Kushe Tincture Ameliorates DNCB-Induced Atopic Dermatitis by Affecting NF-Kb/JAK-STAT3 Pathway: Bioinformatics Analysis and Animal Experiment Verification

Background: Kushe tincture (KSD), an optimized in-hospital preparation of Dongzhimen Hospital of Beijing University of Traditional Chinese Medicine, has been clinically used as an anti-inflammatory and anti-itching topical therapeutic drug for the treatment of eczema, atopic dermatitis (AD) and so on for decades. However, the potential therapeutic mechanisms remained unexplored. Objective: This study aimed to explore the mechanisms underlying the therapeutic effects of KSD against DNCB-induced AD through the combination of single-cell RNA sequencing (scRNA-seq), spatial transcriptomics (ST), and animal experiments. Methods: Firstly, we collected the active components and targets of KSD from the Traditional Chinese Medicine Systems Pharmacology (TCMSP) and obtained AD-associated targets from the Gene Expression Omnibus (GEO) database. Protein-protein interaction (PPI) networks were constructed using Cytoscape with Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. Secondly, core targets were screened and visualized by scRNA-seq and ST technology. Finally, we adopted DNCB-induced AD-like mouse model to observe the therapeutic effect of KSD through lesion scoring, morphological staining, serological tests, skin barrier function measurements, flow cytometry, and Western blotting. Results: Forty-six common drug-disease targets were obtained and top 2 ranked targets were chosen, namely NF-kB1 and STAT3. Combined scRNA-seq and ST suggested that NF-KB and STAT3 had higher and wider expression in most subpopulations, especially in monocytes and CD4+ T-cells. Animal experiments showed that KSD treatment improved the visible symptoms and skin barrier function and decreased mast cell infiltration, IgE levels, Th17 cells, and expression of NF-kB, JAK2, and STAT3. Conclusion: KSD has therapeutic effects on DNCB-induced AD-like skin lesions, and it has effects on NF-kB and JAK-STAT3 proteins, which may be possible targets.