The Role Of Unfolded Protein Response In Cataract Pathogenesis

OBJECTIVESince the first discovery of cell apoptosis by Kerr in 1972, the relationship between cell apoptosis and cataract has always been studied. Now it is belieyed that apoptosis of lens epithelial cells is the cytological basis of cataract except congenital cataract. The research on apoptosis mechanism of LECs has become a hot issue. Endoplasmic reticulum is a principal site for protein synthesis and is where the vast majority of secreted,glycosylated,modified lipid proteins are folded into their teriary and quaternary structure. A lot of unfoled proteins are accumulated in endoplasmic reticulum under the stimulations of hypoxia,glucose imbalance,oxidation and so on. The accumulation of unfolded proteins or mis-folded proteins causes cells to up-regulate transcription of molecular chaperones and modifying enzymes to cope with the high demand for these proteins,known as the unfolded protein response(UPR).Stress signals are translated to cell nuclear and lead to two results:adaptation or apoptosis.So,UPR plays an important role in the outcome of stress cells. This study will investigate the effects of the unfolded protein response(UPR) in endoplasmic reticulum stress on apoptosis of human lens epithelial cells (hLECs) and the relationship between the UPR and cataract formation.METHODSCultured hLECs were divided into control group and stimulated groups-A,B,C,D,which were cultured in 1640 with homocysteine(1,2,5,10mmol/L) for 24h. Inhibition of cell proliferation was determined by MTT assay; cell apoptosis was detected by Hoechst staining;free glutathione was determined using a Glutathione Quantification Kit; levels of Cytosolic ROS were assessed by adding H2-DCFH-DA for 20-30min followed by imaging with a fluorescent microscope;expression of Bip/GRP78, caspase-12 in stimulated cells was observed by Western blot.RESULTS1.Inhibition of cell proliferation by homocysteine(Hcy) with different concentrations:After stimulated by Hcy for 24h,LECs were observed under microscope. The cells of A and B groups grew against the wall of flask, formed a monolayer after fusion and showed a mosaic arrangement. The cells of C and D groups partially floated and the mortality rate of D groups were more than half. Cell vitality of LECs showed dose-dependent decrease,48.2% decline at 5mmol/L concentration of Hcy and 57.7% decline at 10mmol/L concentration of Hcy,which showed significant difference compared with control group(P<0.01).2.The effect on cell apoptotic rate by homocysteine(Hcy) with different concentrations:There was no obvious apoptosis in A and B groups and control groups which cell nuclear showed uniform,diffusion,blue fluorescence under a fluorescent microscope. The cells of C groups shrinkaged and their nuclears presented higher fluorescence intensity.3.ROS production in LECs treated with Hcy of different concentrations: It showed no fluorescence in control groups and various degrees green fluorescence which increased in a dose-dependent manner in stimulated groups under a fluorescent microscope. The statistical analysis of fluorescence intensity detected by flow cytometry showed that there was no significant difference of ROS producttion between A,B and control groups(P>0.05) and there was significant difference of ROS producttion between C,D and control groups(P<0.05).4.Decrease of GSH by Hcy:The content of GSH in LECs decreased with increased concentrations of Hcy. There was no significant difference of the content of GSH between A,B and control groups(P>0.05) and the content of GSH decreased significantly in C and D groups(P<0.05).5.The expression of GRP78 and caspase-12 in LECs: The expression of GRP78 in C and D groups increased significantly which respectively was 4.6 times and 6.9 times of control groups. Simultaneously,the expression of caspase-12 in C and D groups increased significantly which respectively was 2.8 times and 3.1 times of control groups.However,A and B groups showed similar trend with C and D groups,but the difference has no statistical significance(P>0.05).CONCLUSION1 .Hcy can induce endoplamic reticulum stress in LECs.2.Lower concentration of endoplasmic reticulum stressors can disturb the steady-stateof endoplasmic reticulum and cause proteins to unfold or mis-fold in the ERlumen. By the protect of UPR,unfolded protein in cells can be cleared and cells willsurvive.3.Higher concentration of endoplasmic reticulum stressors can induce endoplasmicreticulum and induce apoptosis in LECs through UPR. The conclusion can be drawedthat the UPR is probably one of initiating factors of cataract.