Ameliorative effect of acteoside on high glucose-induced oxidative stress and mitochondrial damage by modulating SIRT1-mediated mitophagy in RPE cells

Authors: Shen Shuhong, Li Yan, Ma Jia, Chen Qianbo, Xi Xiaoting

Corresponding author: Xi Xiaoting, Email:xi_xiao_ting@163.com

Published:2026-09-10

DOI:10.3760/cma.j.cn115989-20260330-00144

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ABSTRACT 

Objective To investigate the regulatory effect and molecular mechanism of acteoside on oxidative stress and mitochondrial damage in high glucose-induced human retinal pigment epithelial cells (ARPE-19).

Methods ARPE-19 cells were divided into normal control group, mannitol group, high-glucose group, high-glucose+Mito-TEMPO (reactive oxygen species [ROS] scavenger) group, high-glucose+acteoside group, high-glucose+acteoside+BafA1 (autophagy inhibitor) group, high-glucose+acteoside+EX-527 (sirtuin 1 [SIRT1] inhibitor) group, and high- glucose+acteoside+si-SIRT1 transfection group. After corresponding treatments according to grouping, the cell viability of different groups was detected using cell counting kit-8; adenosine triphosphate (ATP) content was measured with an ATP level assay kit; mitochondrial membrane potential was detected via JC-1 staining; mitochondrial ROS level was determined by MitoSOX Red staining; autophagic flux was assessed using mRFP-GFP-LC3 dual fluorescence labeling; the relative expression levels of SIRT1, PINK1, Parkin, LC3Ⅱ/Ⅰ and p62 proteins were examined by Western blot.

Results The cell viability, ATP content and mitochondrial membrane potential were significantly lower and the mitochondrial ROS level was significantly higher in the high-glucose group than those in the normal control group, mannitol group and high-glucose+acteoside group (all P<0.05). The cell viability, ATP content and mitochondrial membrane potential were higher and the mitochondrial ROS level was lower in the high-glucose+acteoside group than those in the high-glucose+acteoside+EX-527 group and high-glucose+acteoside+si-SIRT1 transfection group and all differences were statistically significant (all P<0.05). The cell viability and ATP content were significantly lower in the high-glucose group than in the high-glucose+Mito-TEMPO group (both P<0.05). The relative expression levels of PINK1 and Parkin proteins in the high-glucose group were significantly lower than those in the normal control group, mannitol group and high-glucose+acteoside group, whereas the relative expression levels of LC3 Ⅱ/Ⅰ and p62 proteins in the high-glucose group were significantly higher than those in the normal control group and mannitol group (all P<0.05). The relative expression levels of LC3Ⅱ/Ⅰ and p62 proteins were higher in the high-glucose group than in the normal control group (both P<0.05). The relative expression level of LC3Ⅱ/Ⅰ protein in the high-glucose+acteoside group was higher than that in the high-glucose group but lower than that in the high-glucose+acteoside+BafA1 group (both P<0.05). The relative expression level of p62 protein in the high-glucose+acteoside group was lower than that in the high-glucose group and high-glucose+acteoside+BafA1 group (both P<0.05). The relative expression levels of SIRT1, PINK1, Parkin and LC3Ⅱ/Ⅰ proteins were higher and the relative expression level of p62 protein was lower in the high-glucose+acteoside group than in the high-glucose+acteoside+EX-527 group and high-glucose+acteoside+si-SIRT1 transfection group (all P<0.05). The GFP/mRFP fluorescence intensities of the normal control group, high-glucose group, high-glucose+acteoside group and high-glucose+acteoside+BafA1 group were 0.22±0.10, 0.72±0.04, 0.43±0.06 and 0.61±0.04, respectively, with a statistically significant overall difference ( F=33.68, P<0.001). The GFP/mRFP fluorescence intensity was higher in the high-glucose group than in the normal control group and high-glucose+acteoside group, and higher in the high-glucose+acteoside+BafA1 group than in the high-glucose+acteoside group, with statistically significant differences (all P<0.05).

Conclusions Acteoside alleviates high glucose-induced oxidative stress and mitochondrial damage in ARPE-19 cells by activating SIRT1 to promote PINK1/Parkin-mediated mitophagy.

KEYWORDS：

Diabetic retinopathy;Acteoside;Mitophagy;PINK1/Parkin;SIRT1

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All articles published represent the opinions of the authors, and do not reflect the official policy of the Chinese Medical Association or the Editorial Board, unless this is clearly specified.

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Authors Info & Affiliations 

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Figures & Tables

Figure 1 JC-1 and MitoSOX staining images of each group 　A: JC-1 staining image (×400, scale bar=50 μm)　B: MitoSOX staining image (×1 000, scale bar=10 μm)

Table 1 Comparison of cell viability，ATP content and mitochondrial function among different groups (mean±SD) Note: Compared with the normal control group, ^aP<0.05; compared with the mannitol group, ^bP<0.05 (One-way ANOVA, Tukey test)　ATP: adenosine triphosphate; ROS: reactive oxygen species

Figure 2 Electrophoretogram of autophagy-related protein expression in each group

Table 2 Comparison of relative expression levels of autophagy-related proteins among different groups (mean±SD) Note: Compared with the normal control group, ^aP<0.05; compared with the mannitol group, ^bP<0.05 (One-way ANOVA, Tukey test)

Figure 3 Comparison of cell viability and ATP content among different groups after inhibiting mitochondrial ROS 　A: Comparison of cell viability　 F=238.60, P<0.001. Compared with the normal control group, ^aP<0.001; compared with the high-glucose group, ^bP<0.001 (One-way ANOVA, Tukey test; n=3)　B: Comparison of ATP content　 F=890.20, P<0.001. Compared with the normal control group, ^aP<0.001; compared with the high-glucose group, ^bP<0.001 (One-way ANOVA, Tukey test; n=3)　ROS: reactive oxygen species; ATP: adenosine triphosphate

Figure 4 JC-1 and MitoSOX staining images of each group after acteoside treatment 　A: JC-1 staining image (×400, scale bar=50 μm)　B: MitoSOX staining image (×1 000, scale bar=10 μm)

Table 3 Comparison of cell viability，ATP content，and mitochondrial function among different groups after acteoside treatment (mean±SD) Note: Compared with the normal control group, ^a P<0.05; compared with the high-glucose group, ^b P<0.05 (One-way ANOVA, Tukey test)　ATP: adenosine triphosphate; ROS: reactive oxygen species

Figure 5 Electrophoretogram of autophagy-related protein expression in each group after treatment with autophagy inhibitors

Figure 6 Comparison of autophagy flux among different groups after treatment with autophagy inhibitors 　A: mRFP-GFP-LC3 dual fluorescence staining images of each group (mRFP-GFP ×1 000, scale bar=10 μm)　B: Comparison of GFP/mRFP fluorescence intensity among different groups F=33.68, P<0.001. Compared with the normal control group, ^aP<0.05; compared with the high-glucose group, ^bP<0.05; compared with the high-glucose+acteoside group, ^cP<0.05 (One-way ANOVA, Tukey test; n=3)

Table 4 Comparison of relative expression levels of autophagy-related proteins among different groups after treatment with autophagy inhibitors (mean±SD) Note: Compared with the normal control group, ^aP<0.05; compared with the high-glucose group, ^bP<0.05; compared with the high-glucose+acteoside group, ^cP<0.05 (One-way ANOVA, Tukey test)

Figure 7 JC-1 and MitoSOX staining images of different groups after SIRT1 inhibitor treatment 　A: JC-1 staining image (×400, scale bar=50 μm)　B: MitoSOX staining image (×1 000, scale bar=10 μm)　SIRT1: sirtuin 1

Figure 8 Electrophoretogram of SIRT1 protein and autophagy-related protein expression in each group after SIRT1 inhibitor treatment 　SIRT1: sirtuin 1

Table 6 Comparison of relative expression levels of SIRT1 protein and autophagy-related proteins among different groups after treatment with SIRT1 inhibitors (mean±SD) Note: Compared with the normal control group, ^aP<0.05; compared with the high-glucose group, ^bP<0.05; compared with the high-glucose+acteoside group, ^cP<0.05 (One-way ANOVA, Tukey test)　SIRT1: sirtuin 1

Figure 9 JC-1 and MitoSOX staining images of different groups after si-SIRT1 transfection 　A: JC-1 staining image (×400, scale bar=50 μm)　B: MitoSOX staining image (×1 000, scale bar=10 μm)　SIRT1: sirtuin 1

Table 7 Comparison of cell viability，ATP content，and mitochondrial function among different groups after si-SIRT1 transfection (mean±SD) Note: Compared with the normal control group, ^aP<0.05; compared with the high-glucose group, ^bP<0.05; compared with the high-glucose+acteoside group, ^cP<0.05 (One-way ANOVA, Tukey test)　SIRT1: sirtuin 1; ATP: adenosine triphosphate; ROS: reactive oxygen species

Figure 10 Electrophoretogram of SIRT1 protein and autophagy-related protein expression in each group after si-SIRT1 transfection 　SIRT1: sirtuin 1

Table 8 Comparison of the relative expression levels of SIRT1 protein and autophagy-related proteins among different groups after si-SIRT1 transfection (mean±SD) Note: Compared with the normal control group, ^aP<0.05; compared with the high-glucose group, ^bP<0.05; compared with the high-glucose+acteoside group, ^cP<0.05 (One-way ANOVA, Tukey test)　SIRT1: sirtuin 1

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