Protective Effect of Metformin against Hydrogen Peroxide-Induced Oxidative Damage in Human Retinal Pigment Epithelial (RPE) Cells by Enhancing Autophagy through Activation of AMPK Pathway

Figure 4

The protective effect of metformin was inhibited by autophagy blockage. D407 cells were pretreated with or without 2 mM 3-MA or 50 μM CQ for 1 h before 1 mM metformin treatment for 2 h and then incubated with or without 200 μM H₂O₂ for further 24 h. (a) The effect of 3-MA or CQ pretreatment on the expression of LC3B and p62 was assessed using ICC (scale bars, 5 μm). (b) Assessment of the effect of 3-MA pretreatment on cell viability was assessed using the MTT assay. (c) Assessment of the effect of CQ pretreatment on cell viability was assessed using the MTT assay (d) D407 cells were incubated with shBeclin1 plasmid for 24 h before 1 mM metformin treatment for 2 h and then incubated with or without 200 μM H₂O₂ for further 24 h. Cell viability was assessed using the MTT assay. (e) D407 cells were incubated with shLC3B plasmid for 24 h before 1 mM metformin treatment for 2 h and then incubated with or without 200 μM H₂O₂ for further 24 h. Cell viability was assessed using the MTT assay. (f) The effect of Beclin1 or LC3B knockdown on the oxidative damage was assessed by JC-1 and ROS staining (scale bars, 100 μm). (g, h) Quantitative analysis of (d). Data are presented as the ; , , and .

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