İbrahim KARAMAN, Ali Eray GÜNAY, Mükerrem Betül YERER, Eren DEMİRPOLAT, Serap DOĞAN, Arzu Hanım YAY, İBRAHİM HALİL KAFADAR

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/β-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 21st day (Groups F, 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, β-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

___

1. Einhorn TA. The cell and molecular biology of fracture healing. Clin Orthop 1998; 355: 7-21. [Crossref]

2. LaVelle DG. Delayed union and nonunion of fractures. In: Canale S.T. ed. Campbell’s Operative Orthopaedics (10th ed), Philadelphia, Mosby Publishers. 2003; pp.3126.

3. Nusse R, Varmus HE. Many tumors induced by the mouse mammary tumor virus contain a provirus integrated in the same region of the host genome. Cell 1982; 3: 99-109. [Crossref]

4. He S, Lu Y, Liu X, et al. Wnt3a: functions and implications in cancer. Chin J Cancer 2015; 34: 554-62. [Crossref]

5. McDonald BT, Tamai K, He X. Wnt/β-catenin signaling: components, mechanisms, and diseases. Dev Cell 2009; 17: 9-26. [Crossref]

6. Nusse R, Varmus H. Three decades of Wnts: a personal perspective on how a scientific field developed. EMBO J 2012; 31: 2670-84. [Crossref]

7. Byun MR, Hwang JH, Kim AR, et al. Canonical Wnt signalling activates TAZ through PP1A during osteogenic differentiation. Cell Death Differ 2014; 21: 854-63. [Crossref]

8. Shu B, Zhang M, Xie R, et al. BMP2, but not BMP4, is crucial for chondrocyte proliferation and maturation during endochondral bone development. J Cell Sci 2011; 124: 3428-40. [Crossref]

9. Lian JB, Javed A, Zaidi SK, et al. Regulatory controls for osteoblast growth and differentiation: role of Runx/Cbfa/AML factors. Crit Rev Eukaryot Gene Expr 2014; 14: 1-4. [Crossref]

10. Xiao J, Yang R, Yang L, et al. Kirenol attenuates experimental autoimmune encephalomyelitis by ınhibiting differentiation of Th1 and Th17 cells and inducing apoptosis of effector T cells. Sci Rep 2015; 5: doi: 10.1038/srep09022. [Crossref]

11. Jiang Z, Yu QH, Cheng Y, Guo XJ. Simultaneous quantification of eight major constituents in Herba Siegesbeckiae by liquid chromatography coupled with electrospray ionization time-offlight tandem mass spectrometry. J Pharm Biomed Anal 2011; 55: 452-7. [Crossref]

12. Wang JP, Zhou YM, Ye YJ, et al. Topical anti-inflammatory and analgesic activity of kirenol isolated from Siegesbeckia orientalis. J Ethnopharmacol 2011; 137: 1089-94. [Crossref]

13. Bi J, Xin HL, Lin ZF, et al. Effect of kirenol on cytokines in serum and apoptosis proteins expression in synoviocytes of adjuvant arthritis rats. Chinese Traditional and Herbal Drugs 2007; 38: 1207-10.

14. Kim MB, Song Y, Kim C, Hwang JK. Kirenol inhibits adipogenesis through activation of theWnt/β-catenin signaling pathway in T3-L1 adipocytes. Biochem Biophys Res Commun 2014; 445: 433-8. [Crossref]

15. Lua Y, Xiaob J, Wu ZW, et al. Kirenol exerts a potent anti-arthritic effect in collagen-induced arthritis by modifying the T cells balance. Phytomedicine 2012; 19: 882-9. [Crossref]

16. Kim MB, Song Y, Hwang JK. Kirenol stimulates osteoblast differentiation through activation of the BMP and Wnt/β-catenin signaling pathways in MC3T3-E1 cells. Fitoterapia 2014; 98: 59-65. [Crossref]

17. Qian RQ, Zhang CY, Fu HZ. Study on therapeutic mechanism of anti-rheumatism action of Herba siegesbeckiae. Chinese Journal of Integrated Traditional and Western Medicine 2002; 20: 192-5.

18. Augat P, Morgan EF, Lujan TJ, et al. Imaging techniques for the assessment of fracture repair. Injury 2014; 45: 16-22. [Crossref]

19. Huo MH, Troiano NW, Pelker RR, Gundberg CM, Friedlaender GE. The influence of ibuprofen on fracture repair: biomechanical, biochemical, histologic, and histomorphometric parameters in rats. J Orthop Res 1991; 9: 383-90. [Crossref]

20. Baron R, Kineissel M. WNT signaling in bone homeostasis and disease: from human mutations to treatments. Nat Med 2013; 19: 179-92. [Crossref]

21. Colburn NT, Zaal KJ, Wang F, Tuan RS. A role for gamma/delta T cells in a mouse model of fracture healing. Arthritis Rheum 2009; 60: 1694-703. [Crossref]

22. van der Horst G, van der Werf SM, Farih-Sips H, van Bezooijen RL, Löwik CW, Karperien M. Downregulation of Wnt signaling by increased expression of Dickkopf-1 and -2 is a prerequisite for late-stage osteoblast differentiation of KS483 cells. J Bone Miner Res 2005; 20: 1867-77. [Crossref]

23. Henderson CE, Lujan TJ, Kuhl LL, Bottlang M, Fitzpatrick DC, Marsh JL. Mid-America orthopaedic association physician in training award: healing complications are common after locked plating for distal femur fractures. Clin Orthop Relat Res 2011; 469: 1757-65. [Crossref]

24. Welch RD, Jones AL, Bucholz RW, et al. Effect of recombinant human bone morphogenetic protein-2 on fracture healing in a goat tibial fracture model. J Bone Miner Res 1998; 13: 1483-90. [Crossref]

25. Rajfer RA, Kilic A, Neviaser AS, et al. Enhancement of fracture healing in the rat, modulated by compounds that stimulate inducible nitric oxide synthase: Acceleration of fracture healing via inducible nitric oxide synthase. Bone Joint Res 2017; 6: 90- 7. [Crossref]

26. R&D System. Wnt Receptors & Pathways. Available from: https://www.rndsystems.com/pathways/wnt-signaling-pathways- beta-catenin-dependent-wnt-signaling

27. Karner CM, Long F. Wnt signaling and cellular metabolism in osteoblasts. Cell Mol Life Sci 2017; 74: 1649-57. [Crossref]

28. Kato M, Patel MS, Levasseur R, et al. Cbfa1-independent decrease in osteoblast proliferation, osteopenia, and persistent embryonic eye vascularization in mice deficient in Lrp5, a Wnt coreceptor. J Cell Biol 2002; 157: 303-14. [Crossref]

29. Hojo H, Ohba S, Chung UI. Signaling pathways regulating the specification and differentiation of the osteoblast lineage. Regen Ther 2015; 1: 57-62. [Crossref]