硼替佐米与帕米膦酸二钠在大鼠肢体废用性骨质疏松症模型中的疗效比较

Comparison on the Efficacy of Bortezomib and Pamilophosphate in the Rat Model of Disuse Osteoporosis

目的:探讨硼替佐米与帕米膦酸二钠在废用性骨质疏松症(DOP)大鼠模型中的疗效。方法:选取16周龄雄性SD大鼠40只,随机分为对照组、假手术组、模型组、帕米膦酸二钠治疗组和硼替佐米治疗组,每组8只。建立DOP大鼠模型。采用显微-CT测定大鼠后肢胫骨显微结构参数;采用酶联免疫吸附试验测定大鼠血清学指标核因子κB受体激活蛋白配体(RANKL)/骨保护素(OPG)浓度比值,血清骨硬化蛋白(Sclerostin)、碱性磷酸酶(ALP)的水平;采用定量聚合酶链反应测定骨组织中骨吸收相关因子Ⅰ型前胶原氨基端前肽(PINP)、Ⅰ型胶原交联C-末端肽(CTX)-1、基质金属蛋白酶(MMP)-3和MMP-9 mRNA的相对表达水平;采用蛋白质印迹法测定大鼠骨组织中成骨分化关键转录因子钙调磷酸酶(Calcineurin)/活化T细胞核因子(NFAT)信号轴和糖原合成酶激酶-3β(GSK3β)/蛋白激酶B(Akt)信号通路水平。结果:与假手术组比较,模型组大鼠后肢胫骨骨体积分数(BV/TV)、骨小梁数量(Tb.N)、骨小梁厚度(Tb.Th)、连通性密度(Conn.D)、结构模型指数(SMI)和骨密度(BMD)降低,骨小梁分离度(Tb.Sp)升高;血清RANKL/OPG浓度比值、Sclerostin水平升高,血清ALP水平降低;骨组织中PINP、CTX-1 mRNA相对表达水平降低,MMP-3、MMP-9 mRNA相对表达水平升高;Calcineurin相对表达水平降低;p-Akt相对表达水平降低,p-GSK3β相对表达水平升高,上述差异均有统计学意义(P<0.05)。与模型组比较,帕米膦酸二钠治疗组和硼替佐米治疗组部分逆转了上述指标(包括后肢胫骨BV/TV、Tb.N、Tb.Th、Tb.Sp、Conn.D、SMI和BMD,血清RANKL/OPG浓度比值、Sclerostin水平和ALP水平,骨组织中MMP-3、MMP-9 mRNA相对表达水平,骨组织中Calcineurin相对表达水平,骨组织中p-Akt、p-GSK3β相对表达水平),差异均有统计学意义(P<0.05);且硼替佐米治疗组大鼠骨组织中PINP、CTX-1 mRNA相对表达水平升高,NFAT相对表达水平升高,差异均有统计学意义(P<0.05)。与帕米膦酸二钠治疗组比较,硼替佐米治疗组逆转上述指标的效果更为显著,差异均有统计学意义(P<0.05)。结论:硼替佐米改善DOP大鼠骨质疏松症的疗效优于帕米膦酸二钠。

OBJECTIVE:To compare the efficacy of bortezomib and pamilophosphate in the rat model of disuse osteoporosis(DOP).METHODS:Forty 16-week-old male SD rats were randomly divided into the control group,sham surgery group,model group,pamilphosphate treatment group and bortezomib treatment group,with 8 rats in each group.DOP rat model was established.The microstructural parameters of hind limb tibia were determined by micro-CT.Serological indexes receptor activator of nuclear factor-κB ligand(RANKL)/osteoprotegerin(OPG)concentration ratio,serum Sclerostin and alkaline phosphatase(ALP)levels were determined by enzyme linked immunosorbent assay.The mRNA expression levels of bone resorption-related factors type I procollagen amino-terminal prepeptide(PINP),C-terminal crosslinking telopeptide of type I collagen(CTX)-1,matrix metalloproteinase(MMP)-3 were determined by quantitative polymerase chain reaction.Signaling axis of Calcineurin/nuclear factor of activated T-cells isoform(NFAT)and signaling pathway of glycogen synthase kinase-3β(GSK3β)/protein kinase B(Akt),a key transcription factor for osteogenic differentiation,were determined by Western blotting.RESULTS:Compared with sham surgery group,bone volume fraction(BV/TV),trabecular number(Tb.N),trabecular thickness(Tb.Th),connectivity density(Conn.D),structural model index(SMI),bone mineral density(BMD)and trabecular separation(Tb.Sp)of the posterior limb tibia in model group rats decreased,trabecular separation(Tb.Sp)increased;serum RANKL/OPG concentration ratio and serum sclerostin level increased,serum ALP level decreased;the relative expression levels of PINP and CTX-1 mRNA in bone tissues decreased,while the relative expression levels of MMP-3 and MMP-9 mRNA increased,the relative expression levels of Calcineurin decreased;the relative expression levels of p-Akt decreased,and the relative expression levels of p-GSK3 increased,with statistically significant differences(P<0.05).Compared with the model group,the pamilphosphate treatment group and bortezomib treatment group partially reversed the above indicators(including BV/TV,Tb.N,Tb.Th,Tb.Sp,Conn.D,SMI and BMD in posterior limb tibia,serum RANKL/OPG concentration ratio,levels of Sclerostin and ALP,the relative expression levels of MMP-3 and MMP-9 mRNA in bone tissues,the relative expression levels of Calcineurin in bone tissues,and the relative expression levels of p-Akt and p-GSK3βin bone tissues),with statistically significant differences(P<0.05).The relative expression levels of PINP and CTX-1 mRNA and the relative expression levels of NFAT in bone tissues of rats in bortezomib treatment group increased,with statistically significant difference(P<0.05).Compared with the pamilophosphate treatment group,bortezomib treatment group had a more significant effect on reversing the above indicators,the differences were statistically significant(P<0.05).CONCLUSIONS:The efficacy of bortezomib for DOP rat model is better than that of pamilophosphate.