雷帕霉素调节自噬抑制凋亡促进骨折愈合的机制研究

Rapamycin promotes fracture healing by regulating autophagy and inhibition of apoptosis

目的探讨雷帕霉素调节细胞自噬抑制凋亡促进骨折愈合的机制。方法建立SD大鼠右侧股骨干骨折模型,随机分为两组:空白对照组和雷帕霉素组(1 mg/kg/d)。空白对照组无特殊处理,雷帕霉素组在术后12 h、24 h,3 d和7 d腹腔注射1 mg/kg的雷帕霉素。分别在造模后2 w,4 w和6 w应用X线和Micro CT评估骨组织愈合情况,免疫荧光检测LC3-Ⅱ,免疫组化检测Beclin-1的表达水平,western blot检测Bax、VEGF及p-mTOR,HE染色计数骨折后骨组织中成骨细胞数量来评估成骨活性。结果与对照组相比,雷帕霉素组骨折端骨组织中p-mTOR及p-mTOR/mTOR比值降低[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)、6 w(t=6.101,P=0.001;t=6.104,P=0.001)];自噬蛋白LC3-Ⅱ和Beclin-1表达明显增加[2 w(t=9.509,P=0.000;t=9.962,P=0.000)、4 w(t=3.868,P=0.008;t=24.490,P=0.000)、6 w(t=5.071,P=0.002;t=12.454,P=0.001)];抗凋亡蛋白Bcl-2表达增加[2 w(t=-9.099,P=0.000)、4 w(t=-8.128,P=0.000)、6 w(t=-9.497,P=0.000)];促凋亡蛋白Bax表达降低[2 w(t=-4.263,P=0.005)、4 w(t=-2.936,P=0.026)和6 w(t=-8.343,P=0.000)];VEGF表达增加[2 w(t=-5.754,P=0.001)、4 w(t=-5.077,P=0.002)和6 w(t=-12.200,P=0.000)],差异均具有统计学意义。X线结果显示雷帕霉素组术后2 w、4 w和6 w Garrett评分高于对照组(t=4.371,P=0.005;t=5.166,P=0.002;t=4.243,P=0.005),micro-CT结果显示雷帕霉素组术后2 w、4 w和6 w骨密度评分高于对照组(t=8.765,P=0.000;t=25.649,P=0.000;t=11.199,P=0.000),差异具有统计学意义。结论雷帕霉素通过诱导细胞自噬,抑制细胞凋亡促进骨折愈合。

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.