Protections On Lens Epithelial Cells By SIRT1 Gene Expression And Its Mechanism

Age-related cataract (ARC) is the leading cause for blindness worldwide, which makes its prevention and therapy of great importance. Thus, to study its pathologic mechanism on the early stage and to discover the preventive methods for ARC is a main social and economic issue for the whole world.It has been recognized that the oxidative stress is the main risk factor and the beginning link of ARC. The pathological basis of this process is the apoptosis of lens epithelial cells (LECs) and the changes of lens protein. And the apoptosis of LECs is also the cause of lens protein changes. Thus, the pathogenesis of ARC is highly related to the state of LECs. Li et al pointed out in 1995 that the apoptosis of LECs is the common cellular basis for all the non-congenital cataract. Therefore, the apoptosis of LECs has been highlighted in the studies on ARC pathogenesis, and it is of great significance to discover the anti-apoptotic system in LECs to prevent or down-regulate the apoptosis of LECs.SIRT1 (sirtuin type 1) is an important anti-apoptotic factor in human. It is a NAD+dependant classⅢhistone deacetylases (HDAC) and has been under intense scientific investigation recently and shown to be involved in longevity and many age-related diseases. SIRT1 plays its protective role mainly through deacetylation of substrates such as p53, forkhead box class O (FOXOs) family, et al. SIRT1 has been recognized as a promising target for therapy in many age-related diseases. Unfortunately, it has not been studied at all in the field of ARC. Therefore, our study is to observe the expression of SIRT1 in human LECs (HLECs) and its protective role in the onset of ARC and in oxidative stress, and to further study the mechanism of its protection through its downstream P53 pathway.PartⅠChanges in SIRT1 Expression and Its Downstream Pathways in Age-Related Cataract in HumansPurpose To investigate the expression of SIRT1 in HLECs, and to observe the changes in SIRT1 expression and its downstream P53 and FOXOs proteins at the onset of ARC.Methods The anterior lens capsule specimens from normal human donor eyes were divided into 3 groups:group A of young lens (younger than 40 ages without cataract), group B of old but normal lens (50～70 ages without cataract) and group C of ARC lens (50～70 ages with ARC). Real-time quantitative reverse transcription (PT-PCR) of SIRT1 mRNA, western blot (WB) analysis by anti-SIRT1, anti-p53, anti-p53 (acetyl K379), anti-FOXO3a, anti-FOXO4, anti-p27kip1, anti-p130, and anti-Bim, immunofluorescence of SIRT1, and TUNEL assay were performed in each group. Results SIRT1 expression was significantly decreased in group B compared with group A, but increased in group C compared with group B. P53 expression increased with age and topped in group C, however, there was a decrease in active acetyl-P53 expression in group C compared with group B. The expression of both FOXO3a and FOXO4 decreased with age, but in group C, their expression level is equivalent to group A. Accordingly, the downstream p27kipl and p130 showed the similar changes among three groups. In contrast, the expression of Bim was lowest in the ARC lens. And TUNEL assay showed significantly increased apoptosis incidence in group C.Conclusions The expression of SIRT1 increased in ARC in human. Its downstream p53 was inhibited, and FOXOs pathway was activated, indicating that SIRT1 may play a protective role in ARC formation.PartⅡEffects of Up and Down Regulation of the SIRT1 Activity on Apoptosis of HLECs in Oxidative StressPurpose To investigate the effects of up and down regulation of the SIRT1 activity on apoptosis of HLECs in oxidative stress, and to analyze the protection by SIRT1 on HLECs.Methods HLECs was cultured and divided into 4 main groups:(1) control: cultured in normal conditions; (2) H2O2 group:add H2O2 on the concentration of 400μmol/L; (3) SIRT1 activator resveratrol (RES) groups, including 4 different RES concentrations:5,10,20,40μmol/L, and also add H2O2 on the concentration of 400 u mol/L; (4) SIRT1 inhibitor nicotinamide (NAM) groups, including 4 different NAM concentrations:25,50,100,200μmol/L, and also add H2O2 on the concentration of 400μmol/L.24h after the different intervention, immunofluorescence of SIRT1, WB analysis by anti-SIRT1, anti-p53 and anti-p53 (acetyl K379), observations under the upside-down microscopy, MTT assay and TUNEL assay were performed in each group.Results Immunofluorescence and WB analysis of SIRT1 demonstrated that the SIRT1 expression was significantly increased in H2O2 group and further enhanced in RES groups along with the increase of the concentration of RES, while the NAM groups show no significant differences to H2O2 group. The acetyl-P53 was decreased along with the increase of the concentration of RES, and increased along with the increase of the concentration of NAM. Under upside-down microscopy, the control group showed regular hexagonal or round cells with relatively high cell density, while the density in H2O2 group decreased with some fusiform shaped cells. Along with the increase of RES concentration, the fusiform shaped cells decreased with the whole cell density increased. In the contrast, along with the increase of NAM concentration, the irregularity of HLECs increased with a lot of fusiform shaped cells. The OD Value of MTT assay on HLECs increased along with the increase of the concentration of RES, and decreased along with the increase of the concentration of NAM. TUNEL assay found that the apoptosis incidence significantly decreased in RES groups on the concentration of 10,20,40μmol/L, while it significantly increased in all 4 NAM groups.Conclusions The expression of SIRT1 increased in HLECs in oxidative stress. RES reduced the apoptosis of HLECs by H2O2 through the activation of SIRT1, while NAM further increased apoptosis of HLECs by downregulation of SIRT1 activity, which indicated the protective role of SIRT1 on HLECs in oxidative stress.PartⅢImpact of the Blockage of P53 Pathway on the Protective Effect by SIRT1 on HLECsPurpose To investigate the impact of the blockage of p53 pathway on the protective effect by SIRT1 on HLECs, and discover if the p53 pathway is an important downstream pathway of SIRT1 in HLECs.Methods Human lens epithelial cell (HLEC) line was cultured and divided into 4 main groups:(1) control:cultured in normal conditions; (2) H2O2 group:add H2O2 on the concentration of 400μmol/L; (3) NAM groups:add NAM on the concentration of 100μmol/L and H2O2 on the concentration of 400μmol/L; (4) pifithrin-α(p-fifty three inhibitor, PFT-α) groups, including 3 different PFT-αconcentrations:2.5,5,10μmol/L, and also add NAM on the concentration of 100 u mol/L and H2O2 on the concentration of 400μmol/L.24h after the different intervention, immunofluorescence of SIRT1, western blot analysis by anti-SIRT1, anti-p53 and anti-p53 (acetyl K379), observations under the upside-down microscopy, MTT assay and TUNEL assay were performed in each group.Results Immunofluorescence and WB analysis of SIRT1 demonstrated that the SIRT1 expression was significantly increased in H2O2 group, and the NAM group and PFT-αgroups show no significant differences to H2O2 group. The acetyl-P53 was increased in the NAM group and PFT-αgroups. Under upside-down microscopy, the control group showed regular hexagonal or round cells with relatively high cell density, while the density in H2O2 group decreased with some fusiform shaped cells. And the irregularity of HLECs increased with a lot of fusiform shaped cells in the NAM group. Along with the increase of PFT-αconcentration, the fusiform shaped cells decreased with the whole cell density increased. The OD Value of MTT assay on HLECs decreased with the addition of of NAM, but increased along with the increase of the concentration of PFT-α. TUNEL assay found that the apoptosis incidence significantly increased in NAM group, but decreased in PFT-αgroups along with the increase of the concentration of PFT-αsignificantly.Conclusions PFT-αinhibited the apoptosis of HLECs. The blockage of p53 pathway by PFT-αeliminated the inhibition of the protection by SIRT1 on HLECs in the use of NAM, which indicated that the p53 pathway is an important downstream pathway of SIRT1 in HLECs.