Chinese Journal of Dermatology ›› 2023, Vol. 56 ›› Issue (9): 857-861.doi: 10.35541/cjd.20220821

• Research Reports • Previous Articles     Next Articles

Protective effects of isorhamnetin against H2O2-induced mitochondrial structural and functional damage in HaCaT cells

Dai Leheng, Hu Wen, Zhang Kunjie, Kang Xiaojing   

  1. Department of Dermatology and Venereology, People′s Hospital of Xinjiang Uygur Autonomous Region, Xinjiang Clinical Research Center for Dermatologic Diseases, Xinjiang Key Laboratory of Dermatology Research(XJYS1707), Urumqi 830001, China
  • Received:2022-11-21 Revised:2023-02-09 Online:2023-09-15 Published:2023-09-07
  • Contact: Kang Xiaojing
  • Supported by:
    National Natural Science Foundation of China(82173406)

Abstract: 【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, H2O2 group treated with 600 μmol/L H2O2 , and isorhamnetin + H2O2 group pretreated with 60 μmol/L isorhamnetin for 12 hours, followed by medium replacement and 12-hour treatment with 600 μmol/L H2O2 . 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 H2O2 . Compared with the control group, the H2O2 group showed significantly increased ROS levels (10 725.0 ± 845.8 vs. 1 708.0 ± 69.4, t = 18.4, 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 = 0.01, 0.01, 0.003, respectively). After the pretreatment with isorhamnetin followed by H2O2 treatment, the isorhamnetin+ H2O2 group showed significantly decreased ROS levels (7 640.0 ± 922.7) compared with the H2O2 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 H2O2 group (t = 4.59, 4.58, 5.61, P = 0.01, 0.01, 0.005, respectively). Under the electron microscope, the mitochondrial structure was clearer and more complete in the isorhamnetin+ H2O2 group than in the H2O2 group; there was no swelling or slight swelling of mitochondrial cristae, as well as no vacuolization of mitochondria in the isorhamnetin+ H2O2 group; in addition, autophagosomes engulfing damaged mitochondria were observed in the isorhamnetin+ H2O2 group. Conclusion Isorhamnetin may reduce ROS levels by inducing autophagy, and has a protective effect against the H2O2-induced mitochondrial structural and functional damage in HaCaT cells.

Key words: Vitiligo, Oxidative stress, Mitochondria, Reactive oxygen species, Membrane potential, mitochondrial, Adenosine triphosphate, Isorhamnetin, HaCaT cells