SirT1 And STAT3 Protect Retinal Pigmented Epithelium Cells Against Oxidative Stress

Objective:Age-related macular degeneration (age-related macular degeneration,AMD) is the most common cause of central visual field defects in elderly patients, so it will contribute to the clinical treatment on study on the pathogenesis of AMD. Studies have shown that oxidative stress can cause cell dysfunction and impairment. Therefore, this study aims to reveal the interaction of SirTl and STAT3 on retinalpigment epithelial cells under oxidative stress state(retin-al pigmented epithelium cells,RPE) and its significancein pathogenesis of ear-ly AMD.Method1) Establishment and assessment of the model human retinal pigment epithelial cells unjured by oxidative stress. In vitro ARPE-19 cells induced with ox-LDL (200 μg) and H2O2 (50 μm), examine the cell proliferation and apoptosis with annexin V-fluorescein isothiocyanate and propidium iodide (AVFI/PI), cell intrercellular reactive oxygen species level(ROS) with DCFH-DA fluorescent probe, cell’s aging under H2O2 and ox-LDL induced with SA-BETA-GAL analytical assessment.2) Following the former experiment, analysize the SirTl and STAT3 expression during oxidative stress. Learn the expression of mRNA of SIRT1 and STAT3 on RPE cells under H2O2 treat using qRTt-PCR.;3) SirTl protection against oxidative damage on RPE. RPE cells were treated respectively with RSV and NA, then induced by H2O2 (50 μm) and the ox-LDL (200μg/ml), using target to SIRT1 for knock out as negative control, analysize the proliferation, apoptosis, ROS and cell aging state to explore the effect.4) Through the gain and loss functions to seek the effect interaction SirT1/STAT3 on RPE under oxidative stress. Examine the expression of STAT3 after SIRT1 was up-regulated or knocked out.Result1. Oxidative stress can contribute to RPE damage and lead to dysfunction of SirTl and STAT3. H2O2 and ox-LDL induced pathological oxidant stress in AMD. After cultured in H2O2 and ox-LDL 24 hours, check the cell proliferation, apoptosis and endogenous reactive oxygen species.Studies shows that apoptosis rates upregulated, ROS level increased significantly. To evaluate the role of SirTl and STAT3 in oxidative stress, we analyze the S-irTl and STAT3expression in RPE. We found that compared wi- th the control gro-up, after the intervention of H2O2 and ox-LDL RPE SirTl mR-NA expression was si-gnifi-cantly reduced, by contrast STAT3 mRNA expressi-on level was increased significantly. These results prove the RPE injury induc-ed by oxidative stress and cause SirTl and STAT3 mRNA expression disord-er.2. RPE SirTl protection against oxidative stress injury.RPE firstly treated with SirTl inhibitor NA and SirTl activator RSV, then cultured 24 hours in H2O2 (50 μm) and ox-LDL (200 μg), observe the proliferation, apoptosis, ROS level on RPE. Results shows that NA increased toxicity of H2O2, and significantly reduce the rate of proliferation, stimulate apoptosis, so ROS accumulate and aging increased quickly. Compared with NA, SirTl activator RSV shows remarkable resistance to H2O2.OX-LDL induc-ed RPE research findings are similar.3.In the process of oxidative stress, SirTl can negatively regulated expression of STAT3.To explore the correlation between STAT3 activation and SirTl, observe STAT3 expression after the SirTl gene knockout, and expression of STAT3 during oxidative stress. RSV SirTl Activator RSV significantly reduced the expression of STAT3, but after the SirTl gene knockout, we found the H2O2 intervention group increased expression of STAT3 significantly. Further, we found that SirTl knockout can increase the expression of STAT3 protein and phosphorylated STAT3 in oxidative stress; and lack of oxidative stress stimuli RSV and SirTl gene knockouts and will not affect the expression of STAT3.Conversely we also analyzed the expression of SirTl and oxidative stress state of expression of SirTl after STAT3 gene knockout on RPE. Each set of expression of STAT3 protein detected by Western blot analysis.Compared with the C-STAT3OV, STAT3 expression can obviously decreased expression of SirTl. Expression of SirTl on STAT3KO Group increased significantly. These results suggest that oxidative stress state of STAT3 have a negative effect on RPE expression of SirTl.4. STAT3 protects ARPE-19 cells against oxidative stress injury, independent regulation of cell senescence.To better understand how STAT3 and SirTl relevance to the protective effect of RPE cells in oxidative stress state, we investigated STAT3 antioxidant effects of RPE after SirTl inhibitor NA pretreatment. STAT3 enhanced NA cell proliferation and reduce apoptosis, and resulted in an increased endogenous ROS. However, STAT3 and does not affect cellular aging. These results indicate that STAT3 directly protects cells against oxidative stress, rather than rely on SirTl activity. And STAT3 under oxidative stress protective effect is not associated with the regulation of cellular senescence. In conclusion, this study suggests that due to the special interaction between SirTl and STAT3, so there is a balancing mechanism between SirTl and STAT3 during resistance to oxidative stress.Conclusion:1.Exogenous antioxidants H2O2 and ox-LDL can induce oxidative stress on ARPE-19 cells and cause significantly damage, such as a decline in proliferation, increased apoptosis, increased level of ROS, remarkable cellular senescence.2.Oxidative stress can cause SirTl and STAT3 dysfunction that may cause RPE damage.3.STAT3 protects ARPE-19 cells against oxidative stress injury, independently r egulate cellular aging.4. SirTl during oxidative stress plays a negative role on the expression of ST-AT3. SirT1 and STAT3 have role against oxidative stress.These results sug-gest that there is balancing mechanisms between SirTl and STAT3 during resistance to oxidative stress, it indicated that we need combine the use of SirTl and STAT in the treatment of AMD.