Allantoin Enhances Proliferation, Wound Closure, and Nrf2-Associated Antioxidant Defense in Mouse Embryonic Palatal...

Cleft palate is a congenital craniofacial anomaly that originates from disrupted palatogenesis. During palatal shelf morphogenesis, palatal mesenchyme must expand, remodel, and withstand environmental stress, and oxidative imbalance is considered a convergent cellular risk that can compromise developmental robustness. Allantoin is a clinic-friendly small molecule widely used in topical formulations and reported to modulate cell behavior, but its dose-dependent effects and antioxidant mechanism in embryonic palatal mesenchyme remain unclear. Here, we investigated the concentration window and Nrf2-related cytoprotective mechanism of allantoin in mouse embryonic palatal mesenchymal (MEPM) cells.

MEPM cells were isolated from E14.5 mouse palatal shelves and treated with allantoin (0.5-5 mg/mL). Cytotoxicity and time-dependent growth were assessed by CCK-8 and growth index analysis. Mesenchymal motility was evaluated using a scratch assay. For cytoprotection, cells were pretreated with allantoin for 24 hours and challenged with 0.1% (w/v) H2O2 for 2 hours, followed by Annexin V-FITC/7-AAD flow cytometry. Osteogenic and chondrogenic differentiation were induced for 21 days with lower-dose allantoin (0-2 mg/mL) and assessed by Alizarin Red S and Alcian Blue staining with ImageJ quantification. Nrf2 pathway activation was analyzed by RT-qPCR and Western blotting (Nrf2, HO-1, NQO1), and pharmacological inhibition was performed with ML385.

Allantoin was noncytotoxic across 0.5 to 5 mg/mL and enhanced early proliferative capacity, with the strongest growth-promoting effect observed at intermediate concentrations. Allantoin increased wound closure, indicating improved mesenchymal motility. Under oxidative challenge, allantoin reduced apoptosis and restored viability with maximal protection at 3 to 4 mg/mL. Mechanistically, allantoin upregulated Nrf2 and its downstream targets HO-1 and NQO1 at both mRNA and protein levels, while ML385 partially attenuated these responses. In contrast, higher concentrations during lineage induction (1-2 mg/mL) tended to suppress osteogenic mineralization and chondrogenic matrix formation.

Allantoin supports palatogenesis-relevant MEPM cell behaviors, including early growth, mesenchymal motility, and resilience to oxidative stress, within an optimal concentration window (∼3-4 mg/mL). Allantoin's cytoprotective effects are associated with activation of the Nrf2/HO-1/NQO1 antioxidant program. Given the dose-sensitive suppression of osteo-/chondrogenic differentiation at higher exposure, future applications should prioritize dose- and time-controlled designs and validate findings in palatal shelf organ culture and embryonic palatogenesis models.