Abstract：
BACKGROUND: Zoledronic acid (ZOL) is a type of bisphosphonate with good therapeutic effects on orthopaedic diseases. However, the pharmacological functions of ZOL on steroid-induced avascular necrosis of femoral head (SANFH) and the underlying mechanism remain unclear, which deserve further research.
METHODS: SANFH models both in vivo and in vitro were established by dexamethasone (Dex) stimulation. Osteoclastogenesis was examined by TRAP staining. Immunofluorescence was employed to examine autophagy marker (LC3) level. Cell apoptosis was analyzed by TUNEL staining. The interaction between Foxhead box D3 protein (FOXD3) and Annexin A2 (ANXA2) promoter was analyzed using ChIP and dual luciferase reporter gene assays.
RESULTS: Dex aggravated osteoclastogenesis and induced osteoclast differentiation and autophagy in vitro, which was abrogated by ZOL treatment. PI3K inhibitor LY294002 abolished the inhibitory effect of ZOL on Dex-induced osteoclast differentiation and autophagy. FOXD3 overexpression neutralized the downregulation effects of ZOL on Dex-induced osteoclasts by transcriptionally activating ANXA2. ANXA2 knockdown reversed the effect of FOXD3 overexpression on ZOL-mediated biological effects in Dex-treated osteoclasts. In addition, ZOL improved SANFH symptoms in rats.
CONCLUSIONS: ZOL alleviated SANFH through regulating FOXD3 mediated ANXA2 transcriptional activity and then promoting PI3K/AKT/mTOR pathway, revealing that FOXD3 might be a target for ZOL in SANFH treatment.
METHODS: SANFH models both in vivo and in vitro were established by dexamethasone (Dex) stimulation. Osteoclastogenesis was examined by TRAP staining. Immunofluorescence was employed to examine autophagy marker (LC3) level. Cell apoptosis was analyzed by TUNEL staining. The interaction between Foxhead box D3 protein (FOXD3) and Annexin A2 (ANXA2) promoter was analyzed using ChIP and dual luciferase reporter gene assays.
RESULTS: Dex aggravated osteoclastogenesis and induced osteoclast differentiation and autophagy in vitro, which was abrogated by ZOL treatment. PI3K inhibitor LY294002 abolished the inhibitory effect of ZOL on Dex-induced osteoclast differentiation and autophagy. FOXD3 overexpression neutralized the downregulation effects of ZOL on Dex-induced osteoclasts by transcriptionally activating ANXA2. ANXA2 knockdown reversed the effect of FOXD3 overexpression on ZOL-mediated biological effects in Dex-treated osteoclasts. In addition, ZOL improved SANFH symptoms in rats.
CONCLUSIONS: ZOL alleviated SANFH through regulating FOXD3 mediated ANXA2 transcriptional activity and then promoting PI3K/AKT/mTOR pathway, revealing that FOXD3 might be a target for ZOL in SANFH treatment.
摘要：
背景：唑来膦酸（ZOL）是一种双膦酸盐，对骨科疾病具有良好的治疗效果。然而,ZOL对激素性股骨头缺血性坏死(SANFH)的药理作用及潜在机制尚不清楚,这值得进一步研究。
方法：通过地塞米松（Dex）刺激建立体内和体外SANFH模型。通过TRAP染色检查破骨细胞生成。免疫荧光检测自噬标志物(LC3)水平。通过TUNEL染色分析细胞凋亡。使用ChIP和双荧光素酶报告基因测定分析Foxhead盒D3蛋白(FOXD3)与膜联蛋白A2(ANXA2)启动子之间的相互作用。
结果：Dex在体外加重了破骨细胞的形成，诱导破骨细胞分化和自噬，ZOL治疗已废除。PI3K抑制剂LY294002消除了ZOL对Dex诱导的破骨细胞分化和自噬的抑制作用。FOXD3过表达通过转录激活ANXA2来中和ZOL对Dex诱导的破骨细胞的下调作用。在Dex处理的破骨细胞中,ANXA2敲低逆转FOXD3过表达对ZOL介导的生物学效应的影响。此外,ZOL改善大鼠SANFH症状。
结论：ZOL通过调节FOXD3介导的ANXA2转录活性，进而促进PI3K/AKT/mTOR通路，减轻SANFH,揭示FOXD3可能是ZOL在SANFH治疗中的靶标。
方法：通过地塞米松（Dex）刺激建立体内和体外SANFH模型。通过TRAP染色检查破骨细胞生成。免疫荧光检测自噬标志物(LC3)水平。通过TUNEL染色分析细胞凋亡。使用ChIP和双荧光素酶报告基因测定分析Foxhead盒D3蛋白(FOXD3)与膜联蛋白A2(ANXA2)启动子之间的相互作用。
结果：Dex在体外加重了破骨细胞的形成，诱导破骨细胞分化和自噬，ZOL治疗已废除。PI3K抑制剂LY294002消除了ZOL对Dex诱导的破骨细胞分化和自噬的抑制作用。FOXD3过表达通过转录激活ANXA2来中和ZOL对Dex诱导的破骨细胞的下调作用。在Dex处理的破骨细胞中,ANXA2敲低逆转FOXD3过表达对ZOL介导的生物学效应的影响。此外,ZOL改善大鼠SANFH症状。
结论：ZOL通过调节FOXD3介导的ANXA2转录活性，进而促进PI3K/AKT/mTOR通路，减轻SANFH,揭示FOXD3可能是ZOL在SANFH治疗中的靶标。
