阿司匹林体外抑制破骨细胞生成的分子机制研究  

The molecular mechanism of aspirin in the inhibition of osteoclastogenesis in vitro

作　　者：曾艳平[1] 杨超[1] 杨红军[1] 樊勇[1] 吴子祥[1] 刘斌[1] 苏菲[1] 白博[1] 张文强[1] 桑宏勋[1] 

机构地区：[1]第四军医大学西京医院骨科,西安710032

出　　处：《中国骨质疏松杂志》2015年第3期275-280,共6页Chinese Journal of Osteoporosis

基　　金：国家自然科学基金面上项目(81270959)

摘　　要：目的探讨阿司匹林对破骨细胞生成的影响及其分子机制。方法体外培养RAW264.7细胞,以100 ng/ml核激活因子κB(NF-κB)受体配体(RANKL)诱导培养,并同时添加不同溶度的阿司匹林(0,0.25,0.5,1.0,1.5 mmol/L)培养5天。在不同时间点,通过抗酒石酸酸性磷酸酶(TRAP)染色的方法来观察破骨细胞诱导生成能力,用实时荧光PCR方法检测其破骨细胞系标志基因,包括组织蛋白酶K(CTSK)、TRAP、基质金属蛋白酶9(MMP-9)和降钙素受体(CTR)mRNA的表达。裂解不同培养条件的细胞并提取蛋白上样,行western印迹检测NF-κB通道蛋白的表达以及有丝分裂原激活蛋白激酶(MAPKS)通道蛋白的表达,通过免疫荧光的方法分析确定NF-κB的P65的核易位。结果阿司匹林抑制RANKL诱导的破骨细胞生成,随着阿司匹林浓度增加,破骨细胞形成数量明显减少;其标志性基因TRAP、CTSK、MMP9及CTR的mRNA表达均有所下调;磷酸化的P65、P50、IKB-a、P38以及氨基端激酶(C-JNK)和胞外信号调节蛋白激酶(ERK)蛋白表达均有所减少,而其非磷酸化的蛋白表达水平无明显变化。阿司匹林同时对NF-KB P65的核易位也表现出抑制效果。结论在RAW264.7细胞系中,阿司匹林通过抑制NF-κB系统(P65、P50、IKB-a)和MAPKS系统(P38、C-JNK和ERK)通道的激活来抑制破骨细胞的生成,且在一定范围内和阿司匹林浓度呈正相关。阿司匹林可能具有临床预防及治疗骨质疏松的潜能。Objective To explore the influence of aspirin in the generation and differentiation of osteoclasts and its molecular mechanism. Methods RAW264.7 cells were cultured with 100 ng/ml RANKL treatment and additioned with aspirin solution （0, 0.25, 0. 5, 1.0, 1.5 mmol/L） of 5 days. TRAP staining was used to observe the generation of osteoclasts at different time points. Expressions of osteoclast marker mgNAs, including CTSK, MMP-9, and CTR, were detected with real-time PCR. NF-KB and MAPKS protein expressions were detected using Western blotting. Nuclear translocation of NF-KB p65 was analyzed using immunofluorescence technique. Results Aspirin suppressed RANKL-induced osteoclast formation. With the increase of aspirin concentration, the expressions of marker genes including TRAP, CTSK, MMP9, and CTR mRNA decreased, and the expressions of phosphorylated P65, P50, IKB- a, P38, C-JNK, and ERK protein reduced. However, aspirin showed no effect on nonphosphorylated proteins and inhibited P65 dislocation. Conclusion Aspirin inhibits the osteoclastogenesis of RAW264.7 cells by inhibiting the activation of NF-KB and MAPK signaling pathways, indicating that aspirin has the potential of preventing and treating osteoporosis.

关 键 词：阿司匹林 破骨细胞 NF-κB MAPKS 骨质疏松 

分 类 号：R965.2[医药卫生—药理学]