Background/PurposeToll-like receptors (TLRs) are important regulators of innate immunity, and TLR4 pathway can regulate the survival, migration, and differentiation of stem cells, including intestinal stem cells (ISCs). Deferoxamine (DFO), a hypoxia-mimic compound, can activate the proliferation of ISCs. In this study, we investigated the response of TLR4 signaling to DFO-induced hypoxia in cultured ISCs in vitro. MethodsAfter DFO treatment, the crypt organoid number was counted, and the expression levels of Lgr5, Hsp70, HMGB1, HIF-1α, TLR4, MyD88, TRIF, and TRAM in ISCs were examined using QPCR and Western blotting. The chemical inhibitors of different signaling molecules were then used to determine their role in DFO-induced change in ISCs. ResultsThe expression levels of Lgr5, HIF-1α, TLR4, MyD88, and TRIF in ISCs increased after DFO treatment, with peak expression of these molecules 6h after DFO treatment. In addition, DFO-induced gene expression of Lgr5 and HIF-1α was partially reversed by pretreatment with the inhibitor of TLR4 or MyD88, but not TRIF inhibitor. Inhibition of HIF-1α also resulted in partial downregulation of DFO-induced elevation of Lgr5 and TLR4. ConclusionsThese results demonstrated that DFO treatment activated HIF-1α and the TLR4-MyD88 signaling pathway, which might mediate the activation of ISCs.