Effect of kirenol on the interaction between the WNT/β-Catenin and RUNX2/TCF/LEF1 pathways in fracture healing in vivo

Objective: This study aimed to determine the effects of a natural diterpenoid, kirenol, on fracture healing in vivo in an experimental rat model of femur fracture and investigate its potential mechanism of action via the Wnt/beta-catenin pathway. Methods: In this study, 64 male Wistar albino rats aged 5-7 weeks and weighing 261-348 g were randomly divided into 8 groups from A to L, with eight rats in each group. Standardized fractures were created in the right femurs of the rats and then fixed with an intramedullary Kirschner wire. Four experimental groups were administered 2 mg/kg/ day kirenol (Groups C and G) and 4 mg/kg/day (Groups D and H) kirenol by oral gavage.Thereafter, the animals were sacrificed at two time points as follows: on the 10th day (Groups B, C and D) and on the 21 day (Groups 1 7 , G and H) after the surgery; fracture healing in each group was assessed radiologically and histopathologically. The Radiographic Union scale of tibia fracture scoring system was used in the radiological examination; callus volume and density were measured using computed tomography. In the histopathologic examination, the scoring system described by Huo et al. was used. Additionally, the mechanism of action was evaluated based on the analyses of protein expression of Wnt3a, LRP5, TCF -LEF1, beta-catenin, and Runx-2 proteins using western blot analysis. Results: Among the animals sacrificed on the 10th day after the surgery, the highest histopathological and radiological scores were observed in Group D (p<0.05). Furthermore, the callus density (p<0.05) was highest in Group D. Among the animals sacrificed on the 21st day, the highest histopathological and radiological scores were found in Group H, although the differences among the groups were not significant (p>0.05). The callus volume and density were the highest in Groups G and H, respectively, although the differences among groups were not significant. Conclusion: Kirenol may improve fracture healing in a dose-dependent manner with the early activation of the Wnt/beta-catenin pathway and the activation of the Runx-2 pathway.