Method: Mouse salivary glands and cultured human salivary epithelial cells were irradiated by single 15Gy dose. Hedgehog pathway was transiently activated in mouse salivary glands by shortly over-expressing Sonic hedgehog (Shh) transgene or administrating Smoothened Agonist and in human salivary epithelial cells by infecting with adenovirus encoding Gli1. Activity of Hedgehog signaling was examined by expression of Ptch1-lacZ reporter and endogenous Hedgehog target genes. Salivary flow rate was measured following pilocarpine stimulation. Salivary stem/progenitor cells (SSPCs), parasympathetic innervation and expression of related genes were examined by flow cytometry, salisphere assay, IHC, quantitative RT-PCR, Western blot and ELISA.
Result: Irradiation does not activate Hedgehog signaling in mouse salivary glands. Transient Shh over-expression activated Hedgehog pathway in ductal epithelia and that after irradiation rescued salivary function in male mice, which is related with preservation of functional SSPCs and parasympathetic innervation. The preservation of SSPCs was likely mediated by rescue of signaling activities of Bmi1 and Chrm1/HB-EGF pathways. The preservation of parasympathetic innervation was related with rescue of expression of neurotrophic factors such as Bdnf and Nrtn. The expression of genes related with maintenance of salivary stem/progenitor cells and parasympathetic innervation in female salivary glands and cultured human salivary epithelial cells was similarly affected by irradiation and transient Hedgehog activation.
Conclusion: These findings suggest that transient activation of Hedgehog pathway has the potential to restore irradiation-induced salivary gland dysfunction.