Isorhamnetin protects melanocytes against CD8+ T cell-mediated apoptosis and reduces inflammation in vitiligo-afflicted mouse

Abstract

Background: Vernonia anthelmintica (L.) Willd., containing isorhamnetin (ISO) as an active compound, is a conventional treatment for vitiligo within the Xinjiang region. We previously demonstrated the protective role of ISO in mitigating oxidative damage within HaCaT cells, suggesting its therapeutic potential in treating vitiligo. However, its mechanism and effect on melanocytes immune damage remain unclear.

Aim: Here, we examined the effect of ISO on inflammation and apoptosis in melanocytes. Moreover, we comprehensively analyzed key genes in vitiligo treatment.

Methodology: We employed flow cytometry, RT-PCR, ELISA, and western blotting to detect apoptosis and the expression of inflammatory factors and apoptotic proteins. Hematoxylin-eosin staining assays were used to detect infiltration and inflammatory factors in the vitiligo mouse epidermis. We also used RNA sequencing to identify significantly dysregulated differentially expressed genes in vitiligo mice. Additionally, protein-protein interaction network analysis and validation of RNA-seq data were used to identify the core targets associated with vitiligo treatment. Finally, we investigated the mechanism by which ISO binds with four specific target proteins by performing molecular docking and core target and Notch signal function analyses.

Results and conclusion: CD8 + T cells significantly reduced melanocyte viability and increased apoptosis. However, ISO pretreatment improved cell viability and reduced apoptosis. ISO also downregulated proinflammatory cytokines (TNF-α, IFN-γ, IL-6, and IL-8) and chemokines (CXCL9 and CXCL10), as well as p53 and Bcl-2 expression at protein levels in vitiligo mouse models. RNA sequencing identified and molecular docking suggested key differentially expressed genes (PTK7, DLL1, IL21R, and CD226) as potential targets for treatment. KEGG analysis indicated that ISO plays a protective role through the Notch signaling pathway. Further functional experiments suggested that inhibition of PTK7/DLL1 and Notch signals reversed the apoptosis-protective effect of ISO on melanocytes, emphasizing its potential as a therapeutic option for vitiligo treatment.

Keywords: Apoptosis; CD8+ T cells; Isorhamnetin; Melanocyte; Vitiligo.