藁本内酯抑制RANKL诱导RAW264.7向破骨细胞分化及其与GPER相关机制

Ligustilide Inhibits RANKL-Induced Osteoclast Differentiation in RAW264.7 Cells and Its Mechanism Related by GPER

目的观察藁本内酯(LIG)对核因子κB受体活化因子配体(RANKL)诱导RAW264.7向破骨细胞分化的影响,并探讨该作用与G蛋白偶联雌激素受体(GPER)的相关机制。方法体外培养RAW264.7细胞,RANKL诱导破骨细胞分化,并用LIG进行干预。通过抗酒石酸酸性磷酸酶(TRAP)活性检测和TRAP染色法评价破骨细胞形成和分化能力;qPCR法检测GPER及破骨细胞相关基因mRNA水平;Western blot法检测GPER蛋白表达;采用GPER特异性拮抗剂G36进行干预,观察LIG干预破骨细胞分化的作用变化。结果LIG浓度为10μmol/L时,TRAP活性检测结果显示,TRAP活性显著降低(P<0.01);TRAP染色结果显示,与RANKL组相比,LIG组TRAP阳性细胞形成减少(P<0.001);qPCR检测结果显示,与RANKL组相比,LIG组树突状细胞-特异性跨膜蛋白(DC-STAMP)、活化T细胞核因子(NFATc1)、组织蛋白酶K(CTSK)和核因子κB受体(RANK)的mRNA水平显著降低(P<0.05,P<0.001),而GPER mRNA表达明显升高(P<0.001);Western blot结果显示,与RANKL组相比,LIG组GPER蛋白表达升高(P<0.001)。与LIG组比较,LIG+G36组TRAP阳性细胞数升高(P<0.01),TRAP活性增强(P<0.05),DC-STAMP、NFATc1、CTSK、RANK mRNA的表达升高(P<0.05)。结论LIG能够抑制RANKL诱导RAW264.7向破骨细胞分化,其机制可能是促进GPER表达,减少RANK和下游转录因子NFATc1的表达,抑制破骨细胞分化和骨吸收功能。

OBJECTIVE To observe the effect of ligustilide(LIG)on the differentiation of RAW264.7 cells into osteoclasts induced by receptor activator of nuclear factor-κB ligand(RANKL),and to explore the mechanism from the perspective of G protein coupled estrogen receptor(GPER).METHODS RAW264.7 cells were cultured in vitro and osteoclast-like cells differentiation were induced by RANKL.Osteoclast formation and differentiation were identified by tartrate resistant acid phosphatase(TRAP)staining and TRAP enzyme activity.Expressions of GPER and osteoclast differentiation related marker genes were observed by qPCR.GPER protein was assessed by Western blot.The GPER specific antagonist G36 was utilized to detect the effect of LIG on osteoclast differentiation through TRAP staining,TRAP enzyme activity,qPCR and phalloidin staining.RESULTS TRAP enzyme activity of LIG(10μmol/L)group was much lower than that of RANKL group(P<0.01).TRAP staining showed that the number of TRAP positive cells of LIG group was smaller than that of RANKL group(P<0.001).The qPCR results showed that the expressions of DC-STAMP,NFATc1,CTSK and RANK mRNA of LIG group were significantly lower than those of RANKL group(P<0.05,P<0.001),and the expression of GPER mRNA was significantly up-regulated(P<0.001).Western blot showed that the expression of GPER protein of LIG group was higher than that of RANKL group(P<0.001).After the treatment with G36,the effect of LIG on osteoclast-like cells differentiation were attenuated,the number of TRAP positive cells increased(P<0.01),TRAP enzyme activity was enhanced(P<0.05)and the mRNA expressions of DC-STAMP,NFATc1,CTSK and RANK significantly increased(P<0.05).CONCLUSION LIG can promote the expression of GPER,reduce the expression of RANK and downstream transcription factor NFATc1 in RANKL induced osteoclast,which may be one of the mechanisms involved in LIG suppressing osteoclast differentiation and bone resorption.