Fig. 5

Psoralen promoted NRF2 expression and nuclear translocation and increased the production of antioxidants in SSCs by activating the AKT, GSK-3β, and NRF2. The Western Blotting data showed that the expression of NRF2 in SSCs significantly increased at 3 h and reached a peak at 6 h post-irradiation (a and b). Psoralen remarkably stimulated the expression of NRF2 in SSCs at 1 h, and these effects lasted for at least 48 h. Notably, psoralen also increased NRF2 expression in irradiated SSCs (a and b). In addition, irradiation suppressed the expression of KEAP1 in SSCs (a and b). No significant changes in KEAP1 in SSCs after psoralen treatment were observed. The addition of psoralen to irradiated SSCs strongly increased the NRF2 level but did not change the decrease in KEAP1 (a and b). Moreover, the Western Blotting data showed that irradiation significantly suppressed the phosphorylation of GSK-3β, while psoralen promoted the phosphorylation of GSK-3β in SSCs (a and b). Furthermore, the Western Blotting data showed that psoralen significantly decreased cytoplasmic NRF2 but increased nuclear NRF2 in a time-dependent manner (c and d). A specific chemical inhibitor of AKT, LY294002 (5 μM), significantly decreased GSK-3β phosphorylation and NRF2 expression in the presence or absence of psoralen (e). The mRNA expression and protein levels of GCLM and NQO1 were significantly induced, while that of HOMX1 was inhibited by psoralen in SSCs (f–h). ML385, a specific chemical inhibitor of NRF2, significantly suppressed the expression of osteogenic genes, including Runx2, Mepe, and Dmp1, in SSCs in the presence of psoralen (i). All data are shown as the mean ± SD. **P < 0.01, *P < 0.05. SSC: skeletal stem cell; IR: irradiation; and Pso: psoralen