Eslami Mojtaba, Esfandyari Sahar, Aghahosseini Marzieh, Rashidi Zahra, Hosseinishental S Hirzad, Brenjian Samane, Sobhani Aligholi, Amidi Fardin
Department of Anatomy, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran.
Department of Infertility, Shariati Hospital, Tehran University of Medical Sciences, Tehran, Iran.
Cell J. 2021 Aug;23(3):319-328. doi: 10.22074/cellj.2021.7222. Epub 2021 Jul 17.
Astaxanthin (AST) has been introduced as a radical scavenger and an anti-apoptotic factor that acts via regulating the nuclear factor-E2-related factor 2 (NRF2) and related factors. Here, we intended to examine the effect of AST on granulosa cells (GCs) against oxidative stress by examining NRF2 and downstream phase II antioxidant enzymes.
In this experimental study, we used cultured human primary GCs for the study. First, we performed the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test to evaluate cells viability after treatment with hydrogen peroxide (HO) and AST. The apoptosis rate and ROS levels were measured by flow cytometry. To determine NRF2 and phase II enzymes expression, we performed real-time polymerase chain reaction (PCR). Finally, we used western blot to measure the protein levels of NRF2 and Kelch-like ECsH-associated protein 1 (KEAP1). Enzyme activity analysis was also performed to detect NRF2 activity.
This study showed that AST suppressed ROS generation (P<0.01) and cell death (P<0.05) in GCs induced by oxidative stress. AST also elevated gene and protein expression and nuclear localization of NRF2 and had an inhibitory effect on the protein levels of KEAP1 (P<0.05). Furthermore, when we used trigonelline (Trig) as a known inhibitor of NRF2, it attenuated the protective effects of AST by decreasing NRF2 activity and gene expression of phase II enzymes (P<0.05).
Our results presented the protective role of AST against oxidative stress in GCs which was mediated through up-regulating the phase II enzymes as a result of NRF2 activation. Our study may help in improving fertilization (IVF) outcomes and treatment of infertility.
虾青素（AST）已被介绍为一种自由基清除剂和抗凋亡因子，其通过调节核因子E2相关因子2（NRF2）及相关因子发挥作用。在此，我们旨在通过检测NRF2及下游II期抗氧化酶来研究AST对颗粒细胞（GCs）抵抗氧化应激的影响。
在本实验研究中，我们使用培养的人原代GCs进行研究。首先，我们进行了3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐（MTT）试验，以评估用过氧化氢（HO）和AST处理后的细胞活力。通过流式细胞术测量凋亡率和活性氧（ROS）水平。为了确定NRF2和II期酶的表达，我们进行了实时聚合酶链反应（PCR）。最后，我们使用蛋白质印迹法来测量NRF2和 Kelch样ECH相关蛋白1（KEAP1）的蛋白质水平。还进行了酶活性分析以检测NRF2活性。
本研究表明，AST可抑制氧化应激诱导的GCs中ROS的产生（P<0.01）和细胞死亡（P<0.05）。AST还提高了NRF2的基因和蛋白质表达以及核定位，并对KEAP1的蛋白质水平具有抑制作用（P<0.05）。此外，当我们使用胡芦巴碱（Trig）作为已知的NRF2抑制剂时，它通过降低NRF活动和II期酶的基因表达来减弱AST的保护作用（P<0.05）。
我们的结果表明AST对GCs氧化应激具有保护作用，这是通过NRF2激活上调II期酶介导的。我们的研究可能有助于改善体外受精（IVF）结果和不孕症治疗。
