| Objective: This study aim to investigate the effect and potential mechanism of Sirt3 on mitophagy in retinal pigment epithelium cells(RPE)in a high glucose environment.Methods:1.In vitro culture of RPE cells: RPE cells were cultured in DMEM/F12 complete medium and treated under different conditions.2.To detect the effect of high glucose on RPE cells: The m RNA and protein relative expressions of Sirt3,FOXO3 a,PINK1 and LC3 B in normal group(NG)and high glucose group(HG)were determined by RT-PCR and Western blot.DCFH-DA staining was used to determine the ROS level of cells under high glucose condition.Mito Tracker and Lyso Tracker probes were used to label mitochondria and lysosome for fluorescence colocalization analysis to evaluate the effect of high glucose on mitophagy.3.The effect of Sirt3 overexpression on RPE cells: Sirt3 virus vector was transfected into RPE cells to construct Sirt3 overexpression.Groups: NG,Hypertonic,HG,HG+LV-GFP,HG+LV-Sirt3.The m RNA and protein expression levels of Sirt3,FOXO3 a,PINK1,Parkin and LC3 B in each group were compared.The apoptosis rate of each group was determined by flow cytometry.4.Silencing PINK1 further explored the regulation mechanism of Sirt3 on mitophagy: Lentivirus-mediated PINK1 short hairpin RNA(sh RNA)was transfected into cells to silence PINK1.Groups: HG,HG+LV-Sirt3+sh-NC,HG+LV-Sirt3+sh-PINK1.The expression levels of Sirt3,PINK1,Parkin and LC3 B in each group were determined,and it was inversely confirmed that Sirt3 regulates mitophagy through PINK1-Parkin pathway.Results:1.Effects of high glucose on RPE cells: Compared with the NG,the m RNA and protein relative expressions of endogenous Sirt3,FOXO3 a and PINK1 in the HG were significantly decreased(P < 0.05),the mitophagy related proteins LC3 B and LC3Ⅱ/LC3Ⅰ were significantly decreased(P < 0.05),and the mitophagy level was decreased.Fluorescence colocalization analysis showed that the positive point of mitophagy was significantly lower than that of the NG after culture under high glucose for 72 h(P < 0.05).High glucose inhibited mitophagy.While the intracellular ROS level was significantly increased in the HG(P < 0.05).High glucose could cause intracellular ROS accumulation.2.Regulation mechanism of Sirt3 on mitophagy and apoptosis in RPE cells: The expression of GFP was observed under fluorescence microscope 72 hours after transfection of Sirt3 virus vector and empty vector,and over 80% of the cells showed green fluorescence.Meanwhile,the m RNA expression level of Sirt3 virus vector group was significantly increased(P < 0.05).Sirt3 overexpression was successfully constructed.The m RNA and protein levels of Sirt3 were increased in the HG+LV-Sirt3(P < 0.05),and decreased in the HG+LV-GFP(P < 0.05).There was no significant difference between the Hyperotonic and the NG.The expressions of FOXO3 a,PINK1,Parkin,LC3 B and LC3Ⅱ/LC3Ⅰ in the HG+LV-Sirt3 were significantly increased compared with those in the HG+LV-GFP(P < 0.05).Overexpression of Sirt3 can activate FOXO3a/ PINK1-Parkin pathway and enhance mitophagy level.In the HG,the cell apoptosis rate was significantly higher than that in the NG(P < 0.05).High glucose promoted the apoptosis of RPE cells,while the HG+LV-Sirt3 was significantly lower than that in the HG(P < 0.05),and there was no significant difference between the HG+LV-Sirt3 and the NG.Overexpression of Sirt3 could inhibit cell apoptosis.In RPE cells,Sirt3 can regulate mitophagy and affect apoptosis through FOXO3a/ PINK1-Parkin signaling pathway.3.Silencing PINK1 reversely confirmed the mechanism of Sirt3: After transfection of RPE cells with PINK1-sh RNA virus vector and empty vector(MOI=10)to silence PINK1,more than 80% cells showed green fluorescence under fluorescence microscope after transfection 72 hours.Compared with the empty vector transfection group(control group),the m RNA relative expression level of PINK1 in the PINK1-sh RNA virus vector transfection group(PINK1-sh RNA)was significantly decreased(P < 0.05),which successfully silenced PINK1,blocked the PINK1-Parkin pathway,and clarified the silencing efficiency of PINK1-sh RNA virus vector.Compared with the HG,Sirt3 expression in the HG+LV-Sirt3+sh-NC and HG+LV-Sirt3+sh-PINK1 was significantly increased(P < 0.05),and compared with the HG+LV-Sirt3+sh-NC,the expression of PINK1,Parkin,LC3 B,LC3Ⅱ/LC3Ⅰ in HG+LV-Sirt3+sh-PINK1 were significantly decreased(P < 0.05).Silencing PINK1 and blocking PINK1-Parkin pathway can inhibit the activation of Sirt3 on mitophagy in RPE cells,which inversely confirms that Sirt3 regulates mitophagy by mediating PINK1-Parkin signaling pathway.Conclusion: Sirt3 can activate mitophagy through the FOXO3a/PINK1-Parkin pathway and reduce HG-induced apoptosis of RPE cells.This study provides a new direction to understand the pathogenesis and develop a potential therapeutic target for diabetic retinopathy. |
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