| Age-related macular degeneration(AMD)is a major cause of visual impairment and blindness among the elderly.AMD is characterized by retinal pigment epithelial(RPE)cell dysfunction.However,the pathogenesis of AMD is still unclear,and there is currently no effective treatment.Accumulated evidence indicates that oxidative stress and autophagy play a crucial role in the development of AMD.H2S is an effective antioxidant,which can directly remove superoxide anion and hydrogen peroxide in cells.Compared with other classic antioxidants,it has higher removal efficiency.At the same time,as a small gas molecule,H2S can diffuse into cells freely.However,as a good endogenous antioxidant,its intracellular concentration is low,so it is difficult to achieve a good antioxidant effect in vivo.Based on this background,whether exogenous H2S supplementation can improve the antioxidant capacity of RPE cells and what mechanism can protect RPE cells are worth further exploring.Materials and Methods:In this paper,cell biology,molecular biology,and biophysics experiments were carried out,with retinal pigment epithelial ARPE-19 cells as cell model.(1)The proliferation of ARPE-19 cells was detected by MTT assay.(2)The morphological damage of ARPE-19 cells was detected by morphological observation.(3)The level of intracellular reactive oxygen species(ROS)was detected by DCFH-DA staining.(4)Annexin V-FITC/PI double staining was used to detect the level of apoptosis.(5)Hoechst 33342/PI double staining was used to detect cell death.(6)The expression of autophagy markers LC3 and p62 was analyzed by Western blot.(7)The formation and changes of autophagosome and autophagy-lysosome were observed by transmission electron microscope.(8)ARPE-19 cells were transfected with the fusion protein mCherry-EGFP-LC3B adenovirus,and autophagosome formation and autophagy flow were measured.Results:1,In the oxidative damage model of ARPE-19 cells induced by H2O2:(1)exogenous H2S reduced the proliferation inhibition of ARPE-19 cells induced by H2O2.(2)It was observed that exogenous H2S improved the morphological damage of ARPE-19 cells induced by H2O2.(3)Exogenous H2S can reduce the oxidative damage induced by H2O2 by reducing ROS level in ARPE-19 cells.(4)Exogenous H2S protected ARPE-19 cells by reducing apoptosis/death induced by oxidative stress.(5)H2O2 induced autophagy,while exogenous H2S decreased autophagy flow in ARPE-19 cells.(6)Autophagy inhibitor can improve the proliferation activity of H2O2 reduced cells by reducing the level of autophagy.(7)After inhibition of autophagy,the morphological damage induced by H2O2 was improved.(8)Inhibition of autophagy played a protective role by reducing H2O2-induced apoptosis/death.2,In the oxidative damage model of ARPE-19 cells induced by UV irradiation,the similar results showed that:(1)exogenous H2S reduced the inhibition of cell proliferation induced by UV irradiation.(2)Exogenous H2S alleviated the oxidative damage induced by UV irradiation by reducing intracellular ROS level.(3)Exogenous H2S protected cells by reducing apoptosis/death induced by UV irradiation.(4)The increase of autophagy flow induced by UV irradiation was reduced by exogenous H2S.(5)Autophagy inhibitors reduced the level of autophagy in cells,which can alleviate the inhibition of cell proliferation induced by UV irradiation.(6)Autophagy inhibition can reduce the apoptosis/death induced by UV irradiation.Conclusion:Exogenous H2S alleviated the proliferation inhibition of ARPE-19 cells induced by oxidative stress,improved cell morphological damage,reduced apoptosis/death,and decreased autophagy level.After autophagy inhibition,cell proliferation inhibition and apoptosis were significantly restored.Therefore,this study demonstrated the antioxidant effect of H2S in RPE cells and the antioxidant mechanism of autophagy.These findings suggest that autophagy may play a key role in the development of AMD,and exogenous H2S has potential value in the treatment of AMD. |
