Protective role of DDIT4-mediated autophagy in dermal papilla cells after oxidative stress injury

Abstract

Objective To explore the protective role of DNA damage induced transcription factor 4 (DDIT4) in oxidative stress injury in dermal papilla cells and its underlying mechanisms. Methods Dermal papilla cells were exposed to UVA and H2O2 to establish cellular model of oxidative stress. CCK-8 assay was used to detect cell viability under different treatment conditions, and the production of intracellular reactive oxygen species (ROS) was detected using 2′, 7′-dichlorofluorescein diacetate (DCFH-DA). Autophagic vesicles were observed with electron microscopy. Western blotting was employed to measure the expression of DDIT4 and autophagy-related molecules, including microtubule-associated protein 1 light chain 3 (LC3), ubiquitin-binding protein (P62), mammalian target of rapamycin (mTOR), and p-mTOR. Results UVA and H2O2 resulted in more production of ROS (P < 0.05) and decreased viability of dermal papilla cells (P < 0.05). DDIT4 expression was increased in dermal papilla cells under oxidative stress (P < 0.05), and the antioxidant N-acetyl-L-cysteine (NAC) could effectively inhibit this effect (P < 0.05). After treatment with UVA or H2O2, cell autophagy was enhanced in dermal papilla cells, characterized by an increase in the number of autophagosomes and an increased LC3Ⅱ/Ⅰ ratio (P < 0.05), a decrease in P62 expression (P < 0.05), and 3-methyladenine (3-MA) blocking autophagy led to further reduced cell viability (P < 0.05) and increased intracellular ROS production (P < 0.05). Conversely, rapamycin (RAPA) increased autophagy level and improved the viability of dermal papilla cells under oxidative conditions (P < 0.05), and reduced the generation of intracellular ROS (P < 0.05). Additionally, down-regulation of DDIT4 weakened autophagy in dermal papilla cells under oxidative stress, reduced LC3Ⅱ/Ⅰ (P < 0.05), increased p-mTOR/mTOR and P62 (P < 0.05), inhibited cell viability (P < 0.05), and enhanced intracellular ROS production (P < 0.05). Conclusion DDIT4 may alleviate oxidative stress injury in dermal papilla cells through autophagy.

Keywords

• dna damage induced transcription factor 4
• autophagy
• oxidative stress
• dermal papilla cells