司马西特通过Notch/RANKL诱导破骨细胞形成的初步研究

Semagacestat inhibits RANKL-induced osteoclast formation through Notch signaling pathway

目的:探索γ分泌酶抑制剂司马西特通过Notch对核因子κB受体活化因子配体(RANKL)诱导破骨细胞形成以及骨吸收功能的影响及机制。方法:提取5周龄C57/BL6鼠的骨髓腔巨噬细胞进行培养,通过MTT法检测司马西特对巨噬细胞的半抑制浓度(IC50),通过破骨细胞诱导、TRAP染色及骨吸收试验评估司马西特对破骨细胞形成的影响,通过实时荧光定量RT-PCR检测破骨细胞形成相关基因的表达,Western blot法检测Notch信号通路相关蛋白的表达。结果:培养24、48、96 h时,司马西特对巨噬细胞的IC50分别是6.03、5.79、5.18μmol/L。TRAP染色显示,对照组破骨细胞形成数量(209±8)个/孔,100、200、400 nmol/L司马西特组的破骨细胞数量分别为(183±14)、(91±10)、(22±7)个/孔(P<0.05)。体外骨吸收结果显示,对照组骨吸收面积百分比(93.3±3.0)%,100、200及400 nmol/L司马西特组的骨吸收面积百分比分别为(80.6±6.6)%、(52.2±6.6)%、(27.4±5.8)%(P<0.05)。C-fos、TRAP、Cath-K以及NFATc1等破骨相关基因表达量随着司马西特用药浓度的增高而显著降低(P<0.05)。400 nmol/L司马西特组Cleaved-Notch1、NFATc1以及Hes1表达量降低(P<0.05)。结论:司马西特通过抑制Notch信号通路,抑制破骨细胞的分化以及骨吸收,对骨关节炎具有潜在的治疗作用。

Objective:To explore the effect of gamma secretase inhibitor(Semagacestat)on osteoclast formation and bone resorption induced by RANKL and its mechanism.Methods:Bone marrow cavity macrophages of 5 weeks old C57 mice were extracted for culture.IC50 of Semagacestat on macrophages was calculated by MTT to determine the safe drug concentration.The effect of Semagacestat on osteoclast formation was evaluated by osteoclast formation,TRAP staining and bone resorption test.RT-PCR was used to detect gene expression related to osteoclast formation.Western blot analysis of expression of proteins associated with Notch signaling pathway was also tested.Results:The 24 h,48 h and 96 h IC50 of Semagacestat on macrophages were 6.03,5.79 and 5.18μmol/L,respectively.Trap staining showed that the number of osteoclasts formed was(209±8)for each well in the control group,and(183±14)for each well,(91±10)for each well,and(22±7)for each well in the Semagacestat 100,200,and 400 nmol/L dosing groups,respectively(P<0.05).In vitro bone resorption results showed that the percentage of bone resorption area in blank control group was(93.3±3.0)%,and the percentage of bone resorption area in Semagacestat 100,200 and 400 nmol/L dosing groups was(80.6±6.6)%,(52.2±6.6)%,(27.4±5.8)%,respectively(P<0.05).RT-PCR results showed that the expressions of osteoclast related gene:C-fos,TRAP,Cath-K and NFATC1 were significantly decreased with the increase of the concentration of Semagacestat(P<0.05).Western Blot results showed that Cleaved-Notch1,NFATc1,and Hes1 expression decreased in the Semagacestat group compared to the control group(P<0.05).Conclusions:By inhibiting the activation of Notch pathway,Semagacestat inhibits the formation and bone resorption of osteoclasts,and may have a potential therapeutic effect on osteoarthritis.