淫羊藿苷活化β-连环蛋白信号通路促进钛颗粒诱导的小鼠骨髓基质干细胞成骨分化

Icariin attenuates titanium particle inhibition of osteogenic differentiation in mesenchymal stem cells via activation of β-catenin signaling pathway

目的 观察淫羊藿苷（icariin）对钛颗粒（Ti）诱导小鼠骨髓基质干细胞（MSCs）成骨分化的影响.方法 实验分为4组,即对照组、Ti组、icariin组和ICG-001组.MSCs成骨诱导培养.Western blot法检测β-连环蛋白（β-catenin）的表达水平;碱性磷酸酶（ALP）活性检测、茜素红染色及定量分析观察细胞向成骨分化及细胞外基质矿化程度;反转录-聚合酶链反应（RT-PCR）检测Runt相关基因2（Runx2）、Osterix和骨钙素（OCN）基因mRNA的相对表达水平.结果 Western blot结果显示Ti能抑制β-catenin的表达,icariin（10^-8mol/L）加入后Ti的抑制效应不明显.ALP活性检测结果显示icariin组ALP活性为（27.76±5.26） nmol PNP/（min·μg protein）,与Ti组[（19.72±3.28） nmol PNP/（min· μg protein）]比较,差异有统计学意义（P＜0.05）.茜素红染色结果显示,对照组、icariin组细胞外基质矿化程度高,Ti、ICG-001组矿化结节少,定量结果显示icariin组吸光度（A）值为0.52 ±0.06,与Ti组（0.22±0.03）和ICG-001组（0.39±0.07）比较,差异有统计学意义（P＜0.05）.RT-PCR结果显示Ti组Runx2、Osterix和OCN基因mRNA的相对表达量分别为0.65±0.05、0.57 ±0.07和0.60 ±0.07;icariin组上述基因的相对表达量分别为0.95 ±0.03、0.98 ±0.09和0.96 ±0.06,差异有统计学意义（P＜0.05）;ICG-001加入后能抑制icariin对Runx2、Osterix和OCN基因表达的促进作用.结论 Icariin通过活化β-catenin信号通路促进Ti诱导的MSCs成骨分化。

Objective To explore the effect of icariin on titanium particle-induced inhibition of osteogenic differentiation of mesenchymal stem cells （MSCs）.Methods The experiment involved 4 groups：the control group,Ti group,icariin group and ICG-001 group.The protein level of β-catenin was determined by using Western blotting.Alkaline phosphatase （ALP） activity was measured using a p-nitrophenyl phosphate assay.The mineralization of osteoblast,which was differentiation-induced for 21 days,was determined by Alizarin red S （ARS） staining.The mRNA levels of runt related transcription factor-2 （RUNX2）,Osterix and osteocalcin （OCN） were detected by using quantitative reverse transcription-polymerase chain reaction （RT-PCR）.Results Western blotting analysis showed that Ti-particle stimulation reduced the cytosolic and nuclear levels of [β-catenin in MSCs.Pretreatment with icariin inhibits β-catenin degradation induced by Ti particles.After 3 days of incubation,cellular ALP activity was （27.76 ± 5.26） nmol PNW （min· μgprotein） in icariin group,which was higher than （19.72 ±3.28） nmol PNP/（min·μg protein） in Ti group （P 〈0.05）.ARS staining results showed that Ti particles significantly decreased the staining density of ARS in visible observation quantification by spectrophotometry when compared with the control group.However,the inhibitory effect of Ti particles on osteogenic differentiation was potently attenuated by icariin.RT-PCR revealed that icariin also significantly reduced Ti-particle inhibition of Runx2,Osterix,and OCN expression.We also investigated whether the protective effects of icariin were mediated via the β-catenin signaling pathway.ICG-001 treatment abolished icariin-mediated promotion of ALP activity and Runx2,Osterix,and OCN gene expression.Moreover,we found that deletion of β-catenin activity also attenuates icariin-induced osteogenic mineralization mediated by MSCs.Conclusion Icariin attenuates Ti-particle inhibition of MSCs differentiation via the activation of the β-catenin signaling pathway.