Differential Modulation of Keratinocyte Proliferation, Differentiation, and Barrier Function by Hemp Oil, Blackcurrant Seed Oil, and Vitamin D ₃ Under Atopic‐Associated Cytokine Stimulation

ABSTRACT

Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by keratinocyte hyperproliferation, altered differentiation, barrier dysfunction, and oxidative stress. Currently available therapies, including monoclonal antibodies and JAK inhibitors, have improved AD management but do not address all aspects of the disease, supporting the investigation of natural‐product strategies as complementary approaches to long‐term care. An acute in vitro model of canine atopic‐like inflammation was established by exposing canine progenitor epidermal keratinocytes (CPEK) for 24 h to a defined cytokine cocktail (IFN‐γ, IL‐4, IL‐13), followed by 24 h of treatment with hemp oil, blackcurrant seed oil, vitamin D ₃ , or their combination. Proliferation (Ki‐67, cell‐cycle), differentiation (KRT5, KRT10, TGM1, involucrin), tight‐junction organization (CLDN1, TJP1, ZO‐1, TEER), wound closure, nitrosative stress (3‐nitrotyrosine), the NRF2/BACH1/HMOX1 axis, and the secretion of STAT1‐ and NF‐κB‐dependent inflammatory mediators (CXCL9, CXCL10, IL‐8, IL‐6) and the STAT6‐targeted chemokine CCL17 were assessed. The cytokine cocktail induced a coherent AD‐like phenotype: increased Ki‐67, downregulated CLDN1, sustained nitrosative stress, NRF2 elevation paralleled by BACH1 induction, and robust secretion of STAT1‐ and NF‐κB‐dependent mediators. The four treatments modulated this phenotype according to clearly differential, pathway‐specific profiles. Vitamin D ₃ , alone or combined, emerged as the most effective modulator of proliferation and acted preferentially on the downstream redox arm, inducing HMOX1 and reducing BACH1; both vitamin D ₃ ‐containing formulations exerted broad anti‐inflammatory activity across the STAT1 and NF‐κB axes. Hemp oil acted preferentially on tight‐junction integrity (CLDN1 recovery), on the upstream NRF2 arm (NFE2L2 induction), on the STAT1 chemokine arm (CXCL9, CXCL10 reduction), and on wound closure. Blackcurrant seed oil acted preferentially on NF‐κB‐dependent IL‐8 and on early wound‐closure dynamics. This differential pharmacological footprint provides a rational basis for further investigation of these formulations as nutraceutical adjuncts in canine AD.