis and oxidative stress in hLECs. One mechanism may be through regulating intracellular ROS and GSH levels to inhibit apoptosis of the human lens epithelial cells.
Ou
Yu
Y
School of Life Science and Technology, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, China. ouyu1967@163.com
Geng
Pei
P
Liao
Gao-Yong
GY
Zhou
Zhu
Z
Wu
Wu-Tong
WT
D017304:Annexin A5 / Q000737:chemistry; D017209:Apoptosis / Q000187:drug effects*; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004847:Epithelial Cells / Q000187:drug effects* / Q000378:metabolism; D019793:Fluorescein / Q000737:chemistry; D005690:Galactose / Q000633:toxicity*; D005978:Glutathione / Q000032:analysis / Q000378:metabolism*; D006629:Hirudins / Q000145:classification / Q000494:pharmacology*; D006801:Humans; D007908:Lens, Crystalline / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D008856:Microscopy, Fluorescence; D017382:Reactive Oxygen Species / Q000032:analysis / Q000378:metabolism*; D011994:Recombinant Proteins / Q000494:pharmacology; D013194:Staining and Labeling
D017304:Annexin A5; D006629:Hirudins; D017382:Reactive Oxygen Species; D011994:Recombinant Proteins; D005978:Glutathione; D019793:Fluorescein; D005690:Galactose