Avagacestat抑制破骨细胞形成及溶骨功能改善骨关节炎骨破坏

目的 探究γ-分泌酶抑制剂Avagacestat通过抑制破骨细胞的形成及溶骨功能,抑制骨关节炎的进展。方法 提取6周龄C57鼠骨髓细胞诱导为巨噬细胞扩增培养,加入M-CSF及RANKL诱导后通过CCK8测定Avagacestat对该细胞的半数抑制浓度(IC₅₀),设置0、120、240 nmol/L 3种Avagacestat浓度组,对比破骨细胞形成实验、功能实验并检测3组细胞TRAP、C-fos、Cath-K等破骨相关基因的表达。建立LPS诱导关节炎实验动物模型,通过三维骨重建检测软骨下骨溶解情况、通过HE染色及TRAP染色检测破骨细胞分布情况。结果 在24、48、96 h 3个时间点,Avagacestat对破骨细胞的IC₅₀分别为493、426、405 nmol/L。TRAP实验显示Avagacestat抑制破骨细胞形成,骨板骨吸收实验证实Avagacest抑制溶骨功能,且240 nmol/L浓度的Avagacestat对破骨细胞形成及功能的抑制显著高于120 nmol/L;RT-PCR结果提示,加药后溶骨相关基因TRAP、C-fos、Cath-K表达显著下降,差异具有统计学意义。LPS诱导的骨关节炎模型中三维骨重建及TRAP染色都表明破骨细胞数量显著增多、骨溶解增强,而Avagacestat对此有明显的抑制作用,表现为骨密度、骨小梁数量加药后增加、破骨细胞数量加药后减少,差异具有统计学意义。结论 Avagacestat抑制破骨细胞形成、溶骨相关基因表达及溶骨功能,因此可以作为治疗骨关节炎的潜在药物。

Avagacestat inhibits osteoclast formation and attenuates bone destruction in osteoarthritis

Objective To explore the effect of gamma secretase inhibitor (Avagacestat) on inhibiting osteoclast formation and bone resorption to suppress the progress of osteoarthritis. Methods Bone marrow cavity macrophages (BMMs) of 6 weeks old C57 mice were extracted and induced for culture. IC₅₀ of Avagacestat on macrophages induced by M-CSF and RANKL was calculated by CCK8. Osteoclast formation test and function test were conducted to determine the inhibition of Avagacestat on osteoclast by setting 3 groups with drug concentration of 0, 120, 240 nmol/L and osteoclast progress-associated genes such as TRAP, C-fos, Cath-K were checked through RT-PCR in 3 groups. Then osteoarthritis mouse model was constructed by injecting LPS into the Articulatio genus. Bone resorption was examined by 3D bone reconstruction, and distribution of osteoclast was detected through HE staining and TRAP staining. Results At 24 h, 48 h and 96 h, the IC₅₀ of Avagacestat on osteoclasts were 493, 426 and 405 nmol/L, respectively. The cell TRAP and bone resorption test respectively certified that Avagacestat could inhibit the formation of osteoclast and bone resorption, and the inhibition of 240 nmol/L was significantly higher than that of 120 nmol/L. RT-PCR results also indicated that the expression of osteolysis-associated genes TRAP, C-fos, Cath-K was significantly inhibited after the addition of Avagacestat, and with the increasing concentration of Avagacestat, expression of these genes significantly declined. 3D bone reconstruction and TRAP staining showed that the number of osteoclasts increased significantly and osteolysis was enhanced in LPS-induced osteoarthritis model. Avagacestat significantly inhibited the number of osteoclasts, showing bone mineral density and bone trabecular number increasing but osteoclast number decreasing after Avagacestat added, statistically significant. Conclusion Avagacestat can inhibit the proliferation of osteoclasts and the expression of osteolytic genes and resorption in osteoarthritis, so it has the potential to be used as a drug for arthritis treatment.