Bim gene regulation in hypoxic stress response of blunt snout bream (Megalobrama amblycephala): Mechanisms of apoptosis, oxidative stress, and transcriptional control by c-Ets-2

Abstract

Despite its significant economic value, the blunt snout bream (Megalobrama amblycephala) is extremely sensitive to hypoxia. Derived from blunt snout bream gill filament cell lines, Megalobrama amblycephala gill (MAG) cells are closely linked to hypoxia, making them suitable for identifying related genes' hypoxia tolerance. This study explores the roles of the Bcl-2 interacting mediator of cell death gene (Bim) in the gill tissues and MAG cells under hypoxic conditions. Bim expression increased significantly (P < 0.05) after 24 h of hypoxia but decreased following reoxygenation. Overexpression of Bim significantly (P < 0.05) upregulated the expression of pro-apoptotic genes Bax and Caspase 3, while downregulating the expression of the anti-apoptotic gene Bcl-2. Conversely, Bim interference exhibited an opposite trend. Hypoxia led to increased apoptosis and decreased mitochondrial membrane potential (MMP) in MAG cells, which was exacerbated by overexpression of Bim. In contrast, Bim interference attenuated apoptosis and prevented the decrease in MMP. ROS levels significantly (P < 0.05) increased under hypoxic conditions, and Bim overexpression further elevated ROS production, whereas Bim interference reduced ROS levels. Antioxidant enzyme (SOD, CAT) activity decreased after hypoxia, which was exacerbated by Bim overexpression, while Bim interference slowed the decline of enzyme activity. The dual-luciferase reporter assay confirmed that the transcription factor c-Ets-2 regulates the expression of Bim by binding to the -GAGGAA site of the Bim promoter. This study highlights Bim's key role in hypoxic stress and offers new insights into the hypoxic adaptation mechanisms of blunt snout bream.