阿仑膦酸钠联合金天格对泼尼松龙致斑马鱼骨丢失作用机制的研究

Study on Mechanism of Action of Alendronate Combined with Jintiange on Bone Loss Model Using Zebrafish Induced by Prednisolone

目的探究阿仑膦酸钠(Aln)联合金天格对泼尼松龙诱导斑马鱼骨丢失的作用机制。方法将受精后3 d的斑马鱼胚胎随机分为空白对照组(0.5%DMSO)、模型组(25μmol·L^(-1)泼尼松龙)、Aln组、金天格组和Aln+金天格组,每组3份。9 d后处死,用茜素红染色,用于RNA分析、蛋白定量分析和形态学观察。结果与模型组、Aln组及金天格组比较,联用组的脊柱畸形弯曲率明显降低,Wnt的mRNA表达明显升高,RANKL、OC的mRNA表达明显下降,但对OPG的mRNA表达不如泼尼松龙组的影响显著;与两药单用组比较,Aln+金天格组Wnt-3a蛋白的表达量上升显著,而TNF-α、M-CSF和OSTF1蛋白的表达量降低明显。结论阿仑膦酸钠联合金天格对抗斑马鱼骨丢失的作用,可能与激活Wnt/β-catenin信号通路,进而影响RANKL/RANK/OPG系统,提高Wnt-3a蛋白的表达量,抑制RANKL和OC的mRNA表达,减少TNF-α、M-CSF和OSTF1蛋白的表达量有关,而两者对OPG的mRNA表达的影响需进一步研究。

OBJECTIVE To study mechanism of action of Alendronate（ Aln） combined with Jintiange on bone loss model using zebrafish Induced by Prednisolone. METHODS 3 days post fertilization（ d. p. f.） zebrafish larvae were divide into five groups a black group,a model group,Aln group,Jintiange group and Aln combined with Jintiange group.9 d.p.f. zebrafish skeleton was anesthetized and fixed for staining with alizarin red,which were used to as RNA analysis,quantitative analysis of protein and morpholory observation.RESULTS The incidence of spinal curvature of Aln combined with Jintiange group was significantly lower than the model group and Aln group and Jintiange group; compared with the model group,Aln group and Jintiange group,the expression of mRNA in Wnt of Aln combined with Jintiange group were increased,and the expression of mRNA in RANKL and OC had remarkable decreased,while for the expression of mRNA in OPG was not as good as model group; the expression of Wnt-3 a protein of Aln combined with Jintiange group was significantly higher than Aln group and Jintiange group,but the expression levels of protein of TNF-α、M-CSF and OSTF1 were significantly reduced. CONCLUSION Aln combined with Jintiange may be mainly activated Wnt/β-atenin and RANKL/RANK/OPG signaling pathway,and restrain the expression of mRNA in RANKL and OC,influence the expression of protein of Wnt-3 a and osteoclast factors,final achieve the aim of antagonism for zebrafish bone loss.However,the influence of OPG mRMA expression need to further study.