Abstract

Oxidative stress is often initiated by excess reactive oxygen species (ROS) production, resulting in macromolecular damage, which is implicated in many disease states. Glutaredoxin 1 (Grx1) is an antioxidant enzyme that plays an important role in redox signaling and redox homeostasis. In the present study, we generated HeLaS3 cell lines deficient in Grx1 by the CRISPR/CAS9 system to clarify how Grx1 affects the physiological activities of HeLaS3 cells to respond to oxidative stress. First, the survival assay revealed that Grx1-deficient HeLaS3 cells were more sensitive to γ-ray irradiation, heat shock and H₂O₂ exposure than HeLaS3 wild-type cells. Next, the intracellular redox state was investigated using a fluorescent probe (2′-7′dichlorofluorescin diacetate), and the oxidized state of total proteins and a peroxidase Prx2 were measured by Western blot analysis. Exposure to γ-ray irradiation, heat shock and H₂O₂ significantly induced more accumulation of intracellular oxidants including ROS and higher levels of oxidized proteins in Grx1-deficient HeLaS3 cells. Furthermore, MitoSox Red staining demonstrated that Grx1 deficiency causes a higher level of oxidants production in mitochondria. Moreover, Grx1-deficient HeLaS3 cells had a higher cytochrome c level and higher apoptosis rate (Annexin-V/FITC and EthD-III staining assay) upon oxidative stress. These results suggested that Grx1 deficiency lead to mitochondrial redox homeostasis disruption and apoptotic cell death upon oxidative stress. In addition, the results of proliferation assay and MitoTracker staining assay (multinuclear cell formation rate) suggested that oxidative stress exposure inhibits cell proliferation maybe by affecting cytoplasmic division in Grx1-deficient HeLaS3 cells.

摘要

氧化应激通常由过量的活性氧 (ROS) 产生引发，导致大分子损伤，这与许多疾病状态有关。谷氧还蛋白 1 (Grx1) 是一种抗氧化酶，在氧化还原信号传导和氧化还原稳态中起重要作用。在本研究中，我们通过 CRISPR/CAS9 系统生成了缺乏 Grx1 的 HeLaS3 细胞系，以阐明 Grx1 如何影响 HeLaS3 细胞对氧化应激作出反应的生理活动。首先，存活试验表明 Grx1 缺陷型 HeLaS3 细胞对 γ 射线照射、热休克和 H ₂ O ₂更敏感暴露于 HeLaS3 野生型细胞。接下来，使用荧光探针（2'-7'二氯荧光素二乙酸盐）研究细胞内氧化还原状态，并通过蛋白质印迹分析测量总蛋白质和过氧化物酶 Prx2 的氧化状态。暴露于 γ 射线辐射、热休克和 H ₂ O ₂在 Grx1 缺陷的 HeLaS3 细胞中，显着诱导更多的细胞内氧化剂积累，包括 ROS 和更高水平的氧化蛋白。此外，MitoSox Red 染色表明 Grx1 缺乏会导致线粒体中产生更高水平的氧化剂。此外，Grx1 缺陷的 HeLaS3 细胞在氧化应激下具有更高的细胞色素 c 水平和更高的细胞凋亡率（Annexin-V/FITC 和 EthD-III 染色测定）。这些结果表明 Grx1 缺乏导致线粒体氧化还原稳态破坏和氧化应激下的细胞凋亡。此外，增殖测定和 MitoTracker 染色测定（多核细胞形成率）的结果表明，氧化应激暴露可能通过影响 Grx1 缺陷型 HeLaS3 细胞的细胞质分裂来抑制细胞增殖。