Mangiferin-loaded Lactobacillus rhamnosus extracellular vesicles attenuate doxorubicin-induced mesenchymal stem cell senescence and preserve osteogenic and chondrogenic differentiation via multi-module protein cargo remodeling

Chemotherapy-induced mesenchymal stem cell (MSC) senescence impairs tissue repair and limits regenerative therapies. Probiotic-derived extracellular vesicles (EVs) have emerged as biocompatible nanocarriers, yet the molecular determinants of their therapeutic efficacy remain poorly defined.Culturing Lactobacillus rhamnosus PB LR76 with mangiferin (MGF) loads MGF into extracellular vesicles (EVs, 2.66% w/w) and remodels their proteome, generating multimodule anti senescence cargo. PB LR76 MGF EVs (50 μg/mL protein, 1.33 μg/mL MGF) significantly attenuated doxorubicin induced mesenchymal stem cell (MSC) senescence: they reduced ROS, DNA damage (γ-H2AX), and p53/p21 activation, while preserving mitochondrial membrane potential. In an eight group, three strain comparison (PB LR76, probiotic LGG, nonprobiotic ATCC 4356), only probiotic strain EVs preserved osteogenic and chondrogenic differentiation (Alizarin Red S/Alcian Blue; Runx2, ALP, COL1A1, OSX, ACAN, COL2A1, SOX9), confirming strain specificity. DIA Hyper quantitative proteomics (549 proteins, n = 3) identified 134 differentially abundant proteins enriched in PB LR76 MGF EVs, coordinated across four functional modules: redox/stress proteins (thioredoxin, NADH dehydrogenase FC = 30.8), six glycolytic moonlighting enzymes, DnaK GrpE/GroEL chaperones, and metabolic/ribosomal moonlighting proteins. PubMed cross referencing mapped 21 of 26 probiotic EV effector categories, 11 enriched in PB LR76 MGF EVs, to 10 human pathways, including TLR2/NF-κB, integrin/FAK survival, and NAD+/sirtuin dependent p53 deacetylation. These findings establish a proteome to phenotype framework for probiotic EV bioactivity and position PB LR76 MGF EVs as a food grade nanotherapeutic strategy against chemotherapy induced tissue damage.