Selective effects of apocynin and diphenyleneiodonium on COL17 expression and cellular aging in keratinocytes

Summary: Abstract Body: Background: Apocynin, a potent NADPH oxidase inhibitor, has been reported to induce collagen 17 (COL17), a key protein associated with skin health. UV radiation accelerates cellular senescence via oxidative stress and DNA damage, contributing to skin photoaging, while aging is linked to reduced COL17 expression and increased senescence. This study hypothesized that apocynin induces COL17 through its NADPH oxidase inhibitory effect. Methods: hTert-immortalized keratinocytes (KER-CT) were pretreated with apocynin or diphenyleneiodonium (DPI) (10, 20, and 40 µM) for 24 hours prior to UVB exposure (100 J/m^2). Klotho and wild-type mice were administered apocynin in drinking water (2.5 g/l/kg) for 5 weeks. COL17, p16, and γ-H2AX levels were assessed via immunofluorescence; p16, p21, p53, LaminB1, and PAI-1 were quantified by qRT-PCR; and NOX4, p16, γ-H2AX, pP38, p65, and MMP-9 were analyzed using western blot. Cellular senescence was evaluated through SA-β-gal staining and cell cycle analysis with propidium iodide/RNase. Results: UVB exposure decreased COL17 expression while increasing NOX4, pP38, p65, MMP-9, γ-H2AX, and senescence markers (SA-β-gal, PAI-1), alongside reducing LaminB1 and causing cell cycle arrest. Apocynin reversed these effects by restoring COL17, reducing NOX4, pP38, p65, MMP-9, γ-H2AX, and senescence markers, increasing LaminB1, and alleviating cell cycle arrest may be through oxidative stress reduction. In vivo, apocynin upregulated COL17 and reduced senescence markers in Klotho mice. Conversely, DPI reduced DNA damage and p16 expression but did not impact COL17 or other senescence markers. Conclusions: Apocynin induces COL17 expression independently of NADPH oxidase inhibition, suggesting alternative mechanisms of action. Tuba M. Ansary¹, Koji Kamiya¹, Md Razib Hossain¹, Makoto Kuro-o², Mayumi Komine¹ 1. Dermatology, Jichi Ika Daigaku, Shimotsuke, Tochigi Prefecture, Japan. 2. Division of Anti-aging Medicine, Jichi Ika Daigaku, Shimotsuke, Tochigi Prefecture, Japan. UV Biology/Injury and Non-melanoma Cancers