Hesperetin Protects Against UVB-Induced Skin Damage

Cisd2KO mice were generated as previously described [25 (link)]. All mice used in this study are males with pure or congenic C57BL/6 backgrounds. All mice were bred and housed in a specific pathogen-free facility at a constant room temperature (20–22 °C) with a 12 h light and 12 h dark cycle (7 a.m–7 p.m.). For the anti-aging study, the mice were fed ad libitum with AIN-93G (TestDiet, St. Louis, MO, USA) diet mixed with hesperetin (0.07% [w/w]; Sigma-Aldrich, H4125; 100 mg/kg/day) or mixed with vehicle (3.04% propylene glycol [w/w]; Sigma-Aldrich, 16033). To evaluate the protective effect of hesperetin on UVB-induced skin damage, the mice were treated with hesperetin (30 mg/kg/day) or vehicle by a feeding tube. For the UVB treatment, the UVB apparatus consisted of four UVB lamps (G4T5E, SANKYO DENKI, Hiratsuka, Kanagawa, Japan); the spectral wavelength range of the UVB lamps was 280–360 nm, and peak light source intensity was 306 nm. Mice under anesthesia were placed individually in a plastic box with UVB lamps; the fluence of UVB on the mouse dorsal surface was 349 mJ/cm² for 75 s [31 (link), 32 (link)]. Mice were treated with hesperetin or vehicle for 7 days before UVB irradiation followed by UVB irradiation for 5 consecutive days as hesperetin or vehicle treatment continued. The dorsal skins were dissected two days after the final UVB treatment. After each specific treatment, the mice were sacrificed by CO₂ inhalation, which is a humane method of euthanasia. The animal protocol was approved by the Institutional Animal Care and Use Committee (No. 1040103) of National Yang Ming Chiao Tung University. The animal protocols were designed to follow the associated guidelines and the 3R principles (Replacement, Reduction and Refinement) in accordance with the “Animal Protection Act” of Taiwan.