Resveratrol alleviates alveolar epithelial cell injury induced by hyperoxia by reducing apoptosis and mitochondrial dysfunction

Abstract

Bronchopulmonary dysplasia is a severe and long-term pulmonary disease in premature infants. Hyperoxia-induced acute lung injury plays a critical role in bronchopulmonary dysplasia. Resveratrol is a polyphenolic phytoalexin and a natural agonist of Sirtuin 1. Many studies have shown that resveratrol has a protective effect on hyperoxia-induced lung damage, but its specific protective mechanism is still not clear. Further exploration of the possible protective mechanism of resveratrol was the main goal of this study. In this study, human alveolar epithelial cells were used to establish a hyperoxia-induced acute lung injury cell model, and resveratrol (Res or R), the Sirtuin 1 activator SRT1720 (S) and the Sirtuin 1 inhibitor EX-527 (E) were administered to alveolar epithelial cells, which were then exposed to hyperoxia to investigate the role of Res in mitochondrial function and apoptosis. We divided human alveolar epithelial cells into the following groups: (1) the control group, (2) hyperoxia group, (3) hyperoxia+Res20 group, (4) hyperoxia+Res20+E5 group, (5) hyperoxia+Res20+E10 group, (6) hyperoxia+S2 group, (7) hyperoxia+S2+E5 group, and (8) hyperoxia+S2+E10 group. Hyperoxia-induced cell apoptosis and mitochondrial dysfunction were alleviated by Res and SRT1720. Res and SRT1720 upregulated Sirtuin 1, PGC-1α, NRF1, and TFAM but decreased the expression of acetyl-p53 in human alveolar epithelial cells that were exposed to hyperoxia. These findings revealed that Res may alleviated hyperoxia-induced mitochondrial dysfunction and apoptosis in alveolar epithelial cells through the SIRT1/PGC-1a signaling pathway. Thus, Sirtuin 1 upregulation plays an important role in lung protection.

Keywords: Bronchopulmonary dysplasia; EX-527; SRT1720; apoptosis; mitochondrial dysfunction; resveratrol.

Figure 1.

Effects of Res, SRT1720 and EX-527 on the viability of HPAEpiC cells. (a) Res inhibits the growth of HPAEpiC cells in a dose-dependent manner. (b) SRT1720 inhibits the growth of HPAEpiC cells in a dose-dependent manner. (c) EX-527 inhibits the growth of HPAEpiC cells in a dose-dependent manner. Data from four independent experiments. *P < 0.05; **P < 0.01; and ***P < 0.001.

Figure 2.

The effects of Res, SRT1720, and EX-527 on the total ROS of HPAEpiC cells. (a) Effects of Res at different concentrations on the total ROS of HPAEpiC cells induced by hyperoxia. (b) Effects of EX-527 at different concentrations on the total ROS of HPAEpiC cells induced by hyperoxia. (c) Effects of SRT1720 at different concentrations on the total ROS of HPAEpiC cells induced by hyperoxia. (d) Effects of Res and EX-527 on the total ROS of HPAEpiC cells induced by hyperoxia. (e) Effects of SRT1720 and EX-527 on the total ROS of HPAEpiC cells induced by hyperoxia. Data from four independent experiments. *P < 0.05; **P < 0.01; and ***P < 0.001.

Figure 3.

Res reduced mtROS production in HPAEpiC cells that were exposed to hyperoxia. (a) The effects of Res on mtROS induced by hyperoxia. The blue fluorescence represents the nucleus, while the red fluorescence represents mtROS (400× magnification, Scale bar: 50 μm). (b) The relative level of mtROS compared to the control group. Data from four independent experiments. *P < 0.05; **P< 0.01; and ***P< 0.001.(A color version of this figure is available in the online journal.)

Figure 4.

Res alleviated the decrease in mitochondrial membrane potential of alveolar epithelial cells when exposed to hyperoxia. (a) The effects of Res on the decrease in mitochondrial membrane potential in HPAEpiC cells induced by hyperoxia (400× magnification, Scale bar: 50 μm). (b) The red/green ratio of each group. Data from four independent experiments. *P < 0.05; **P< 0.01; and ***P< 0.001. (A color version of this figure is available in the online journal.)

Figure 5.

Res reduced the apoptosis of alveolar epithelial cells that were exposed to hyperoxia. (a) The effects of Res and EX-527 on apoptosis of HPAEpiC cells induced by hyperoxia. (b) The apoptotic rates of each group. Data from four independent experiments. *P < 0.05; **P< 0.01; and ***P< 0.001. (A color version of this figure is available in the online journal.)

Figure 6.

Res and SRT1720 upregulated the expression of SIRT1, PGC-1α, NRF1, and TFAM in alveolar epithelial cells and downregulated Ac-p53 when exposed to hyperoxia. (a, b) The effects of Res on the protein expression of SIRT1, PGC-1α, NRF1, TFAM, and Ac-p53 in HPAEpiC cells during hyperoxia. Data from four independent experiments. *: Control group versus hyperoxia (H+R0) group, P < 0.05; #: Hyperoxia (H+R0) group versus hyperoxia+Res20 (H+R20) group, P < 0.05; ^: Hyperoxia+Res (H+R20) group versus hyperoxia+Res20 + 5 μM EX-527 (H+R20+E5) group, P < 0.05; &: Hyperoxia+Res20 (H+R20) group versus hyperoxia+Res20 + 5 μM EX-527 (H+R20+E5) group, P < 0.05. (c, d) The effects of SRT1720 on the protein expression of SIRT1, PGC-1α, NRF1, TFAM and Ac-p53 in HPAEpiC cells during hyperoxia. Data from four independent experiments. *: Control group versus hyperoxia (H+S0) group, P < 0.05; #: Hyperoxia (H+S0) group versus hyperoxia+SRT1720 (H+S2) group, P < 0.05; ^: Hyperoxia+SRT1720 (H+S2) group versus hyperoxia+SRT1720 + 5 μM EX-527 (H+S2+E5) group, P < 0.05; &: Hyperoxia+SRT1720 (H+S2) group versus hyperoxia+SRT1720 + 10 μM EX-527 (H+S2+E10) group, P < 0.05.

Figure 7.

Protective mechanism of resveratrol against hyperoxia-induced cell damage. (A color version of this figure is available in the online journal.)