Abstract: 【Abstract】 Objective To investigate the effects of chronic psychological stress on atopic dermatitis （AD）-like skin inflammation, and to elucidate the potential mechanisms from the perspectives of lipid metabolism and skin immune molecular changes. Methods Male C57BL/6 mice aged 6 - 8 weeks were randomly assigned to 4 groups （6 mice in each group）: a control group, a chronic unpredictable stress group （CUS group）, a calcipotriol group, and a chronic unpredictable stress + calcipotriol group （CUS + calcipotriol group）. A chronic unpredictable stress mouse model was established from day 0 to day 14, and AD-like skin inflammation was induced by the topical application of calcipotriol from day 15 onwards. The open-field test was used to assess the time spent in the center area and total travel distance. Skin lesion severity was dynamically evaluated using a dermatitis severity score, and plasma corticosterone levels were detected by enzyme-linked immunosorbent assay. Histopathological changes were observed, and epidermal thickness was measured. Flow cytometry was performed to assess immune cell infiltration in skin lesions. The relative mRNA expression levels of interleukin-4 （IL-4） and thymic stromal lymphopoietin （Tslp） were determined by real-time quantitative PCR （qPCR）. Untargeted plasma lipidomics analysis was performed using liquid chromatography-mass spectrometry. In addition, bulk RNA sequencing was conducted on lesional mouse ear skin tissues, followed by differential gene expression and pathway enrichment analyses. Spearman rank correlation analysis was conducted to assess the correlation between differential lipids and the expression of key genes. Statistical analyses were performed using the t test, Mann-Whitney U test, and analysis of variance （ANOVA）. Changes in lesion severity scores over time were analyzed using a two-way repeated-measures ANOVA. Results As the open-field test showed, compared with the control group, the CUS group exhibited reduced time spent in the center area （7.20 ± 1.60 s vs. 19.53 ± 1.13 s, t = 15.43, P < 0.001） and decreased total travel distance （10 649.00 ± 761.90 mm vs. 15 848.00 ± 1 513.00 mm, t = 7.52, P < 0.001）, along with elevated plasma corticosterone levels （55.66 ± 7.26 ng/ml vs. 20.57 ± 6.38 ng/ml, t = 8.90, P < 0.001）. Two-way repeated-measures ANOVA of lesion severity scores revealed a significant interaction between time and group （F = 61.67, P < 0.001）, and the main effects of both time and group were also statistically significant （both P < 0.001）, indicating the successful establishment of the stress model. No significant differences in phenotype or epidermal thickness were observed between the control group and the CUS group; compared with the calcipotriol group, the CUS + calcipotriol group exhibited more severe skin lesions, greater epidermal thickening （153.20 ± 10.08 μm vs. 106.30 ± 5.06 μm, q = 14.41, P < 0.001）, and more pronounced immune infiltration, as evidenced by an increased proportion of CD45? cells among live cells in skin lesions （q = 8.28, P < 0.001）; among these infiltrating immune cells, the proportion of neutrophils was significantly higher （t = 17.08, P < 0.001）, but that of dendritic cells was significantly lower （t = 3.35, P = 0.007） in the CUS + calcipotriol group than in the calcipotriol group, while no significant differences were observed in the proportions of CD4? T cells or macrophages between the two groups （both P > 0.05）. qPCR showed that the relative mRNA expression levels of IL-4 and Tslp in skin lesions were significantly higher in the CUS + calcipotriol group than in the calcipotriol group （both P < 0.05）. Lipidomics analysis demonstrated clear separation of lipid profiles between the calcipotriol group and the CUS + calcipotriol group, with significant alterations in multiple glycerophospholipid and sphingolipid species. Specifically, the levels of phosphatidylserine （18∶0/20∶4）, phosphatidyl-inositol （16∶0/16∶1）, and ceramide （18∶0/24∶1） were decreased after stress exposure, whereas several inflammation-related lipids, including lysophosphatidic acid （18∶2）, lysophosphatidyl-ethanolamine （16∶0）, and lysophosphatidylcholine （18∶4）, were increased. Differential lipids were enriched in glycerophospholipid metabolism and multiple inflammation-related signaling pathways. Transcriptomic analysis showed clear separation of transcriptional profiles between the two groups, with significant enrichment of pathways related to leukocyte chemotaxis, inflammatory responses, IL-4 response, and skin barrier function. Spearman rank correlation analysis suggested correlations between key genes and differential lipid species. qPCR further demonstrated downregulated expression of lipid metabolism-related genes and upregulated expression of inflammation-related genes in skin lesions in the CUS + calcipotriol group compared with the calcipotriol group （all P < 0.05）. Conclusion Chronic psychological stress could significantly aggravate AD-like skin inflammation, accompanied by abnormalities in lipid metabolic profiles and remodeling of the skin immune microenvironment, suggesting that the “neuroendocrine-lipid metabolism-immune inflammation” axis may be involved in the stress-associated exacerbation of AD.

Key words: Dermatitis, atopic, Stress, psychological, Lipidomics, Transcriptomics, Skin immune microenvironment