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Abstract
Purpose: 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/F12-10% 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 galatose-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.
Declaration of interest: This study is part of a research project funded by Ministry of Education of the People’s Republic of China (100,000 RMB).