Activity of adenosine monophosphate-activated protein kinase and autophagy during the fracture healing and nonunion process

Abstract

Objective To investigate the correlation between adenosine monophosphate-activated protein kinase (AMPK), autophagy and fracture healing by comparing the activity of AMPK and autophagy during the fracture healing and nonunion process. Methods The fracture healing and nonunion animal models were established and verified by x-ray imaging. Bone maturation was measured by Masson staining and alizarin red staining. The expression of AMPK, p-AMPK, LC3B-Ⅱ, and p62 in the fracture ends was detected by immunohistochemical staining and subsequently semi-quantified by Image-pro plus (IPP 6.0) software. Autophagosome was observed by transmission electron microscope and density of autophagosome was calculated. Results X-ray showed animals in the healing group achieved osseous union while the nonunion group had fracture end sclerosis, and medullary cavity closed. Masson staining and alizarin red staining indicated bone maturation disorder in the nonunion group. Immunohistochemical staining of the fracture ends 4 weeks after surgery showed higher expression of p-AMPK (mean density, healing group vs. nonunion group: 0.031 20±0.002 00 vs. 0.005 79±0.001 00, P=0.001), LC3B-Ⅱ (healing group vs. nonunion group: 0.037 50±0.002 00 vs. 0.003 17±0.000 20, P=0.000) and lower expression of p62 (healing group vs. nonunion group: 0.013 80±0.001 00 vs. 0.041 10±0.005 00, P=0.004) in the healing group, while no significant difference in AMPK was found between two groups (healing group vs. nonunion group: 0.041 41±0.002 00 vs. 0.039 40±0.002 00, P=0.572). The transmission electron microscopy revealed higher density of autophagosome in the healing group (healing group vs. nonunion group: 4.6±0.5 vs. 2.6±0.5, P=0.024). Conclusion AMPK and autophagy may play an important role in promoting fracture healing, and low activity of AMPK and autophagy may be associated with the occurrence of nonunion. Key words: Fracture healing; Nonunion; Adenosine monophosphate-activated protein kinase; Autophagy