芹甙元抑制破骨细胞分化的机制研究

Mechanism of Apigetrin Ihibiting Osteoclast Differentiation

目的探讨芹甙元抑制破骨细胞分化机制。方法小鼠骨髓巨噬细胞(BMMs)分不同浓度的芹甙元组,通过CCK-8法检测芹甙元对BMMs的细胞毒性,抗酒石酸酶(TRAP)染色法检测各组成熟破骨细胞数量,荧光定量聚合酶法(qPCR)检测各组破骨细胞分化标志基因(NFATC1、TRAP、CTSK、Atp6v0d2、Dc-stamp、C-Fos)mRNA的表达,Western blot法检测核转录因子κB p65(NF-κB p65)中磷酸化蛋白的水平。结果CCK-8检测结果显示,芹甙元在50μmol/L范围内对BMMs无明显的细胞毒性;TRAP染色结果显示,TRAP染色阳性的破骨细胞数目随着芹甙元浓度的升高而减少;qPCR检测结果显示,随着芹甙元浓度的升高,破骨细胞分化基因(NFATC1、TRAP、CTSK、Atp6v0d2、Dc-stamp、C-Fos mRNA)mRNA的表达水平明显降低(P<0.05);Western blot法检测结果显示,芹甙元组p65的磷酸化水平相对于对照组明显降低(P<0.05)。结论芹甙元可以通过NF-κB通路抑制破骨细胞分化与成熟,有望作为治疗骨质疏松骨溶解的潜在药物。

Objective To investigate the mechanism of apigetrin inhibiting osteoclast differentiation.Methods Mouse bone marrow macrophages(BMMs)were divided into different concentrations of apigetrin.The cytotoxicity of apigetrin to BMMs was detected by CCK-8 method.The number of mature osteoclasts in each group was detected by anti tartrate enzyme(TRAP)staining.The mRNA expression of osteoclast differentiation marker genes(NFATc1,TRAP,CTSK,Atp6v0d2,DC-stamp,C-Fos)was detected by fluorescent quantitative polymerase chain reaction(qPCR).Western blot was used to detect the levels of phosphorylated proteins in nuclear transcription factorκBp65(NF-κB-p65).Results CCK-8 showed that apigentrin had no obvious cytotoxicity to BMMs even the concentration of apigetrin is up to 50μmol/L;TRAP staining:the number of trap positive osteoclasts decreased with the increase of apigetrin concentration.qPCR detection:with the increase of apigetrin concentration,the mRNA expression levels of osteoclast differentiation genes(NFATc1,TRAP,CTSK,Atp6v0d2,Dc-stamp,C-Fos)were significantly decreased(P<0.05).Western blot showed that the phosphorylation level of p65 in apigetrin group was significantly lower than that of the control group(P<0.05).Conclusion Apigetrin can inhibits the differentiation and maturation of osteoclasts by inhibiting the activation of NF-κB signaling pathway,which may be a potential drug for the treatment of osteolysis in osteoporosis.