Abstract:
Objective To investigate the changes in cell autophagy and the molecularmechanism of rapamycin affecting the fracture healing.
Methods Sprague-Dawley (SD) rats were used to establish the right femoral shaft fracture models. After model establishment, SD rats were divided into two groups, the control group and the rapamycin group (1 mg/kg/d). There was no special treatment in the blank control group. The rapamycin group was intraperitoneally injected with 1 mg/kg rapamycin at 12 h, 24 h, 3 d and 7 d after surgery. X-ray and Micro CT were used to evaluate the healing of bone tissue at 2 w, 4 w and 6 w after model establishment. Immunofluorescence was used to detect LC3-Ⅱ. Immunohistochemistry was used to detect the expression level of Beclin-1. Western blot was used to detect Bax and VEGF. p-mTOR, hematoxylin and eosin staining (HE staining) to evaluate theosteoblastic activity through count of osteoblast in bone tissue at the end of fracture.
Results Compared with the control group, the ratio of p-mTOR and p-mTOR/mTOR in the fractured bone tissue of the Papamycin group were decreased [2 w (t=6.703, P=0.004; t=16.346, P=0.000), 4 w (t=16.407, P=0.000; t=16.738, P=0.000) and 6 w (t=6.101, P=0.001, t=6.104, P=0.001)]. The expression of autophagy proteins LC3-II and Beclin-1 were significant increased[2 w (P=0.000, t=9.509; P=0.000, t=9.962), 4 w (t=3.868, P=0.008, t=24.490, P=0.000,) and 6 w(t=5.071, P=0.002, t=12.454, P=0.001,)]. Expression of anti-apoptotic protein Bcl-2 were increased [2 w (t=-9.099, P=0.000), 4 w (t=-8.128, P=0.000, And 6 w (t=-9.497, P=0.000)]. Pro-apoptotic protein Bax expression were decreased [2 w (t=-4.263, P=0.005), 4 w (t=-2.936, P=0.026) and 6 w (t=-8.343, P=0.000)]. VEGF expression were increased, 2 w [(t=-5.754, P=0.001), 4 w (t=-5.077, P=0.002) and 6 w (t=-12.200, P=0.000)], all the differences were statistically significant. X-ray results showed that Garrett scores were significantly higher in the rapamycin group than in the control group at 2 w, 4 w and 6 w after surgery (t=4.371, P=0.005, t=5.166, P=0.002, t=4.243, P=0.005), micro-CT results showed that the bone density scores at 2 w, 4 w and 6 w after rapamycin group were significantly higher than those in the control group (t=8.765, P=0.000, t=25.649, P=0.000, t=11.199, P=0.000), the difference was statistically significant.
Conclusion Rapamycin promotes fracture healing by inducing autophagy and inhibiting apoptosis.
Key words:
Sirolimus,
Autophagy,
Apoptosis,
Fracture healing
Zhaoyang Yin, Yongfeng Huo, Xinhui Liu, Jian Yin. Rapamycin promotes fracture healing by regulating autophagy and inhibition of apoptosis[J]. Chinese Journal of Geriatric Orthopaedics and Rehabilitation(Electronic Edition), 2019, 05(01): 25-32.