Resveratrol Attenuates Hydrogen Peroxide-induced Injury of Rat Ovarian Granulosa-lutein Cells by Resisting Oxidative Stress via the SIRT1/Nrf2/ARE Signaling Pathway

Abstract

Introduction: This paper aims to reveal the molecular mechanism of resveratrol against oxidative stress and cell injury. The ovarian granulosa-lutein cell injury and apoptosis induced by oxidative stress may be responsible for female luteal phase deficiency. The antioxidant function of resveratrol has been confirmed; however, its effect on the expression of antioxidant enzymes and regulatory mechanisms in ovarian granulosa-lutein cells remains unclear.

Objective: This study aimed to investigate the role of the SIRT1/Nrf2/ARE signaling pathway in the effect of resveratrol on the hydrogen peroxide-induced injury of rat ovarian granulosa-lutein cells.

Methods: In this study, ovarian granulosa-lutein cells extracted from 3-week female SD rats were treated with 200 μM H₂O₂ in the presence or absence of 20 μM resveratrol. siRNA-SIRT1 and siRNA-Nrf2 were used to inhibit the expression of SIRT1 and Nrf2, respectively. Cell counting kit 8 (CCK-8), cellular morphology, progesterone secretion, and estradiol were used to evaluate cell injury. Hoechst 33258 staining was used to measure cell apoptosis. DHE staining, DCFH-DA staining, malondialdehyde content, protein carbonyl content, total antioxidant capacity and SOD viability were used to estimate the levels of oxidative stress. Western blot analysis was used to detect the levels of apoptosis-related proteins, and SIRT1/Nrf2/ARE signaling pathway-related proteins.

Results: The H₂O₂ treatment-induced rat ovarian granulosa-lutein cells injury was shown as decreased cell viability, impaired cellular morphology, and decreased levels of progesterone and estradiol. The H₂O₂ treatment also exacerbated cell apoptosis demonstrated as more apoptotic cells stained by Hoechst staining, decreased level of anti-apoptosis protein Bcl-2 and increased level of pro-apoptosis protein Bax. These effects of cell injury and apoptosis induced by H₂O₂ can be ameliorated by resveratrol. Resveratrol also alleviated oxidative stress induced by H₂O₂, supported by decreased superoxide anion and cellular total ROS, decreased malondialdehyde and protein carbonyl levels, and increased total antioxidant capacity and SOD viability. Western blot results demonstrated resveratrol reversed the H₂O₂-induced decrease in levels of antioxidant enzymes containing ARE sequences and activated SIRT1/Nrf2 pathway. Further treatment by siRNA-Nrf2 suggested resveratrol could not activate the expression of antioxidant enzymes under a condition of inhibition of Nrf2.

Conclusion: This study demonstrates that resveratrol attenuated oxidative stress to protect H₂O₂-induced rat ovarian granulosa-lutein cell injury and apoptosis via SIRT1/Nrf2/ARE signaling pathway.

Keywords: H₂O₂; Ovarian granulosa-lutein cells; SIRT1/Nrf2/ARE signaling pathway; oxidative stress; protein Bcl-2; resveratrol.