Protective Effects Of Tauroursodeoxycholic Acid On Human Lens Epithelial Cells In High Glucose Environment

ObjectiveEndoplasmic reticulum (ER) is a principal site where proteins are modified and folded. Unfolded or misfolded proteins accumulating in endoplasmic reticulum and the disturbance of cells homeostasis will result in the endoplasmic reticulum stress (ERS). ERS to a certain extent lead to the restoration of cells internal environment. But long-term strong ER stress will induce cell apoptosis. Recently, it is found that apoptosis of lens epithelial cells caused by cataract is associated with the signal pathway of ERS. Many of cataractogenic stresses induce ERS and apoptosis in lens epithelial cells, such as glucose deprivation, hyperglycemia, homocysteine, tunicamycin. A recent study suggested that tauroursodeoxycholic acid (TUDCA) does inhibit apoptosis by preventing endoplasmic reticulum stress and protect hepatic cells against bile acid-induced apoptosis. This study will establish a model of apoptotic HLECs and explore the effects of TUDCA on apoptosis of HLECs, as wells as level of GRP78 which were expressed in ER.To investigate the effects and mechanisms of TUDCA on HLECs in high glucose medium.MethodsThe line SRA01/04 of the human lens epithelial cells (HLECs) were cultured in DMEM medium. HLECs were incubated in DMEM with different concentrations of glucose each for 24h. A model of apoptotic HLECs was established and exposed to TUDCA (0.2,0.5,1.0,2.0mmol/L) for 24h. Inhibition of cell proliferation was determined by MTT assay. Morphologic evaluation of apoptotic cells was performed by Hoechst 33258 staining and apoptosis rate was measured by flow cytometry (FCM) after the staining of Annexin V-FITC and PI. Western blotting was used to determine the expression of GRP78.Results1. Inhibition of cell proliferation by glucose with different concentrations After glucose treatment for 24h, the inhibition of cell proliferation was increased accordingly with glucose concentration increasing. There are a lot of apoptotic cells which nuclear presented higher fluorescence intensity under a fluorescent microscope at 24h treatment with 250mmol/L high glucose.The frequency of apoptotic cells increased to (57.94±1.29)%. And a model of apoptotic HLECs was established by 250mmol/L high glucose for 24h.2. Effects of TUDCA on inhibition of HLECs treated in high glucose medium According to MTT assay, incubating the cells with high concentration of glucose (250mmol/L) for 24h inhibited the proliferation of HLECs. The inhibition of cell proliferation was increased with 250mmol/L high glucose.and decreased significantly after coincubation with TUDCA (P<0.01).3. Effect of TUDCA on the morphology of HLECs treated in high glucose medium There was no obvious apoptosis in control groups which cell nuclear showed uniform, diffusion, blue fluorescence under a fluorescent microscope. There are lots of apoptotic cells in 250mmol/L groups and their nuclear presented higher fluorescence intensity. The number of apoptotic cells was decreased markedly after coincubation with TUDCA (2.0mmol/L).4. Effect of TUDCA on the apoptosis rate of HLECs treated in high glucose medium The apoptosis rate of HLECs was measured by flow cytometry. The apoptosis rate of HLECs increase significantly in model group compared with the control group and was inhibited after coincubation with TUDCA (P<0.01).5. Effects of TUDCA on Expression of GPR78 protein in HLECs treated in high glucose mediumThe densitometric analysis of bands for GRP78. The expression of GRP78 was significant increase after treatment with 250mmol/L high glucose compared with control group and inhibited markedly after coincubation with TUDCA (P<0.05).ConclusionsEndoplasmic reticulum stress is involved in HLECs apoptosis induced by high concentration of glucose. TUDCA inhibits HLECs apoptosis, thus protecting HLECs in vitro. The signal transduction mechanisms are related to endoplasmic reticulum stress.