Integrative multi-omics and single-cell dissection of the immune-fibrotic microenvironment in Duchenne muscular dystrophy reveals therapeutic potential of hesperidin.

BACKGROUND: Duchenne muscular dystrophy (DMD) is a severe pediatric, X-linked muscle-wasting disorder marked by chronic inflammation, incomplete regeneration, and progressive fibrosis. The immune-fibrotic microenvironment and its regulatory targets remain insufficiently defined. We sought to identify hub genes central to DMD pathology, delineate their functional and immune associations, and evaluate the therapeutic potential of hesperidin.

METHODS: Bulk transcriptomes from GEO GSE38417 were analyzed with Limma to identify differentially expressed genes (DEGs). Weighted gene co-expression network analysis (WGCNA) defined disease-related modules, and intersecting these genes with GeneCards entries yielded potential candidates. Hub genes were prioritized using protein-protein interaction topology (MCC, MNC, EPC, Degree). Functional enrichment employed Gene Ontology (GO) and KEGG. Immune infiltration was quantified by single-sample GSEA. A mouse single-cell dataset (GSE273343) mapped hub-gene expression across cell types. Gene Set Enrichment Analysis (GSEA) profiled pathway associations. Hesperidin, nominated by network pharmacology, was docked against hub proteins and tested in vitro in TNF-induced inflammatory myoblasts using CCK-8, qRT-PCR, and Western blot.

RESULTS: We identified 2187 DEGs; integration with WGCNA and GeneCards highlighted five hub genes: SPP1, PTPRC, CD86, CXCL12, and COL1A1. Enrichment analyses implicated immune activation, extracellular-matrix organization, leukocyte transendothelial migration, focal adhesion, complement cascades, and PI3K/AKT signaling. Immune infiltration revealed increased macrophages, neutrophils, and T-cell subsets in DMD muscle, correlating with hub-gene expression. Single-cell analysis showed hub genes concentrated in immune cells, fibroblasts, and proliferating muscle stem cells, indicating a pathological niche of persistent inflammation and incomplete repair. Docking predicted strong binding between hesperidin and all five hub proteins (binding energy -6.9 to -10.1 kcal/mol). In vitro, hesperidin improved cell viability and significantly reduced TNF-induced upregulation of SPP1, PTPRC, CXCL12, and COL1A1 at protein and mRNA levels, with a modest decrease in CD86.

CONCLUSIONS: An integrative multi-omics framework identified five hub genes driving immune-fibrotic remodeling in pediatric DMD. Hesperidin attenuates inflammatory injury by downregulating these targets, supporting its promise as a candidate therapy to modulate the DMD muscle microenvironment.