利拉鲁肽通过介导O3a/Wnt信号通路改善骨质疏松大鼠骨密度的研究

Liraglutide improves bone mineral density in osteoporotic rats by mediating O3a/Wnt signaling

目的研究利拉鲁肽对骨质疏松大鼠叉头框蛋白O3a(FoxO3a)/Wnt信号通路及椎骨密度的影响。方法将雌性Sprague-dawley大鼠根据随机数字表法分成假手术组、模型组、利拉鲁肽干预组,每组10只,模型组、干预组均采用双侧卵巢切除术建立骨质疏松症模型。干预组每天分别皮下注射利拉鲁肽,假手术组、模型组给予等体积的生理盐水。连续给药12周后,检测骨密度、骨生物力学,采用酶联免疫吸附法血清骨保护素、抗酒石酸酸性磷酸酶及骨钙素水平,采用Real-time PCR技术检测O3a/Wnt途径中相关mRNA表达水平,采用免疫印迹法检测O3a/Wnt途径中相关蛋白表达水平。结果造模成功大鼠的L4~5(0.33±0.04 vs 0.18±0.03)及左、右股骨骨密度(0.37±0.05 vs 0.23±0.04,0.35±0.04 vs 0.24±0.03)水平明显低于假手术组(P<0.05)。治疗12周后3组大鼠骨最大载荷、三点弯曲应力、骨密度及弹性模量差异有统计学意义,其中假手术组各指标水平最高(36.53±5.23,154.13±6.27,0.34±0.04,3102.34±160.44),其次为依次干预组(19.37±4.32,141.54±6.58,0.18±0.03,2270.18±145.53)、模型组(26.17±4.68,147.56±5.84,0.28±0.03,2804.24±140.42)(P<0.05)。3组大鼠血清骨保护素、抗酒石酸酸性磷酸酶及骨钙素水平差异有统计学意义,其中假手术组的骨保护素(假手术组vs模型组vs干预组:7.53±0.63 vs 4.57±0.42 vs 6.15±0.61)明显高于干预组、模型组,抗酒石酸酸性磷酸酶(假手术组vs模型组vs干预组:14.34±2.87 vs 19.53±3.52 vs 15.96±3.14)、骨钙素(假手术组vs模型组vs干预组:0.84±0.09 vs 1.13±0.12 vs 0.95±0.08)明显低于干预组、模型组(P<0.05)。3组大鼠FoxO3a、Wnt2及β-catenin mRNA和蛋白表达水平差异有统计学意义,其中假手术组的Wnt2、β-catenin明显高于干预组、模型组,FoxO3a明显低于干预组、模型组(P<0.05)。结论利拉鲁肽通过活化O3a/Wnt信号通路而增加骨密度,改善骨生物力学状态,从而有效治疗骨质疏松。

Objective To study the effect of liraglutide on the forkhead box O3a(forkhead box O3a,FoxO3a)/Wnt signaling pathway and vertebral bone density in osteoporotic rats.Methods Female Sprague-dawley rats were divided into sham operation group,model group,and liraglutide intervention group according to the random number table method,with 10 rats in each group.Both the model group and the intervention group used bilateral oophorectomy to establish an osteoporosis model.The intervention group was injected subcutaneously with liraglutide every day,and the sham operation group and the model group were given an equal volume of normal saline.After 12 weeks of continuous administration,the bone mineral density and bone biomechanics were measured,the serum osteoprotegerin,anti-tartrate acid phosphatase,and osteocalcin levels were detected by enzyme-linked immunosorbent assay,and the O3a/Wnt pathway was detected by Real-time PCR technology.The expression level of mRNA was detected by Western blot to detect the expression level of related proteins in the O3a/Wnt pathway.Results The bone mineral density levels of L4,5(0.33±0.04 vs 0.18±0.03)and left and right femurs(0.37±0.05 vs 0.23±0.040.35±0.04 vs 0.24±0.03)of the successfully modeled rats were significantly lower than those of the sham operation group(P<0.05).After 12 weeks of treatment,the differences in the maximum bone load,three-point bending stress,bone density and elastic modulus of the three groups of rats were statistically significant.Among them,the sham operation group had the highest level of each index(36.53±5.23,154.13±6.27,0.34±0.04,3102.34±160.44),followed by the intervention group(19.37±4.32,141.54±6.58,0.18±0.03,2270.18±145.53)and the model group in turn(26.17±4.68,147.56±5.84,0.28±0.03,2804.24±140.42)(P<0.05).There were statistically significant differences in the levels of serum osteoprotegerin,anti-tartrate acid phosphatase and osteocalcin among the three groups.Among them,the osteoprotegerin(Sham operation group vs model group vs intervention group:7.53±0.63 vs 4.57±0.42 vs 6.15±0.61)of the sham operation group was significantly higher than that of the intervention group and the model group.The anti-tartrate acid phosphatase(Sham operation group vs model group vs intervention group:14.34±2.87 vs 19.53±3.52 vs 15.96±3.14)and osteocalcin levels(Sham operation group vs model group vs intervention group:0.84±0.09 vs 1.13±0.12 vs 0.95±0.08)of rats The factor was significantly lower than that of the intervention group and model group(P<0.05).The mRNA and protein expression levels of FoxO3a,Wnt2,andβ-catenin in the three groups of rats were significantly different.Among them,Wnt2 andβ-catenin in the sham operation group were significantly higher than the intervention group and model group,and FoxO3a was significantly lower than the intervention group and model Group(P<0.05).Conclusion Liraglutide can increase bone density and improve bone biomechanics by activating O3a/Wnt signal,thereby effectively treating osteoporosis.