Rutin targets PD-L1 for the treatment of atopic dermatitis: network pharmacological analysis and experimental evidence.

[BACKGROUND] Atopic dermatitis (AD) is a chronic inflammatory skin disease with high prevalence, recalcitrant nature, and significant socio-economic burden. Rutin, a bioactive flavonoid abundant in traditional medicinal plants, has documented ethnomedical uses in inflammatory conditions and exhibits antioxidant, cytoprotective, and anti-inflammatory properties. Evidence suggests that topical application of rutin can ameliorate the symptoms of atopic dermatitis. However, its underlying therapeutic mechanism remains elucidated, necessitating further in-depth investigation.

[AIM OF THE STUDY] To explore the potential mechanisms of rutin on AD through network pharmacology and experiments.

[MATERIALS AND METHODS] Potential targets and signaling pathways of rutin in AD treatment were identified via network pharmacology. In vitro, cell viability of rutin was evaluated using the CCK8 assay (Cell Counting Kit-8), and the expression of inflammatory factors was detected by RT-qPCR. In vivo, the therapeutic efficacy of rutin was assessed in calcipotriol (MC903)-induced AD-like mouse models. Immunofluorescence was performed to confirm PD-L1 as the core target. The binding interaction between rutin and PD-L1 was verified by Cellular Thermal Shift Assay (CETSA) and Surface Plasmon Resonance (SPR). Finally, shPD-L1 lentiviral infection of Normal Human Epidermal Keratinocytes (NHEK) was conducted to validate that rutin exerts anti-inflammatory effects through PD-L1.

[RESULTS] Network pharmacology identified PD-L1 signaling pathway as rutin's core pathway in AD treatment. In vitro, CCK-8 assay revealed that the IC₅₀ of rutin in NHEKs was 17.51 μM, 10 μM rutin significantly downregulated TSLP/CCL17 mRNA in TII-stimulated NHEKs. In vivo, 1.5% rutin cream best ameliorated MC903-induced AD-like lesions in mice, reducing EASI scores, scratching bouts, and epidermal thickness, comparable to desonide; IHC showed upregulated epidermal PD-L1. Target validation: immunofluorescence confirmed PD-L1 co-localization with cytokeratin 14 (CK14), CETSA enhanced PD-L1 stability, SPR verified specific binding (KD = 2.53 × 10 M). shPD-L1 lentiviral infection of NHEKs confirmed rutin's anti-inflammatory effect is PD-L1-dependent.

[CONCLUSION] In summary, this study elucidates a novel mechanism by which rutin ameliorates AD-like inflammation through direct binding and upregulation of PD-L1, leading to enhanced epidermal expression and subsequent immunomodulation. Moreover, compared to conventional medications, rutin effectively reduces TEWL and strengthens the skin barrier. These findings not only advance our understanding of rutin's pharmacological activity but also support the continued investigation of natural compounds as targeted therapeutic agents for inflammatory skin diseases. The convergence of traditional knowledge and modern mechanistic validation presented here underscores the enduring value of the ethnopharmacological approach to in drug discovery.