The effect of tail suspension and treadmill exercise on LRP6 expression, bone mass and biomechanical properties of hindlimb bones in SD rats

Abstract

Purpose: To investigate whether mechanical load regulates LRP6 expression and whether different intensities of treadmill exercise have different effects on LRP6 expression and the biomechanical properties of hindlimb bones in Sprague-Dawley (SD) rats. Methods: Fifty-six three-month-old virgin female SD rats were randomly divided into seven groups (n=8). Each group was subjected to tail suspension, free physiological activity or different intensities of treadmill exercise according to the experimental design for four or eight weeks. Rats were sacrificed after the intervention based on experimental design, and fresh femurs, tibias and fibulas were harvested for molecular biological analysis, biomechanical testing and micro-CT analysis. Results: LRP6 expression and the Wnt/β-catenin pathway activity decreased, and bone mass and biomechanical properties decreased after loss of mechanical stimulation. For disuse osteoporosis, even physiological activity could improve LRP6 expression, Wnt/β-catenin pathway activity, bone mass and biomechanical properties. Compared with physiological activity, treadmill exercise had better and faster effects on bone recovery. Compared with the Low intensity Exercise Group (LE group), the Medium intensity Exercise Group (ME group) and High intensity Exercise Group (HE group) had higher LRP6 expression, bone mass and biomechanical properties, while there were no significant difference between the ME group and HE group. Conclusions: Mechanical load appears to be a regulator of LRP6 expression, and it further affects the Wnt/β-catenin pathway activity and bone mass. The LRP6 expression, bone mass and biomechanical properties gradually improve as treadmill exercise intensity increases, while there is no significant difference between the ME group and HE group.

Keywords: LRP6; Mechanical load; Wnt/β-catenin pathway; biomechanical property; disuse osteoporosis; treadmill exercise.

Figure 1

The experimental design (A) and LRP6 (B), β-catenin (C), c-myc (D) and Runx2 (E) mRNA expression in each group relative to the NN group calculated using the 2^-ΔΔCt method. a: P<0.05 compared with the NN group. b: P<0.05 compared with the SN group. c: P<0.05 compared with the LE group. d: P<0.05 compared with the ME group. e: P<0.05 compared with the HE group.

Figure 2

LRP6-positive osteocyte distribution according to immunohistochemical staining in each group (A-E) and Western blot analysis of LRP6 protein (F). (A) The NN group, (B) The SN group, (C) The LE group, (D) The ME group, (E) The HE group. Original magnification ×200.

Figure 3

β-catenin-positive osteocyte distribution by immunohistochemical staining in each group (A-E) and Western blot analysis of β-catenin (F). (A) The NN group, (B) The SN group, (C) The LE group, (D) The ME group, (E) The HE group. Original magnification ×200.

Figure 4

Trabecular structural measurement results of the ROI of the distal femur in the seven groups. a: P<0.05 compared with the C4W group. b: P<0.05 compared with the NN group. c: P<0.05 compared with the SN group. d: P<0.05 compared with the LE group.

Figure 5

Representative 3D reconstruction images of the distal femur and distal femoral growth plate.

Figure 6

Biomechanical parameters of the femoral diaphysis and neck. (A-C) Maximum load (A), Stiffness (B) and Energy to maximum load (C) of the femoral shaft; (D-F) Maximum load (D), Stiffness (E) and Energy to maximum load (F) of the femoral neck. a: P<0.05 compared with the C4W group. b: P<0.05 compared with the NN group. c: P<0.05 compared with the SN group. d: P<0.05 compared with the LE group. e: P<0.05 compared with the MM group.