3.10 | Cyclopamine prevents mechanical stress-induced LF fibrosis and hypertrophy in vivo
As stated previously, we built a rabbit model in which mechanical stress was concentrated on the level of L3-4 segment with fixation of adjacent segments (L2-3 and L4-5, Fig. 10A). We found that the L3-4 level LF in group B was more thicker than that in group A (Fig. 10B). In addition, it was shown that the structure of the hypertrophic LF in rabbits was almost similar to that of the LF in humans (Fig. 10C, D) (Hayashi et al., 2017). Those results demonstrated the degeneration and hypertrophy of LF under the condition with continuously mechanical stress. Briefly, the L3-4 level LF in Group B showed a significant decrease in the density of elastic fibres, the disruption of elastic fibres and an increase in collagen fibres (Fig. 10C, D). The levels of mRNA expression in each group were evaluated by RT-PCR. The expression of fibrosis-related genes on the L3-4 level LF in Group B, including WISP-1, Gli1, Col1a2, Col2a1, and Col3a1 was significantly elevated as compared with that on the same level in group A, whereas elastin expression was significantly decreased (p<0.01) (Fig. 10 E, F). These data indicated the activation of WISP-1/Hedgehog-Gli1 signaling in hypertrophic LF induced by mechanical stress in rabbit model, which were similar to the results observed in hypertrophic LF in patients with LSCS.
Moreover, our in vitro experiment suggested that cyclopamine suppressed WISP-1-induced fibrogenesis. Therefore, we proposed that Hedgehog signaling may serve as a therapeutic target for the prevention of LF fibrosis and hypertrophy. To confirm this hypothesis, the in vivo effect of cyclopamine was also analyzed in a rat LF hypertrophy model. Rat LF sections were evaluated using immunohistochemistry and RT-PCR. The results showed that LF thickness was significantly lower in cyclopamine-treated LF group (Group C) compared to the Group B, whereas the Group C showed approximately the same thickness as the group A (Fig. 10B). Cyclopamine treatment of LF in Group C attenuated the upregulation of fibrosis-related genes such as collagen, Gli1 and a-SMA, and the downregulation of elastin compared to the Group B (Fig. 10E, F). In addition to these changes of ECM component, we detected the cellular changes using our rabbit samples. The number of BrDu-positive proliferating cells was significantly higher in the Group B as compared with Group A, whereas the number of proliferating cells was reduced by cyclopamine-treated LF in Group C as compared with Group B (Fig. 10G). These data suggest that LF thickness is positively correlated with mechanical stress/WISP-1/Hedgehog-Gli1/a-SMA axis and Hedgehog-Gli1 signaling may serve as a therapeutic target for the prevention and treatment of LF fibrosis and hypertrophy.