木犀草素通过Wnt/β-catenin通路抑制RAW264.7细胞向破骨细胞分化

Luteolin inhibits the differentiation of RAW264.7 cells into osteoclasts through Wnt/β-catenin pathway

目的:探究木犀草素(Luteolin)调控Wnt/β-连环蛋白(β-catenin)通路对RAW264.7细胞向破骨细胞分化的抑制作用。方法:检测不同浓度木犀草素(1、2.5、5、10μmol/L)对RAW264.7细胞活性和破骨分化的影响;RAW264.7细胞分为Blank组、Osteolysis induction组、Luteolin组、Luteolin+siRNA NC组、Luteolin+Wnt-3a siRNA组、Luteolin+NC组、Luteolin+Wnt-3a组。除Blank组外,其余各组细胞均进行破骨诱导分化,并相应进行5μmol/L木犀草素及细胞转染处理。诱导7 d后,TRAP染色、罗丹明-鬼笔环肽染色检测RAW264.7细胞向破骨细胞分化的能力;RT-qPCR、Western blot检测破骨细胞分化标志物(NFATc1、CTSK、c-Fos、MMP9、RANKL)和Wnt/β-catenin通路(Wnt-3a、β-catenin)mRNA和蛋白水平。结果:浓度1、2.5、5μmol/L的木犀草素对RAW264.7细胞活性无明显影响(P>0.05),但能够明显抑制RAW264.7细胞破骨分化。破骨诱导分化后,细胞TRAP阳性多核细胞数量增多,F-actin环和细胞核数量增多,NFATc1、CTSK、c-Fos、MMP9、RANKL mRNA和蛋白水平升高,Wnt-3a、β-catenin mRNA和蛋白水平降低(均P<0.05);木犀草素处理后,细胞TRAP阳性多核细胞数量减少,F-actin环和细胞核数量减少,NFATc1、CTSK、c-Fos、MMP9、RANKL mRNA和蛋白水平降低,Wnt-3a、β-catenin mRNA和蛋白水平升高(均P<0.05);敲低Wnt-3a表达能够减弱木犀草素对上述指标的影响(均P<0.05);过表达Wnt-3a能够增强木犀草素对上述指标的影响(均P<0.05)。结论:木犀草素通过Wnt/β-catenin通路抑制RAW264.7细胞向破骨细胞分化。

Objective:To explore the inhibitory effects of Luteolin on the differentiation of RAW264.7 cells into osteoclasts through Wnt/β-catenin pathway.Methods:The effects of different concentrations of Luteolin(1,2.5,5,10μmol/L)on the activity and osteoclast differentiation of RAW264.7 cells were detected.RAW264.7 cells were divided into Blank group,Osteoclast induction group,Luteolin group,Luteolin+siRNA NC group,Luteolin+Wnt-3a siRNA group,Luteolin+NC group and Luteolin+Wnt-3a group.Except for Blank group,cells in all other groups were induced to differentiate by osteoclastogenesis and subsequently treated with 5μmol/L luteolin and cell transfection.After 7 days of induction,TRAP staining and Rhodamine-phalloidin staining were used to detect the ability of RAW264.7 cells to differentiate into osteoclasts.RT-qPCR and Western blot were used to detect osteoclast differentiation markers(NFATc1,CTSK,c-Fos,MMP9,RANKL)and Wnt/β-catenin pathway(Wnt-3a,β-catenin)mRNA and protein levels.Results:Concentrations of 1,2.5,5μmol/L Luteolin had no significant effect on the activity of RAW264.7 cells(P>0.05),but could significantly inhibit the osteogenic differentiation of RAW264.7 cells.After osteoclast induced differentiation,the number of TRAP positive multinucleated cells was increased,F-actin rings and number of cell nuclei were increased,NFATc1,CTSK,c-Fos,MMP9 and RANKL mRNA and protein levels were increased,while Wnt-3a andβ-catenin mRNA and protein levels were decreased(all P<0.05).After treatment with Luteolin,the number of TRAP positive multinucleated cells was decreased,F-actin rings and number of cell nuclei were decreased,NFATc1,CTSK,c-Fos,MMP9 and RANKL mRNA and protein levels were decreased,while Wnt-3a andβ-catenin mRNA and protein levels were increased(all P<0.05).Knocking down Wnt-3a expression could weaken the effect of magnolol on the above indicators(all P<0.05).Overexpression of Wnt-3a could enhance the effect of magnolol on the above indicators(all P<0.05).Conclusion:Luteolin inhibited the differentiation of RAW264.7 cells into osteoclasts through Wnt/β-catenin pathway.