3.8 ISX9 promotes hair regrowth in mice via the activation of Wnt/β-catenin signaling
The Wnt/β-catenin pathway has been shown to play a crucial role in the development of new hair follicles and initiation of hair growth. We next evaluated the effect of ISX9 on the hair regrowth in mice. Minoxidil was exploited as a positive control. ISX9 and minoxidil were topically applied daily to the shaved dorsal skin of 7-week‐old C57BL/6 mice. On day 14 and day 21, ISX9 was more effective than minoxidil in promoting hair regrowth in mice. After treatment for 25 days, both ISX9 and minoxidil exhibited strong hair regrowth-promoting activity compared to control mice (Figure 6A and B). Similar results were obtained by measuring the weight of freshly grown hair (Figure 6C). Histopathology with H&E staining showed significantly increased numbers of hair follicles in the anagen phase on day 25 in ISX9 and minoxidil group compared with that in control mice (Figure 6D), while hair follicles in control mice were still in the telogen phase. Consistently, quantitative analysis of hair follicles also confirmed that the number of hair follicles were markedly increased in ISX9 and minoxidil-treated group compared with control mice (Figure 6E). These results suggest that treatment with ISX9 or minoxidil induces hair follicle transition from telogen to anagen.
We next examined the effect of ISX9 on the Wnt/β-catenin pathway in mice. The results of immunohistochemical staining indicated that ISX9 remarkedly increased the expression of active β-catenin, total β-catenin and the Wnt signaling target gene LGR5, concomitant with expression of the proliferation marker Ki-67 (Figure 6F). Moreover, real‐time PCR analysis showed that ISX9 treatment significantly upregulated the expression of Wnt target genes (LEF1, Axin2, Fibronectin and Survivin) and stemness marker genes (LGR5, Twist1, SOX2 and OCT4) (Figure 7A and B). Consistent with the above results, ISX9-treated mice exhibited obviously increased protein levels of active β-catenin, total β-catenin, LEF1, Axin2, Survivin, LGR5, and SOX2, while no apparent differences in protein levels of phosphorylated LRP6, total LRP6, CK1ε, phosphorylated GSK3β and total GSK3β were observed in ISX9, minoxidil and control groups (Figure 7C). Interestingly, we noted that Keratin17 (Waters, Richardson, & Jahoda, 2007), a known-marker of cells with stem cell properties in hair follicle, was also upregulated in response to ISX9 treatment (Figure 7C).