Protective effect of citrus flavone against acute hypoxia in cultured human intestinal smooth muscle cells and the mechanism

Abstract

Objective To investigate the protective effect of citrus flavone on cultured human intestinal smooth muscle cells (HISMCs) against acute hypoxia and explore the mitochondrial mechanism. Methods Cultured HISMCs were exposed to acute hypoxia with 1%, 3%, or 5% O2 (5% CO2, 94% N2) for 6 h or with 1% O2 for 3, 6, and 9 h. The cells cultured in 1% O2 (5% CO2, 94% N2) were treated with citrus flavonoids (300 or 600 μg/mL) for 6 h. Mitochondrial membrane potential and the cell apoptotic rate were determined with flow cytometry, and the activities of caspase-3 and caspase-8 were detected using Ac-DEVD-pNA and AC-IETD-pNA colorimetry, respectively. The total content of TR3 nuclear orphan receptor (TR3) in the hypoxic cells and its content in the mitochondria were determined with Western blotting. We also tested the effect of TR3 overexpression on the protective effect of 600 μg/mL citrus flavonoids in the HISMCs exposed to 1% O2. Results Acute hypoxia resulted in significantly decreased mitochondrial membrane potential, enhanced caspase-3 and caspase-8 activities, and increased apoptosis in cultured HISMCs (P < 0.05). Citrus flavonoids effectively enhanced mitochondrial membrane potential level, suppressed the mitochondrial apoptotic pathway (P < 0.05), and inhibited the translocation of TR3 to the mitochondria in the hypoxic cells. Overexpression of TR3 significantly attenuated the protective effect of citrus flavonoids on the hypoxic cells (P < 0.05). Conclusion Citrus flavonoids protect intestinal smooth muscle cells against hypoxic injury by regulating mitochondrial translocation of TR3.

Keywords

• citrus flavonoids
• hypoxia
• intestinal smooth muscle cells
• mitochondria
• apoptosis