Expression and role of ubiqutin carboxyl-terminal hydrolase L1 in lens with age-related cataract under oxidative stress / 中华实验眼科杂志

ABSTRACT

Background Oxidative damage is a major cause of age-related cataracts,and the ubiquitinproteasome system is involved in lens differentiation and development.Ubiquitin carboxy-terminal hydrolase L1 (UCHL1),one of key enzymes of ubiquitin-proteasome system,was discovered to participate in the age related diseases and oxidative stress damage. Objective This study was to investigate the effects of UCHL1 on the formation and development of age-related cataract. Methods Lens capsule were collected from 24 patients with age-related cataract(including 12 cases of cortical cataract and 12 cases of nuclear cataract) during the surgery.Five normal lens capsule membranes were obtained from eye bank of Tongji University.Human lens epithelial cells (LECs) line (SRA01/04) was also collected in this study.Expression of UCHL1 in the lens epithelial layer of different samples was assayed using immunofluorescence technology.UCHL1 eukaryotic expressing vector was constructed and transfected into cultured SRA01/04 by liposome,and green fluorescent protein (GFP) eukaryotic expressing vector was transfected at the same method as the control group.UCHL1 over-expressing cells were then exposed to different concentrations (0.2,0.3,0.4 and 0.5 mol/L) of tert-butyl hydroperoxide (TBHP) for 24 hours and subsequently monitored for cell viability evaluation by MTT assay. Results Immunofluorescence showed that UCHL1 was expressed in human lens epithelial layer,but significantly different expressing levels were seen among normal lens capsular membrane,cortical cataract and nuclear cataract ( F =13.411,P =0.000),and UCHL1 expressing levels were lower in cortical cataract and nuclear cataract than the normal lens (P =0.000,P =0.000).No significant difference was found in UCHL1 expressing level between cortical cataract and nuclear cataract ( P =0.164).Western blot analysis verified that UCHL1 exhibited a stranger expression in the UCHL1 transfected group compared with the GFP transfected group,illuminating a successful transfection of UCHL1 in SRA01/04 cells.MMT assay revealed that the A570/630 value in UCHL1 transfected cells was significantly elevated in comparison with GFP transfected cells following the treatment of 0.3 mol/L TBHP. Conclusions UCHL1 has an antioxidative ability,and it might plays an important role in the progress of age-related cataract.