Protective effects of isorhamnetin against H 2O 2-induced mitochondrial structural and functional damage in HaCaT cells / 中华皮肤科杂志

ABSTRACT

Objective:To evaluate protective effects of isorhamnetin on mitochondrial structure and function in HaCaT cells under oxidative stress.Methods:HaCaT cells served as the research object, and were divided into 4 groups: control group receiving conventional culture, isorhamnetin group treated with 60 μmol/L isorhamnetin, H 2O 2 group treated with 600 μmol/L H 2O 2, and isorhamnetin + H 2O 2 group pretreated with 60 μmol/L isorhamnetin for 12 hours followed by medium replacement and 12-hour treatment with 600 μmol/L H 2O 2. Flow cytometry was performed to detect cellular reactive oxygen species (ROS) levels, transmission electron microscopy to observe mitochondrial ultrastructure, confocal fluorescence microscopy to evaluate mitochondrial membrane potential, real-time fluorescence-based quantitative PCR (qRT-PCR) to determine the mitochondrial DNA copy number, and adenosine triphosphate (ATP) assay kit was used to determine the mitochondrial ATP content. One-way analysis of variance was used for comparisons among multiple groups, and least significant difference- t test for multiple comparisons. Results:Oxidative stress was provoked in HaCaT cells after the treatment with H 2O 2. Compared with the control group, the H 2O 2 group showed significantly increased ROS levels (10 725.0 ± 845.8 vs. 1 708.0 ± 69.4, t = 18.40, P < 0.001), but significantly decreased fluorescence intensity of mitochondrial membrane potential, mitochondrial ATP content, and expression of ND-1 (a characteristic gene of mitochondrial DNA) ( t = 4.58, 4.48, 6.11, P = 0.010, 0.010, 0.003, respectively). After the pretreatment with isorhamnetin followed by H 2O 2 treatment, the isorhamnetin+ H 2O 2 group showed significantly decreased ROS levels (7 640.0 ± 922.7) compared with the H 2O 2 group ( t = 4.27, P = 0.013), but significantly increased fluorescence intensity of mitochondrial membrane potential, mitochondrial ATP content, and expression of ND-1 compared with the H 2O 2 group ( t = 4.59, 4.58, 5.61, P = 0.010, 0.010, 0.005, respectively). Under the electron microscope, the mitochondrial structure was clearer and more complete in the isorhamnetin+ H 2O 2 group than in the H 2O 2 group; there was slight or no swelling of mitochondrial cristae, and no vacuolization of mitochondria in the isorhamnetin+ H 2O 2 group; in addition, autophagosomes engulfing damaged mitochondria were observed in the isorhamnetin+ H 2O 2 group. Conclusion:Isorhamnetin may reduce ROS levels by inducing autophagy, and has a protective effect against the H 2O 2-induced mitochondrial structural and functional damage in HaCaT cells.