Resveratrol attenuates hydrogen peroxide-induced aging through upregulation of autophagy in human umbilical vein endothelial cells

Abstract

Purpose: Resveratrol (RESV; trans-3,5,4'-trihydroxystilbene) has emerged as a potential new therapeutic for age-related atherosclerotic diseases. However, the effect of RESV on cellular aging and its underlying mechanisms remain unknown. Therefore, the aim of this study was to examine whether RESV can delay cellular aging through upregulation of autophagy.

Materials and methods: Human umbilical endothelial vein cells (HUVECs) were divided into four groups: the control group, and the hydrogen peroxide (H₂O₂) alone, H₂O₂ + RESV pretreatment, and H₂O₂ + 3-methyladenine (3-MA) + RESV pretreatment intervention groups. The cell viability was evaluated by a cell counting kit-8 assay. Superoxide dismutase (SOD) activity and intracellular reactive oxygen species (ROS) levels were tested using commercial kits. Senescence-related β-galactosidase activities were detected by immunohistochemical staining. The expression levels of aging-related and autophagy-related markers, including phosphorylated Rb (p-Rb), LC3, and p62, with or without RESV were measured by Western blotting.

Results: Pretreatment with 10 µM RESV increased the cell viability and SOD levels. The remarkably higher positive rate of senescence-associated β-galactosidase and increased intracellular ROS levels in the H₂O₂ treatment group were reversed by treatment with 10 µM RESV. As compared to the H₂O₂ treatment group, 10 µM RESV could upregulate autophagy through the regulation of p-Rb, LC3, and p62 levels. The anti-aging effect of RESV via an autophagy regulation mechanism was further confirmed by the suppression of these effects with 3-MA treatment.

Conclusion: RESV may reverse and delay the aging process of HUVECs via upregulation of autophagy and could be a candidate therapeutic for age-related atherosclerotic diseases.

Keywords: LC3; oxidative stress; p-Rb; p62; senescence.

Figure 1

Effects of RESV on cell viability and SOD levels in HUVECs treated with H₂O₂. Notes: (A) Morphology of HUVECs under normal culture condition (100×). (B) Effect of different concentrations of H₂O₂ on HUVECs viability. (C) Effects of different concentrations of RESV on HUVECs viability in the presence of 100 µM H₂O₂. (D) SOD level of HUVECs treated with different concentrations of RESV and in the presence of 100 µM H₂O₂. N=5. **P<0.01, ***P<0.001 vs the control group; ^##P<0.01 vs the H₂O₂ group. Abbreviations: HUVECs, human umbilical endothelial vein cells; RESV, resveratrol; SOD, superoxide dismutase.

Figure 2

Percentage of SA-β galactosidase-positive HUVECs cells treated with H₂O₂, RESV, and 3-MA. N=5. Notes: ***P<0.001 vs control group; ^###P<0.001 vs H₂O₂ group; ^ΔΔΔP<0.001 vs RESV group. Abbreviations: HUVECs, human umbilical endothelial vein cells; 3-MA, 3-methyladenine; RESV, resveratrol.

Figure 3

Detections of ROS in various groups. Notes: (A) Detection of ROS in various groups by DCFH-DA probes and representative images taken under a fluorescence microscope (100×). The green fluorescence intensity reflects the presence of ROS. (B) Comparison of ROS levels across different treatment groups. N=5. ***P<0.001 vs control group; ^###P<0.001 vs H₂O₂ group; ^ΔΔΔP<0.001 vs RESV group. Abbreviations: DCFH-DA, 2′7′-dichlorodihydrofluorescein diacetate; 3-MA, 3-methyladenine; RESV, resveratrol; ROS, reactive oxygen species.

Figure 4

Expression levels of p-Rb in response to different concentrations of H₂O₂. Notes: A total of 50 µg lysates collected from cells treated with or without H₂O₂ were separated on 6% SDS-PAGE. Quantification of band intensity was carried out using ImageJ software. According to the results, treatment of 100 µM of H₂O₂ for 24 hours was selected for subsequent experiments. N=3. *P<0.05, **P<0.01, ***P<0.001 vs the control group.

Figure 5

Anti-aging effect and autophagy-related markers of H₂O₂-induced HUVECs treated with different concentrations of RESV. Notes: (A) Expression levels of p-Rb and Rb in HUVECs treated with different concentrations of RESV and in the presence of 100 µM H₂O₂. ^#P<0.05, ^##P<0.01, ^###P<0.001 vs H₂O₂ group (N=3). (B) LC3 II/LC3 I ratios and expression levels of p62 in HUVECs treated with different concentrations of RESV and in the presence of 100 µM H₂O₂. #P<0.05, ^##P<0.01, ^###P<0.001 vs H₂O₂ group (N=3). Abbreviations: HUVECs, human umbilical endothelial vein cells; LC3, microtubule light chain 3; RESV, resveratrol.

Figure 6

Effect of 3-MA on the anti-aging effect of RESV by inhibiting autophagy. Notes: (A) Expression levels of p-Rb and Rb in different treatment groups. (B) LC3 II/LC3 I ratios and expression levels of p62 proteins in different treatment groups. N=3. *P<0.05 vs control group; ^#P<0.05, ^###P<0.001 vs H₂O₂ group; ^ΔP<0.05, ^ΔΔP<0.01 vs RESV group. Abbreviations: LC3, microtubule light chain 3; 3-MA, 3-methyladenine; RESV, resveratrol.