补肾活血法预防芳香化酶抑制剂所致骨丢失的机制

Mechanism of tonifying kidney and activating blood circulation to prevent bone loss caused by aromatase inhibitor based on proteomic sequencing

目的探讨补肾活血法预防芳香化酶抑制剂(aromatase inhibitors,AIs)所致骨丢失的关键靶标和通路。方法将小鼠随机分为假手术组、模型组和给药组,模型组和给药组去势后予以10μg/d来曲唑溶液皮下注射构建绝经后AIs所致骨质疏松动物模型,给药组同时予以黄芪补肾活血汤(HQD)19.24 g/(kg·d)灌胃预防骨丢失,假手术组与模型组予以等量生理盐水灌胃。给药三个月后取小鼠股骨进行骨密度检测验证造模效果,并借助TMT蛋白组学测序分析获取其组学特征。结果与假手术组相比,模型组小鼠骨小梁结构疏松紊乱,数量减少,证明造模成功。与模型组相比,给药组骨小梁粗大致密,数量增多,证明补肾活血法预防作用显著。模型组与假手术组相比,上调蛋白275个,下调189个;给药组与模型组相比,上调蛋白286个,下调蛋白898个。三组对比后共有18个交集蛋白,分别为Ptbp1、Epx、Ear1、Ear2、Col6a5、Mta2、Alox15、Lztfl1、Ccl6、Diaph2、Wfdc21、Smarcb1、Strip1、C19orf12、Pex14、Akr1b7、Fahd1、Nr1d2。差异蛋白主要参与了细胞发育和分化的调节、脂质代谢、核酸内切酶活性等生物过程,并在铁死亡、花生四烯酸代谢通路、肌钙蛋白细胞骨架调节、PI3K-AKT等信号通路显著富集。结论补肾活血法可有效预防AIs所致骨丢失的发生,其关键靶标和作用机制可能与Ptbp1、Alox15及铁死亡、PI3K-AKT等通路相关。

Objective To investigate the key targets and pathways for preventing bone loss caused by aromatase inhibitors(AIs)by tonifying the kidney and activating the blood.Methods Thirty mice were randomly divided into sham operation group,model group,and drug administration group.After castration,mice in the model group and drug administration group were subcutaneously injected with letrozole solution of 10μg/d to establish an animal model of postmenopausal osteoporosis caused by AIs.Mice in the drug administration group was also given 19.24 g/(kg·d)of Huangqi Bushen Huoxue decoction(HQD)to prevent bone loss,while mice in the sham operation group and model group were given the same amount of normal saline.After three months of drug administration,the femurs of the mice were collected to test the bone mineral density and to verify the model.Results Compared to those in the blank group,the trabecular structure of the model group was loose and disordered,and the number was reduced,which proved that the model was successful.Compared to those in the model group,the trabecular bone in the drug group was thick and dense,and the number increased,which proved that the method of tonifying kidney and promoting blood circulation had a significant preventive effect.Compared to those in the blank group,275 proteins were up-regulated and 189 proteins were down-regulated in the model group.Compared to those in the model group,286 proteins were up-regulated and 898 proteins were down-regulated.There were 18 intersecting proteins among the three groups after comparison,including Ptbp1,Epx,Ear1,Ear2,Col6a5,Mta2,Alox15,Lztfl1,Ccl6,Diaph2,Wfdc21,Smarcb1,Strip1,C19orf12,Pex14,Akr1b7,Fahd1,and Nr1d2.Differential proteins were mainly involved in the regulation of cell development and differentiation,lipid metabolism,endonuclease activity,and other biological processes,and were significantly enriched in iron death,arachidonic acid metabolism pathway,troponin cytoskeleton regulation,and PI3K-AKT and other signal pathways.Conclusion Tonifying kidney and activating the blood effectively prevents bone loss caused by AIs.Its key target and mechanism may be related to Ptbp1,Alox15,iron death,and PI3K-AKT and other pathways.