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).