阿米洛利对破骨细胞分化的影响及其机制的分析word论文.docxVIP

中文摘要目的 阿米洛利（Amiloride）作为利尿剂已经在临床中广泛应用，本文旨在研 究在单核巨噬细胞系 RAW264.7 细胞系中阿米洛利对由 RANKL 诱导的破骨细胞分 化形成的作用及阐明其机制。方法 通过计数 TRAP 阳性的多核细胞和计算骨板骨吸收面积，以及用 qRT-PCR 检测破骨细胞特异性基因及 NFATc1 的表达，并通过 Western Blot 检测 NF-κB 和 MAPK 信号通路的蛋白表达。结果 我们发现阿米洛利能够显著的抑制 TRAP 阳性多核细胞的形成和骨吸收 的面积，同时能够明显下调 NFATc1 的信使 RNA 表达，抑制破骨细胞特异性基因 的表达，而其可能的机制是通过抑制 NF-κB 的 IκB 的降解和 MAPK 信号通路 ERK、 JNK 和 p38 的磷酸化。结论 综上所述，阿米洛利通过 NF-κB 和 MAPK 信号通路抑制破骨细胞分化， 可以作为治疗破骨细胞相关的骨病的潜在手段。关键词：阿米洛利; 破骨细胞; RAW264.7; MAPKs; NF-κBAbstractObjective: Amiloride is widely used as a diuretic and known to interact with the epithelial sodium channel and acid-sensing ion channel proteins, as well as Na+/H+ antiporters and Na+/Ca2+ exchangers. The aim of this study was to examine the effects of amiloride on receptor activator of nuclear factor kappaB (NF-κB) ligand (RANKL)-induced osteoclastogenesis and to elucidate the related mechanisms in RAW264.7 cell lines.Methods: We counted the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells and measured the bone resorption area. We also examined the expression of NFATc1 mRNA and osteoclast-specific genes by real-time reverse transcription-polymerase chain reaction. Additionally, we analyzed the NF-κB and mitogen-activated protein kinase (MAPK) signaling pathways by western blotting. Results:WefoundthatamiloridesignificantlydecreasedtheTRAP-positive multinucleated cells and the bone resorption area. Amiloride also downregulated NFATc1 mRNA expression and inhibited the expression of osteoclast-specific genes. As a possible mechanism, amiloride suppressed the degradation of inhibitor of kappaB and blocked the activation of c-Jun N-terminal kinase, extracellular signal-regulated kinase, and p38, implicating the NF-κB and MAPK pathways.Conclusion: In conclusion, our data indicated that amiloride was a strong inhibitor of osteoclast differentiation, suggesting that amiloride could have a new indication in the treatment of bone-loss related di